genes the environment and disease

National Center for Biotechnology Information NCBI http://www.ncbi.nlm.nih.gov/ Broad access to biomedical and genomic information, literature PubMed, sequence databases, software for a

principles of human Genetics

J Larry Jameson, Peter Kopp

IMPACT OF GENETICS AND GENOMICS ON MEDICAL PRACTICE

The prevalence of genetic diseases, combined with their potential

severity and chronic nature, imposes great human, social, and financial

burdens on society Human genetics refers to the study of individual

genes, their role and function in disease, and their mode of inheritance

Genomics refers to an organism’s entire genetic information, the

genome, and the function and interaction of DNA within the genome,

as well as with environmental or nongenetic factors, such as a person’s

lifestyle With the characterization of the human genome, genomics

complements traditional genetics in our efforts to elucidate the

etiol-ogy and pathogenesis of disease and to improve therapeutic

inter-ventions and outcomes Following impressive advances in genetics,

genomics, and health care information technology, the consequences

of this wealth of knowledge for the practice of medicine are profound

and play an increasingly prominent role in the diagnosis, prevention,

Personalized medicine, the customization of medical decisions to an

individual patient, relies heavily on genetic information For example,

a patient’s genetic characteristics (genotype) can be used to optimize

drug therapy and predict efficacy, adverse events, and drug dosing of

profile of a malignancy allows the selection of therapies that target

mutated or overexpressed signaling molecules Although still

inves-tigational, genomic risk prediction models for common diseases are

beginning to emerge

Genetics has traditionally been viewed through the window of

rela-tively rare single-gene diseases These disorders account for ~10% of

pediatric admissions and childhood mortality Historically, genetics

has focused predominantly on chromosomal and metabolic disorders,

reflecting the long-standing availability of techniques to diagnose

these conditions For example, conditions such as trisomy 21 (Down’s

syndrome) or monosomy X (Turner’s syndrome) can be diagnosed

(e.g., phenylketonuria, familial hypercholesterolemia) are diagnosed

using biochemical analyses The advances in DNA diagnostics have

extended the field of genetics to include virtually all medical

special-ties and have led to the elucidation of the pathogenesis of numerous

monogenic disorders In addition, it is apparent that virtually every

medical condition has a genetic component As is often evident from

a patient’s family history, many common disorders such as

hyperten-sion, heart disease, asthma, diabetes mellitus, and mental illnesses are

significantly influenced by the genetic background These polygenic or

multifactorial (complex) disorders involve the contributions of many

different genes, as well as environmental factors that can modify

elucidated numerous disease-associated loci and are providing novel

insights into the allelic architecture of complex traits These studies

have been facilitated by the availability of comprehensive catalogues of

human single-nucleotide polymorphism (SNP) haplotypes generated

through the HapMap Project The sequencing of whole genomes or

exomes (the exons within the genome) is increasingly used in the

clini-cal realm in order to characterize individuals with complex

undiag-nosed conditions or to characterize the mutational profile of advanced

malignancies in order to select better targeted therapies

Cancer has a genetic basis because it results from acquired somatic

mutations in genes controlling growth, apoptosis, and cellular

can-cers is associated with a hereditary predisposition Characterization

of the genome (and epigenome) in various malignancies has led to fundamental new insights into cancer biology and reveals that the genomic profile of mutations is in many cases more important in determining the appropriate chemotherapy than the organ in which the tumor originates Hence, comprehensive mutational profiling of malignancies has increasing impact on cancer taxonomy, the choice

of targeted therapies, and improved outcomes

Genetic and genomic approaches have proven invaluable for the detection of infectious pathogens and are used clinically to identify agents that are difficult to culture such as mycobacteria, viruses, and parasites, or to track infectious agents locally or globally In many cases, molecular genetics has improved the feasibility and accuracy of diagnostic testing and is beginning to open new avenues for therapy,

genetics has also provided the opportunity to characterize the

microbi-ome, a new field that characterizes the population dynamics of

bacte-ria, viruses, and parasites that coexist with humans and other animals

(Chap 86e) Emerging data indicate that the microbiome has cant effects on normal physiology as well as various disease states

signifi-Molecular biology has significantly changed the treatment of human disease Peptide hormones, growth factors, cytokines, and vaccines can now be produced in large amounts using recombinant DNA technol-ogy Targeted modifications of these peptides provide the practitioner with improved therapeutic tools, as illustrated by genetically modified insulin analogues with more favorable kinetics Lastly, there is reason

to believe that a better understanding of the genetic basis of human disease will also have an increasing impact on disease prevention

The astounding rate at which new genetic information is being generated creates a major challenge for physicians, health care provid-ers, and basic investigators Although many functional aspects of the genome remain unknown, there are many clinical situations where sufficient evidence exits for the use of genetic and genomic informa-tion to optimize patient care and treatment Much genetic information resides in databases or is being published in basic science journals

Databases provide easy access to the expanding information about the

example, several thousand monogenic disorders are summarized in a

large, continuously evolving compendium, referred to as the Online

Mendelian Inheritance in Man (OMIM) catalogue (Table 82-1) The

ongoing refinement of bioinformatics is simplifying the analysis and access to this daunting amount of new information

THE HUMAN GENOME Structure of the Human Genome • Human Genome Project The Human Genome Project was initiated in the mid-1980s as an ambitious effort

to characterize the entire human genome Although the prospect of determining the complete sequence of the human genome seemed daunting several years ago, technical advances in DNA sequencing and bioinformatics led to the completion of a draft human sequence

in 2000 and the completion of the DNA sequence for the last of the human chromosomes in May 2006 Currently, facilitated by rapidly decreasing costs for comprehensive sequence analyses and improve-ment of bioinformatics pipelines for data analysis, the sequencing

of whole genomes and exomes is used with increasing frequency in the clinical setting The scope of a whole genome sequence analysis can be illustrated by the following analogy Human DNA consists of

~3 billion base pairs (bp) of DNA per haploid genome, which is nearly

1000-fold greater than that of the Escherichia coli genome If the

human DNA sequence were printed out, it would correspond to about

120 volumes of Harrison’s Principles of Internal Medicine.

In addition to the human genome, the genomes of numerous organisms have been sequenced completely (~4000) or partially (~10,000) (Genomes Online Database [GOLD]; Table 82-1) They

include, among others, eukaryotes such as the mouse (Mus musculus),

82

part 3: Genes, the Environment, and Disease

National Center for

Biotechnology Information

(NCBI)

http://www.ncbi.nlm.nih.gov/ Broad access to biomedical and genomic information, literature (PubMed), sequence

databases, software for analyses of nucleotides and proteins Extensive links to other databases, genome resources, and tutorialsNational Human Genome

Research Institute

http://www.genome.gov/ An institute of the National Institutes of Health focused on genomic and genetic research;

links providing information about the human genome sequence, genomes of other organisms, and genomic research

Catalog of Published

Genome-Wide Association Studies

http://www.genome.gov/

GWAStudies/ Published high-resolution genome-wide association studies (GWAS)Ensembl Genome browser http://www.ensembl.org Maps and sequence information of eukaryotic genomes

Online Mendelian Inheritance

in Man http://www.ncbi.nlm.nih.gov/ omim Online compendium of Mendelian disorders and human genes causing genetic disorders

Office of Biotechnology

Activities, National Institutes

of Health

http://oba.od.nih.gov/oba Information about recombinant DNA and gene transfer; medical, ethical, legal, and

social issues raised by genetic testing; medical, ethical, legal, and social issues raised by xenotransplantation

American College of Medical

Genetics and Genomics

http://www.acmg.net/ Extensive links to other databases relevant for the diagnosis, treatment, and prevention of

genetic diseaseAmerican Society of Human

Genetics http://www.ashg.org Information about advances in genetic research, professional and public education, social and scientific policies

Cancer Genome Anatomy

MITOMAP, a human

mitochon-drial genome database http://www.mitomap.org/ A compendium of polymorphisms and mutations of the human mitochondrial DNA

International HapMap Project http://www.hapmap.org/ Catalogue of haplotypes in different ethnic groups relevant for association studies and

pharmacogenomicsENCODE http://www.genome.gov/10005107 Encyclopedia of DNA Elements; catalogue of all functional elements in the human genome

Dolan DNA Learning

Center, Cold Spring Harbor

Laboratories

http://www.dnalc.org/ Educational material about selected genetic disorders, DNA, eugenics, and genetic origin

The Online Metabolic and

Molecular Bases of Inherited

Disease (OMMBID)

http://www.ommbid.com/ Online version of the comprehensive text on the metabolic and molecular bases of

inherited diseaseOnline Mendelian Inheritance

in Animals (OMIA) http://omia.angis.org.au/ Online compendium of Mendelian disorders in animals

The Jackson Laboratory http://www.jax.org/ Information about murine models and the mouse genome

http://www.informatics.jax.org Mouse genome informatics

Note: Databases are evolving constantly Pertinent information may be found by using links listed in the few selected databases.

Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila

melanogaster; bacteria (e.g., E coli); and Archaea, viruses, organelles

(mitochondria, chloroplasts), and plants (e.g., Arabidopsis thaliana)

Genomic information of infectious agents has significant impact for

the characterization of infectious outbreaks and epidemics Other

ram-ifications arising from the availability of genomic data include, among

others, (1) the comparison of entire genomes (comparative genomics),

(2) the study of large-scale expression of RNAs (functional genomics)

and proteins (proteomics) to detect differences between various tissues

in health and disease, (3) the characterization of the variation among

individuals by establishing catalogues of sequence variations and SNPs

(HapMap Project), and (4) the identification of genes that play critical

roles in the development of polygenic and multifactorial disorders

cHromosomes The human genome is divided into 23 different

chro-mosomes, including 22 autosomes (numbered 1–22) and the X and

contain two homologous sets of 22 autosomes and a pair of sex

chro-mosomes Females have two X chromosomes (XX), whereas males

have one X and one Y chromosome (XY) As a consequence of meiosis,

germ cells (sperm or oocytes) are haploid and contain one set of 22

autosomes and one of the sex chromosomes At the time of

fertiliza-tion, the diploid genome is reconstituted by pairing of the homologous

chromosomes from the mother and father With each cell division

(mitosis), chromosomes are replicated, paired, segregated, and divided into two daughter cells

structure of Dna DNA is a double-stranded helix composed of four different bases: adenine (A), thymidine (T), guanine (G), and cyto-sine (C) Adenine is paired to thymidine, and guanine is paired to cytosine, by hydrogen bond interactions that span the double helix (Fig 82-1) DNA has several remarkable features that make it ideal for the transmission of genetic information It is relatively stable, and the double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division Complementarity also allows the transmission of genetic information

so-called sense or coding strand of the DNA double helix and is lated into proteins by ribosomes

trans-The presence of four different bases provides surprising genetic diversity In the protein-coding regions of genes, the DNA bases are arranged into codons, a triplet of bases that specifies a particular amino acid It is possible to arrange the four bases into 64 different

acids, or a regulatory signal such as initiation and stop of translation Because there are more codons than amino acids, the genetic code is degenerate; that is, most amino acids can be specified by several differ-ent codons By arranging the codons in different combinations and in

various lengths, it is possible to generate the tremendous diversity of

primary protein structure

DNA length is normally measured in units of 1000 bp (kilobases,

kb) or 1,000,000 bp (megabases, Mb) Not all DNA encodes genes In

fact, genes account for only ~10–15% of DNA Much of the

remain-ing DNA consists of sequences, often of highly repetitive nature, the

function of which is poorly understood These repetitive DNA regions,

along with nonrepetitive sequences that do not encode genes, serve, in

part, a structural role in the packaging of DNA into chromatin (i.e.,

DNA bound to histone proteins, and chromosomes) and exert

regula-tory functions (Fig 82-1)

Genes A gene is a functional unit that is regulated by transcription

(see below) and encodes an RNA product, which is most commonly,

but not always, translated into a protein that exerts activity within or

they conferred specific traits that are transmitted from one generation

to the next Increasingly, they are characterized based on expression in

various tissues (transcriptome) The size of genes is quite broad; some

genes are only a few hundred base pairs, whereas others are

extraordi-narily large (2 Mb) The number of genes greatly underestimates the

complexity of genetic expression, because single genes can generate

multiple spliced messenger RNA (mRNA) products (isoforms), which

are translated into proteins that are subject to complex

posttransla-tional modification such as phosphorylation Exons refer to the portion

of genes that are eventually spliced together to form mRNA Introns

refer to the spacing regions between the exons that are spliced out of

precursor RNAs during RNA processing The gene locus also includes

Metaphasechromosome Nucleosomefiber

Nucleosome coreHistone H2A, H2B, H4

Double-strand DNAwithout histones

Cytosine Guanine

Thymine Adenine

Supercoiledchromatin

p, short arm

HistoneH1

q, long arm

Centromere

SolenoidTelomere

C

H H

C P

H N

N N C C

H 3 C O

C H

H

C C

N C

N H O O O

N H

H

C C

C N

N N C C N

H

A

A T

T

G G

FIGURE 82-1 Structure of chromatin and chromosomes

Chromatin is composed of double-strand DNA that is wrapped

around histone and nonhistone proteins forming nucleosomes

The nucleosomes are further organized into solenoid structures

Chromosomes assume their characteristic structure, with short (p)

and long (q) arms at the metaphase stage of the cell cycle

regions that are necessary to control its expression (Fig 82-2) Current estimates predict 20,687 protein-coding genes in the human genome with an average of about four different coding transcripts per gene

Remarkably, the exome only constitutes 1.14% of the genome In tion, thousands of noncoding transcripts (RNAs of various length such

addi-as microRNAs and long noncoding RNAs), which function, at leaddi-ast

in part, as transcriptional and posttranscriptional regulators of gene expression, have been identified Aberrant expression of microRNAs has been found to play a pathogenic role in numerous diseases

sinGle-nucleotiDe PolymorPHisms An SNP is a variation of a single base pair in the DNA The identification of the ~10 million SNPs estimated to occur in the human genome has generated a catalogue

of common genetic variants that occur in human beings from distinct ethnic backgrounds (Fig 82-3) SNPs are the most common type of sequence variation and account for ~90% of all sequence variation

They occur on average every 100 to 300 bases and are the major source

of genetic heterogeneity Remarkably, however, the primary DNA sequence of humans has ~99.9% similarity compared to that of any other human SNPs that are in close proximity are inherited together

HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations The haplotype map information, referred to as HapMap,

is greatly facilitating GWAS designed to elucidate the complex tions among multiple genes and lifestyle factors in multifactorial dis-orders (see below) Moreover, haplotype analyses are useful to assess

interac-variations in responses to medications (pharmacogenomics) and

envi-ronmental factors, as well as the prediction of disease predisposition

coPy number variations Copy number variations (CNVs) are tively large genomic regions (1 kb to several Mb) that have been

been estimated that as many as 1500 CNVs, scattered throughout the genome, are present in an individual When comparing the genomes

of two individuals, approximately 0.4–0.8% of their genomes differ in terms of CNVs Of note, de novo CNVs have been observed between monozygotic twins, who otherwise have identical genomes Some CNVs have been associated with susceptibility or resistance to disease, and CNVs can be elevated in cancer cells

Replication of DNA and Mitosis Genetic information in DNA is mitted to daughter cells under two different circumstances: (1) somatic

trans-cells divide by mitosis, allowing the diploid (2n) genome to replicate

itself completely in conjunction with cell division; and (2) germ cells

(sperm and ova) undergo meiosis, a process that enables the reduction

of the diploid (2n) set of chromosomes to the haploid state (1n).

undergo DNA synthesis (S phase), during which the DNA in each chromosome is replicated, yielding two pairs of sister chromatids

(2n → 4n) The process of DNA synthesis requires stringent fidelity in

order to avoid transmitting errors to subsequent generations of cells

Genetic abnormalities of DNA mismatch/repair include xeroderma pigmentosum, Bloom’s syndrome, ataxia telangiectasia, and hereditary nonpolyposis colon cancer (HNPCC), among others Many of these disorders strongly predispose to neoplasia because of the rapid acquisi-

before entering mitosis At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase The two identical sister chromatids, held together at the centromere, divide and migrate

to opposite poles of the cell After formation of a nuclear membrane around the two separated sets of chromatids, the cell divides and two

daughter cells are formed, thus restoring the diploid (2n) state.

Assortment and Segregation of Genes During Meiosis Meiosis occurs only

in germ cells of the gonads It shares certain features with mitosis but involves two distinct steps of cell division that reduce the chromosome number to the haploid state In addition, there is active recombina-tion that generates genetic diversity During the first cell division, two

isease sister chromatids (2n → 4n) are formed for each chromosome pair

and there is an exchange of DNA between homologous paternal and

maternal chromosomes This process involves the formation of

chias-mata, structures that correspond to the DNA segments that cross over

there is at least one crossover on each chromosomal arm;

recombina-tion occurs more frequently in female meiosis than in male meiosis

Subsequently, the chromosomes segregate randomly Because there

of chromosomes Together with the genetic exchanges that occur

dur-ing recombination, chromosomal segregation generates tremendous

diversity, and each gamete is genetically unique The process of

recom-bination and the independent segregation of chromosomes provide

the foundation for performing linkage analyses, whereby one attempts

to correlate the inheritance of certain chromosomal regions (or linked

genes) with the presence of a disease or genetic trait (see below)

After the first meiotic division, which results in two daughter cells

(2n), the two chromatids of each chromosome separate during a

sec-ond meiotic division to yield four gametes with a haploid state (1n)

When the egg is fertilized by sperm, the two haploid sets are combined,

thereby restoring the diploid state (2n) in the zygote.

REGULATION OF GENE EXPRESSION

Regulation by Transcription Factors The expression of genes is regulated

by DNA-binding proteins that activate or repress transcription The

number of DNA sequences and transcription factors that regulate

transcription is much greater than originally anticipated Most genes

contain at least 15–20 discrete regulatory elements within 300 bp of

the transcription start site This densely packed promoter region often

contains binding sites for ubiquitous transcription factors such as

CAAT box/enhancer binding protein (C/EBP), cyclic AMP response

element–binding (CREB) protein, selective promoter factor 1 (Sp-1),

or activator protein 1 (AP-1) However, factors involved in cell-specific expression may also bind to these sequences Key regulatory elements may also reside at a large distance from the proximal promoter The

globin and the immunoglobulin genes, for example, contain locus

con-trol regions that are several kilobases away from the structural sequences

of the gene Specific groups of transcription factors that bind to these promoter and enhancer sequences provide a combinatorial code for regulating transcription In this manner, relatively ubiquitous factors interact with more restricted factors to allow each gene to be expressed and regulated in a unique manner that is dependent on developmental state, cell type, and numerous extracellular stimuli Regulatory factors also bind within the gene itself, particularly in the intronic regions The transcription factors that bind to DNA actually represent only the first

level of regulatory control Other proteins—co-activators and

co-repres-sors—interact with the DNA-binding transcription factors to generate

large regulatory complexes These complexes are subject to control by numerous cell-signaling pathways and enzymes, leading to phosphory-lation, acetylation, sumoylation, and ubiquitination Ultimately, the recruited transcription factors interact with, and stabilize, components

of the basal transcription complex that assembles at the site of the TATA box and initiator region This basal transcription factor complex consists of >30 different proteins Gene transcription occurs when RNA polymerase begins to synthesize RNA from the DNA template A large number of identified genetic diseases involve transcription factors

(Table 82-2)

The field of functional genomics is based on the concept that

under-standing alterations of gene expression under various physiologic and pathologic conditions provides insight into the underlying functional role of the gene By revealing specific gene expression profiles, this knowledge may be of diagnostic and therapeutic relevance The large-scale study of expression profiles, which takes advantage of microar-

ray and bead array technologies, is also referred to as transcriptomics

FIGURE 82-2 Flow of genetic information Multiple extracellular signals activate intracellular signal cascades that result in altered regulation

of gene expression through the interaction of transcription factors with regulatory regions of genes RNA polymerase transcribes DNA into RNA

that is processed to mRNA by excision of intronic sequences The mRNA is translated into a polypeptide chain to form the mature protein after

undergoing posttranslational processing CBP, CREB-binding protein; CoA, co-activator; COOH, carboxyterminus; CRE, cyclic AMP responsive ment; CREB, cyclic AMP response element–binding protein; GTF, general transcription factors; HAT, histone acetyl transferase; NH2, aminotermi-

ele-nus; RE, response element; TAF, TBP-associated factors; TATA, TATA box; TBP, TATA-binding protein

Transcription factor

RNA polymerase II

Nuclear receptor

GTF

factors Hormoneslight mechanical stressUV-light

Regulation of Gene Expression

mRNA Protein

NH2–

because the complement of mRNAs transcribed by the cellular genome

is called the transcriptome.

Most studies of gene expression have focused on the regulatory DNA elements of genes that control transcription However, it should

be emphasized that gene expression requires a series of steps, including mRNA processing, protein translation, and posttranslational modifi-cations, all of which are actively regulated (Fig 82-2)

Epigenetic Regulation of Gene Expression Epigenetics describes

mecha-nisms and phenotypic changes that are not a result of variation in the primary DNA nucleotide sequence, but are caused by second-ary modifications of DNA or histones These modifications include heritable changes such as X-inactivation and imprinting, but they can also result from dynamic posttranslational protein modifications in response to environmental influences such as diet, age, or drugs The epigenetic modifications result in altered expression of individual genes or chromosomal loci encompassing multiple genes The term

epigenome describes the constellation of covalent modifications of

DNA and histones that impact chromatin structure, as well as coding transcripts that modulate the transcriptional activity of DNA

non-Although the primary DNA sequence is usually identical in all cells of

an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell (transcriptome) and hence the protein expression profile (proteome)

Mechanistically, DNA and histone modifications can result in the

methyla-tion involves the addimethyla-tion of a methyl group to cytosine residues This is

FIGURE 82-4 The origin of haplotypes is due to repeated

recombi-nation events occurring in multiple generations Over time, this leads

to distinct haplotypes These haplotype blocks can often be

character-ized by genotyping selected Tag single-nucleotide polymorphisms

(SNPs), an approach that facilitates performing genome-wide

associa-tion studies (GWAS)

Splice site

Coding region, frameshift

FIGURE 82-3 Chromosome 7 is shown with the density of single-nucleotide polymorphisms (SNPs) and genes above A 200-kb region

in 7q31.2 containing the CFTR gene is shown below The CFTR gene contains 27 exons More than 1900 mutations in this gene have been found

in patients with cystic fibrosis A 20-kb region encompassing exons 4–9 is shown further amplified to illustrate the SNPs in this region

dinucleo-also exist in so-called CpG islands, stretches of DNA

characterized by a high CG content, which are found in the majority of human gene promoters CpG islands in promoter regions are typically unmethylated, and the lack of methylation facilitates transcription

Histone methylation involves the addition of a methyl group to lysine residues in histone proteins (Fig 82-7) Depending on the specific lysine residue being methyl-ated, this alters chromatin configuration, either making

it more open or tightly packed Acetylation of histone proteins is another well-characterized mechanism that results in an open chromatin configuration, which favors active transcription Acetylation is generally more dynamic than methylation, and many transcriptional activation complexes have histone acetylase activity, whereas repressor complexes often contain deacetylases and remove acetyl groups from histones Other histone modifications, whose effects are incompletely character-ized, include phosphorylation and sumoylation Lastly, noncoding RNAs that bind to DNA can have a signifi-cant impact on transcriptional activity

Physiologically, epigenetic mechanisms play an important role in several instances For example, X-inactivation refers to the relative silencing of one

of the two X chromosome copies present in females The inactivation process is a form of dosage compen-sation such that females (XX) do not generally express twice as many X-chromosomal gene products as males (XY) In a given cell, the choice

of which chromosome is inactivated occurs randomly in humans But once the maternal or paternal X chromosome is inactivated, it will remain inactive, and this information is transmitted with each cell

division The X-inactive specific transcript (Xist) gene encodes a large

noncoding RNA that mediates the silencing of the X chromosome from which it is transcribed by coating it with Xist RNA The inactive X chro-mosome is highly methylated and has low levels of histone acetylation.Epigenetic gene inactivation also occurs on selected chromosomal

regions of autosomes, a phenomenon referred to as genomic imprinting

Through this mechanism, a small subset of genes is only expressed in

a monoallelic fashion Imprinting is heritable and leads to the ential expression of one of the parental alleles, which deviates from the usual biallelic expression seen for the majority of genes Remarkably, imprinting can be limited to a subset of tissues Imprinting is medi-ated through DNA methylation of one of the alleles The epigenetic marks on imprinted genes are maintained throughout life, but during zygote formation, they are activated or inactivated in a sex-specific

expres-sion pattern in the fertilized egg and the subsequent mitotic diviexpres-sions Appropriate expression of imprinted genes is important for normal development and cellular functions Imprinting defects and uniparental disomy, which is the inheritance of two chromosomes or chromosomal regions from the same parent, are the cause of several developmental disorders such as Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman’s syndrome, and Prader-Willi syndrome (see

below) Monoallelic loss-of-function mutations in the GNAS1 gene lead

to Albright’s hereditary osteodystrophy (AHO) Paternal transmission

of GNAS1 mutations leads to an isolated AHO phenotype

(pseudop-seudohypoparathyroidism), whereas maternal transmission leads to AHO in combination with hormone resistance to parathyroid hor-mone, thyrotropin, and gonadotropins (pseudohypoparathyroidism type IA) These phenotypic differences are explained by tissue-specific

imprinting of the GNAS1 gene, which is expressed primarily from the

maternal allele in the thyroid, gonadotropes, and the proximal renal

tubule In most other tissues, the GNAS1 gene is expressed biallelically

1 2

Normal

Deleted Area

Duplicated Area

of the genome that have been duplicated or deleted Chromosome 8 is shown with

CNV detected by genomic hybridization An increase in the signal strength indicates a

duplication, a decrease reflects a deletion of the covered chromosomal regions

Homologous chromosomes

A B C D

a b c d

a b c d

a b c d

Chromatids

A B C D

A B C D

a b c d

A B C D

a b C d

Double cross-over

A B C D

a b c d

A B c D

a b C D

Cross-over

A B C D

a b c d

A B c d

a b c d

A B C D

a b C D

Recombination

in gametes

A B C D

a b c d

A B c d

a b C d

Recombination

in gametes

A B C D

a b c d

A B c D

FIGURE 82-6 Crossing-over and genetic recombination During

chiasma formation, either of the two sister chromatids on one

mosome pairs with one of the chromatids of the homologous

chro-mosome Genetic recombination occurs through crossing-over and

results in recombinant and nonrecombinant chromosome segments

in the gametes Together with the random segregation of the

mater-nal and patermater-nal chromosomes, recombination contributes to genetic

diversity and forms the basis of the concept of linkage

Transcription Factor Class Example Associated Disorder

Nuclear receptors Androgen receptor Complete or partial androgen insensitivity (recessive missense mutations)

Spinobulbar muscular atrophy (CAG repeat expansion)Zinc finger proteins WT1 WAGR syndrome: Wilms’ tumor, aniridia, genitourinary malformations, mental retardation

Basic helix-loop-helix MITF Waardenburg’s syndrome type 2A

Homeobox IPF1 Maturity onset of diabetes mellitus type 4 (heterozygous mutation/haploinsufficiency)

Pancreatic agenesis (homozygous mutation)Leucine zipper Retina leucine zipper

(NRL) Autosomal dominant retinitis pigmentosaHigh mobility group (HMG)

Forkhead HNF4α, HNF1α, HNF1β Maturity onset of diabetes mellitus types 1, 3, 5

T-box TBX5 Holt-Oram syndrome (thumb anomalies, atrial or ventricular septum defects, phocomelia)

Cell cycle control proteins P53 Li-Fraumeni syndrome, other cancers

Co-activators CREB binding protein

General transcription

factors TATA-binding protein (TBP) Spinocerebellar ataxia 17 (CAG expansion)

Transcription elongation

factor VHL von Hippel–Lindau syndrome (renal cell carcinoma, pheochromocytoma, pancreatic tumors, hemangioblastomas)

Autosomal dominant inheritance, somatic inactivation of second allele (Knudson two-hit model)Runt CBFA2 Familial thrombocytopenia with propensity to acute myelogenous leukemia

Chimeric proteins due to

translocations PML-RAR Acute promyelocytic leukemia t(15;17)(q22;q11.2-q12) translocation

Abbreviations: CREB, cAMP responsive element–binding protein; HNF, hepatocyte nuclear factor; PML, promyelocytic leukemia; RAR, retinoic acid receptor; SRY, sex-determining region

Y; VHL, von Hippel–Lindau.

In patients with isolated renal resistance to parathyroid hormone

(pseu-dohypoparathyroidism type IB), defective imprinting of the GNAS1

gene results in decreased Gsα expression in the proximal renal tubules

Rett’s syndrome is an X-linked dominant disorder resulting in

devel-opmental regression and stereotypic hand movements in affected girls

It is caused by mutations in the MECP2 gene, which encodes a

methyl-binding protein The ensuing aberrant methylation results in abnormal gene expression in neurons, which are otherwise normally developed

Remarkably, epigenetic differences also occur among monozygotic twins Although twins are epigenetically indistinguishable during the

early years of life, older monozygotic twins exhibit differences in the overall content and genomic distribution of DNA methyla-tion and histone acetylation, which would

be expected to alter gene expression in ous tissues

vari-In cancer, the epigenome is characterized

by simultaneous losses and gains of DNA methylation in different genomic regions,

as well as repressive histone modifications

Hyper- and hypomethylation are associated with mutations in genes that control DNA methylation Hypomethylation is thought to remove normal control mechanisms that pre-vent expression of repressed DNA regions

It is also associated with genomic ity Hypermethylation, in contrast, results

instabil-in the silencinstabil-ing of CpG islands instabil-in promoter regions of genes, including tumor-suppressor genes Epigenetic alterations are considered

to be more easily reversible compared to genetic changes, and modification of the epig-enome with demethylating agents and histone deacetylases is being explored in clinical trials

MODELS OF GENETIC DISEASE

Several organisms have been studied

exten-sively as genetic models, including M

mus-culus (mouse), D melanogaster (fruit fly),

C elegans (nematode), S cerevisiae (baker’s

yeast), and E coli (colonic bacterium) The

ability to use these evolutionarily distant organisms as genetic models that are relevant

Histone Modifications

Cytosine Methylation

Acetylation Phosphorylation Sumoylation Methylation

NH 2

FIGURE 82-7 Epigenetic modifications of DNA and histones Methylation of cytosine

resi-dues is associated with gene silencing Methylation of certain genomic regions is inherited

(imprinting), and it is involved in the silencing of one of the two X chromosomes in females

(X-inactivation) Alterations in methylation can also be acquired, e.g., in cancer cells Covalent

posttranslational modifications of histones play an important role in altering DNA

accessibil-ity and chromatin structure and hence in regulating transcription Histones can be reversibly

modified in their amino-terminal tails, which protrude from the nucleosome core particle, by

acetylation of lysine, phosphorylation of serine, methylation of lysine and arginine residues, and

sumoylation Acetylation of histones by histone acetylases (HATs), e.g., leads to unwinding of

chromatin and accessibility to transcription factors Conversely, deacetylation by histone

deacet-ylases (HDACs) results in a compact chromatin structure and silencing of transcription

or oocytes); these can be transmitted to progeny Alternatively, mutations can occur during embryogenesis or in somatic tissues Mutations that occur during development

lead to mosaicism, a situation in which tissues

are composed of cells with different genetic constitutions If the germline is mosaic, a mutation can be transmitted to some progeny but not others, which sometimes leads to con-fusion in assessing the pattern of inheritance Somatic mutations that do not affect cell survival can sometimes be detected because

of variable phenotypic effects in tissues (e.g., pigmented lesions in McCune-Albright syn-drome) Other somatic mutations are asso-ciated with neoplasia because they confer a growth advantage to cells Epigenetic events may also influence gene expression or facili-tate genetic damage With the exception of triplet nucleotide repeats, which can expand (see below), mutations are usually stable

Mutations are structurally diverse—they can involve the entire genome, as in triploidy (one extra set of chromosomes), or gross numerical or structural alterations in chro-

Large deletions may affect a portion of a gene or an entire gene, or, if several genes are

involved, they may lead to a contiguous gene

syndrome Unequal crossing-over between

homologous genes can result in fusion gene mutations, as illustrated by color blindness Mutations involving single nucleotides are

referred to as point mutations Substitutions are called transitions if a purine is replaced by

another purine base (A ↔ G) or if a pyrimidine

is replaced by another pyrimidine (C ↔ T) Changes from a purine to a pyrimidine, or

vice versa, are referred to as transversions

If the DNA sequence change occurs in a coding region and alters an amino acid, it is

called a missense mutation Depending on the

functional consequences of such a missense mutation, amino acid substitutions in differ-ent regions of the protein can lead to distinct phenotypes

Mutations can occur in all domains of a

within the coding region leads to an amino acid substitution if the

pre-mature stop codon result in a truncated protein Large deletions may affect a portion of a gene or an entire gene, whereas small deletions and insertions alter the reading frame if they do not represent a mul-tiple of three bases These “frameshift” mutations lead to an entirely altered carboxy terminus Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites Mutations may also be found in the regulatory sequences of genes, resulting in reduced or enhanced gene transcription

Certain DNA sequences are particularly susceptible to esis Successive pyrimidine residues (e.g., T-T or C-C) are subject

mutagen-to the formation of ultraviolet light–induced phomutagen-toadducts If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation In this case, methylation of the cytosine is associated with

to human physiology reflects a surprising conservation of genetic

pathways and gene function Transgenic mouse models have been

particularly valuable, because many human and mouse genes exhibit

similar structure and function and because manipulation of the mouse

genome is relatively straightforward compared to that of other

mam-malian species Transgenic strategies in mice can be divided into two

main approaches: (1) expression of a gene by random insertion into the

genome, and (2) deletion or targeted mutagenesis of a gene by

homolo-gous recombination with the native endogenous gene (knock-out,

knock-in) Previous versions of this chapter provide more detail about

the technical principles underlying the development of genetically

modi-fied animals Several databases provide comprehensive information

about natural and transgenic animal models, the associated phenotypes,

and integrated genetic, genomic, and biologic data (Table 82-1)

TRANSMISSION OF GENETIC DISEASE

Origins and Types of Mutations A mutation can be defined as any

change in the primary nucleotide sequence of DNA regardless of

Active Unmethylated

mat pat

Inactive Methylated

Maternal somatic cell

Inactive Methylated

pat mat

Active Unmethylated Paternal somatic cell

Inactive Methylated

pat mat

Active Unmethylated Zygote

Active

Unmethylated

mat pat

Active Unmethylated

Inactive Methylated

pat mat

Inactive Methylated

Germline development:

Imprint reset

FIGURE 82-8 A few genomic regions are imprinted in a parent-specific fashion The

unmethylated chromosomal regions are actively expressed, whereas the methylated regions

are silenced In the germline, the imprint is reset in a parent-specific fashion: both

chromo-somes are unmethylated in the maternal (mat) germline and methylated in the paternal (pat)

germline In the zygote, the resulting imprinting pattern is identical with the pattern in the

somatic cells of the parents

it is often unclear whether it creates

a mutation with functional quences or a benign polymorphism In this situation, the sequence alteration

conse-is described as variant of unknown

sig-nificance (VUS).

mutation rates Mutations represent

an important cause of genetic sity as well as disease Mutation rates are difficult to determine in humans because many mutations are silent and because testing is often not adequate

diver-to detect the phenotypic consequences

Mutation rates vary in different genes but are estimated to occur at a rate of

mutation rates (as opposed to somatic mutations) are relevant in the trans-mission of genetic disease Because the population of oocytes is estab-lished very early in development, only

~20 cell divisions are required for pleted oogenesis, whereas spermato-genesis involves ~30 divisions by the time of puberty and 20 cell divisions each year thereafter Consequently, the probability of acquiring new point mutations is much greater in the male germline than the female

Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan’s syn-drome, neurofibromatosis) It is estimated that about 1 in 10 sperm carries a new deleterious mutation The rates for new mutations are calculated most readily for autosomal dominant and X-linked

monogenic diseases are relatively rare, new mutations account for

a significant fraction of cases This is important in the context of genetic counseling, because a new mutation can be transmitted

to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children An exception to this is when the new mutation occurs early in germline

development, leading to gonadal mosaicism.

FIGURE 82-9 Point mutations causing β thalassemia as example of allelic heterogeneity The

β-globin gene is located in the globin gene cluster Point mutations can be located in the promoter,

the CAP site, the 5’-untranslated region, the initiation codon, each of the three exons, the introns,

or the polyadenylation signal Many mutations introduce missense or nonsense mutations, whereas

others cause defective RNA splicing Not shown here are deletion mutations of the β-globin gene or

mutations; A, Poly A signal

an enhanced rate of deamination to uracil, which is then replaced with

thymine This C → T transition (or G → A on the opposite strand)

accounts for at least one-third of point mutations associated with

polymorphisms and mutations In addition to the fact that certain

types of mutations (C → T or G → A) are relatively common, the

nature of the genetic code also results in overrepresentation of certain

amino acid substitutions

Polymorphisms are sequence variations that have a frequency of

at least 1% Usually, they do not result in a perceptible phenotype

Often they consist of single base-pair substitutions that do not alter

the protein coding sequence because of the degenerate nature of the

genetic code (synonymous polymorphism), although it is possible

that some might alter mRNA stability, translation, or the amino

acid sequence (nonsynonymous polymorphism) (Fig 82-10) The

detection of sequence variants poses a practical problem because

Wild-type

AA

DNAAGCA

CTAS

TCGH

CACA

GCTR

CGGE

GAGG

GGCE

GAAN

AATE

GAGSAGC

Silent mutation

AA

DNAA

GCAL

CTCL

CTAS

TCGH

CACA

GCTR

CGT

E

GAGG

GGCE

GAAN

AATE

GAGSAGC

Missense mutation

AA

DNAA

GCAL

CTCL

CTAS

TCGH

CACA

GCTP

C GE

GAGG

GGCE

GAAN

AATE

GAGSAGC

Nonsense mutation

AA

DNAA

GCAL

CTCL

CTAS

TCGH

CACA

GCTR

CGGE

GAGG

GGCX

TAA AAT GAG AGC

1 bp Deletion with frameshift

AA

DNAA

GCAL

CTCL

CTAR

CGCT

ACGL

CTCG

GGGR

AGGA

GCGK

AAAM

ATGRAGA GC

FIGURE 82-10 A Examples of mutations The coding strand is shown with the encoded amino acid sequence B Chromatograms of sequence

analyses after amplification of genomic DNA by polymerase chain reaction

unequal crossinG-over Normally, DNA recombination in germ cells

occurs with remarkable fidelity to maintain the precise junction sites

for the exchanged DNA sequences (Fig 82-6) However,

mispair-ing of homologous sequences leads to unequal crossover, with gene

duplication on one of the chromosomes and gene deletion on

the other chromosome A significant fraction of growth hormone

(GH) gene deletions, for example, involve unequal crossing-over

(Chap 402) The GH gene is a member of a large gene cluster that

includes a GH variant gene as well as several structurally related

cho-rionic somatomammotropin genes and pseudogenes (highly

homolo-gous but functionally inactive relatives of a normal gene) Because such

gene clusters contain multiple homologous DNA sequences arranged

in tandem, they are particularly prone to undergo recombination and,

consequently, gene duplication or deletion On the other hand,

dupli-cation of the PMP22 gene because of unequal crossing-over results

in increased gene dosage and type IA Charcot-Marie-Tooth disease

Unequal crossing-over resulting in deletion of PMP22 causes a distinct

Glucocorticoid-remediable aldosteronism (GRA) is caused by a

gene fusion or rearrangement involving the genes that encode

aldo-sterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1),

normally arranged in tandem on chromosome 8q These two genes

are 95% identical, predisposing to gene duplication and deletion by

unequal crossing-over The rearranged gene product contains the

regulatory regions of 11β-hydroxylase fused to the coding sequence of

aldosterone synthetase Consequently, the latter enzyme is expressed

in the adrenocorticotropic hormone (ACTH)–dependent zona

fas-ciculata of the adrenal gland, resulting in overproduction of

Gene conversion refers to a nonreciprocal exchange of homologous

genetic information It has been used to explain how an internal portion

of a gene is replaced by a homologous segment copied from another

allele or locus; these genetic alterations may range from a few

nucleo-tides to a few thousand nucleonucleo-tides As a result of gene conversion, it is

possible for short DNA segments of two chromosomes to be identical,

even though these sequences are distinct in the parents A practical

consequence of this phenomenon is that nucleotide substitutions can

occur during gene conversion between related genes, often altering the

function of the gene In disease states, gene conversion often involves

intergenic exchange of DNA between a gene and a related pseudogene

For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a

non-functional pseudogene (CYP21A1P) Many of the nucleotide

substitu-tions that are found in the CYP21A2 gene in patients with congenital

adrenal hyperplasia correspond to sequences that are present in the

CYP21A1P pseudogene, suggesting gene conversion as one cause of

mutagenesis In addition, mitotic gene conversion has been suggested

as a mechanism to explain revertant mosaicism in which an inherited

mutation is “corrected” in certain cells For example, patients with

autosomal recessive generalized atrophic benign epidermolysis bullosa

have acquired reverse mutations in one of the two mutated COL17A1

alleles, leading to clinically unaffected patches of skin

insertions anD Deletions Although many instances of insertions and

deletions occur as a consequence of unequal crossing-over, there is

also evidence for internal duplication, inversion, or deletion of DNA

sequences The fact that certain deletions or insertions appear to occur

repeatedly as independent events indicates that specific regions within

the DNA sequence predispose to these errors For example, certain

regions of the DMD gene, which encodes dystrophin, appear to be hot

Some regions within the human genome are rearrangement hot spots

and lead to CNVs

errors in Dna rePair Because mutations caused by defects in DNA

repair accumulate as somatic cells divide, these types of mutations

are particularly important in the context of neoplastic disorders

(Chap 102e) Several genetic disorders involving DNA repair enzymes

underscore their importance Patients with xeroderma pigmentosum

have defects in DNA damage recognition or in the nucleotide excision

and is extraordinarily sensitive to the mutagenic effects of ultraviolet irradiation More than 10 different genes have been shown to cause the different forms of xeroderma pigmentosum This finding is consistent with the earlier classification of this disease into different complemen-tation groups in which normal function is rescued by the fusion of cells derived from two different forms of xeroderma pigmentosum

Ataxia telangiectasia causes large telangiectatic lesions of the face, cerebellar ataxia, immunologic defects, and hypersensitivity to ion-

mutated (ATM) gene reveals that it is homologous to genes involved

in DNA repair and control of cell cycle checkpoints Mutations in

the ATM gene give rise to defects in meiosis as well as increasing

susceptibility to damage from ionizing radiation Fanconi’s anemia

is also associated with an increased risk of multiple acquired genetic abnormalities It is characterized by diverse congenital anomalies and

a strong predisposition to develop aplastic anemia and acute

chromosomal breaks caused by a defect in genetic recombination At least 13 different complementation groups have been identified, and the loci and genes associated with Fanconi’s anemia have been cloned HNPCC (Lynch’s syndrome) is characterized by autosomal dominant transmission of colon cancer, young age (<50 years) of presentation, predisposition to lesions in the proximal large bowel, and associated malignancies such as uterine cancer and ovarian cancer HNPCC is predominantly caused by mutations in one of several different mis-

match repair (MMR) genes including MutS homologue 2 (MSH2), MutL homologue 1 and 6 (MLH1, MLH6), MSH6, PMS1, and PMS2

(Chap 110) These proteins are involved in the detection of tide mismatches and in the recognition of slipped-strand trinucleotide repeats Germline mutations in these genes lead to microsatellite instability and a high mutation rate in colon cancer Genetic screening tests for this disorder are now being used for families considered to be

colonoscopy and the implementation of prevention strategies using nonsteroidal anti-inflammatory drugs

unstable Dna sequences Trinucleotide repeats may be unstable and

expand beyond a critical number Mechanistically, the expansion is thought to be caused by unequal recombination and slipped mispair-ing A premutation represents a small increase in trinucleotide copy number In subsequent generations, the expanded repeat may increase further in length and result in an increasingly severe phenotype, a

process called dynamic mutation (see below for discussion of

antici-pation) Trinucleotide expansion was first recognized as a cause of the fragile X syndrome, one of the most common causes of intel-lectual disability Other disorders arising from a similar mechanism

Malignant cells are also characterized by genetic instability, indicating

a breakdown in mechanisms that regulate DNA repair and the cell cycle

Functional Consequences of Mutations Functionally, mutations can

be broadly classified as gain-of-function and loss-of-function tions Gain-of-function mutations are typically dominant (e.g., they result in phenotypic alterations when a single allele is affected) Inactivating mutations are usually recessive, and an affected indi-vidual is homozygous or compound heterozygous (e.g., carrying two different mutant alleles of the same gene) for the disease-causing

muta-mutations Alternatively, mutation in a single allele can result in

hap-loinsufficiency, a situation in which one normal allele is not sufficient

to maintain a normal phenotype Haploinsufficiency is a commonly observed mechanism in diseases associated with mutations in tran-scription factors (Table 82-2) Remarkably, the clinical features among patients with an identical mutation in a transcription factor often vary significantly One mechanism underlying this variability consists in the influence of modifying genes Haploinsufficiency can also affect the expression of rate-limiting enzymes For example, haploinsuf-ficiency in enzymes involved in heme synthesis can cause porphyrias

(Chap 430)

An increase in dosage of a gene product may also result in disease,

as illustrated by the duplication of the DAX1 gene in dosage-sensitive

loss of function due to a dominant-negative effect In this case, the

mutated allele interferes with the function of the normal gene product

by one of several different mechanisms: (1) a mutant protein may

interfere with the function of a multimeric protein complex, as

illus-trated by mutations in type 1 collagen (COL1A1, COL1A2) genes in

binding sites on proteins or promoter response elements, as illustrated

by thyroid hormone resistance, a disorder in which inactivated thyroid

hormone receptor β binds to target genes and functions as an

the abnormally folded proteins are trapped within the endoplasmic

reticulum and ultimately cause cellular damage

Genotype and Phnotype • alleles, GenotyPes, anD HaPlotyPes An

observed trait is referred to as a phenotype; the genetic information

defining the phenotype is called the genotype Alternative forms of a gene

or a genetic marker are referred to as alleles Alleles may be polymorphic

variants of nucleic acids that have no apparent effect on gene expression

or function In other instances, these variants may have subtle effects

on gene expression, thereby conferring adaptive advantages associated

with genetic diversity On the other hand, allelic variants may reflect

mutations that clearly alter the function of a gene product The common

Glu6Val (E6V) sickle cell mutation in the β-globin gene and the ΔF508

deletion of phenylalanine (F) in the CFTR gene are examples of allelic

variants of these genes that result in disease Because each individual has

two copies of each chromosome (one inherited from the mother and

one inherited from the father), he or she can have only two alleles at a

given locus However, there can be many different alleles in the

popula-tion The normal or common allele is usually referred to as wild type

When alleles at a given locus are identical, the individual is homozygous

Inheriting identical copies of a mutant allele occurs in many autosomal

recessive disorders, particularly in circumstances of consanguinity or

isolated populations If the alleles are different on the maternal and the

paternal copy of the gene, the individual is heterozygous at this locus

(Fig 82-10) If two different mutant alleles are inherited at a given locus,

the individual is said to be a compound heterozygote Hemizygous is used

to describe males with a mutation in an X chromosomal gene or a female

with a loss of one X chromosomal locus

Genotypes describe the specific alleles at a particular locus For

example, there are three common alleles (E2, E3, E4) of the

apolipo-protein E (APOE) gene The genotype of an individual can therefore

be described as APOE3/4 or APOE4/4 or any other variant These

des-ignations indicate which alleles are present on the two chromosomes

in the APOE gene at locus 19q13.2 In other cases, the genotype might

be assigned arbitrary numbers (e.g., 1/2) or letters (e.g., B/b) to

distin-guish different alleles

A haplotype refers to a group of alleles that are closely linked

together at a genomic locus (Fig 82-4) Haplotypes are useful for

tracking the transmission of genomic segments within families and

for detecting evidence of genetic recombination, if the crossover event

occurs between the alleles (Fig 82-6) As an example, various alleles

at the histocompatibility locus antigen (HLA) on chromosome 6p

are used to establish haplotypes associated with certain disease states

For example, 21-hydroxylase deficiency, complement deficiency, and

hemochromatosis are each associated with specific HLA haplotypes It

is now recognized that these genes lie in close proximity to the HLA

locus, which explains why HLA associations were identified even

before the disease genes were cloned and localized In other cases,

specific HLA associations with diseases such as ankylosing spondylitis

(HLA-B27) or type 1 diabetes mellitus (HLA-DR4) reflect the role of

specific HLA allelic variants in susceptibility to these autoimmune

dis-eases The characterization of common SNP haplotypes in numerous

populations from different parts of the world through the HapMap

Project is providing a novel tool for association studies designed

to detect genes involved in the pathogenesis of complex disorders (Table 82-1) The presence or absence of certain haplotypes may also become relevant for the customized choice of medical therapies (pharmacogenomics) or for preventive strategies

Genotype-phenotype correlation describes the association of a

spe-cific mutation and the resulting phenotype The phenotype may differ depending on the location or type of the mutation in some genes For example, in von Hippel–Lindau disease, an autosomal dominant mul-tisystem disease that can include renal cell carcinoma, hemangioblas-tomas, and pheochromocytomas, among others, the phenotype varies greatly and the identification of the specific mutation can be clinically useful in order to predict the phenotypic spectrum

allelic HeteroGeneity Allelic heterogeneity refers to the fact that

differ-ent mutations in the same genetic locus can cause an iddiffer-entical or similar phenotype For example, many different mutations of the β-globin locus

hetero-geneity reflects the fact that many different mutations are capable of ing protein structure and function For this reason, maps of inactivating mutations in genes usually show a near-random distribution Exceptions include (1) a founder effect, in which a particular mutation that does not affect reproductive capacity can be traced to a single individual;

alter-(2) “hot spots” for mutations, in which the nature of the DNA sequence predisposes to a recurring mutation; and (3) localization of mutations to certain domains that are particularly critical for protein function Allelic heterogeneity creates a practical problem for genetic testing because one must often examine the entire genetic locus for mutations, because these can differ in each patient For example, there are currently 1963 reported

mutations in the CFTR gene (Fig 82-3) Mutational analysis may initially

focus on a panel of mutations that are particularly frequent (often taking the ethnic background of the patient into account), but a negative result does not exclude the presence of a mutation elsewhere in the gene One should also be aware that mutational analyses generally focus on the cod-ing region of a gene without considering regulatory and intronic regions

Because disease-causing mutations may be located outside the coding regions, negative results need to be interpreted with caution The advent

of more comprehensive sequencing technologies greatly facilitates comitant mutational analyses of several genes after targeted enrichment,

con-or even mutational analysis of the whole exome con-or genome However, comprehensive sequencing can result in significant diagnostic challenges because the detection of a sequence alteration alone is not always suf-ficient to establish that it has a causal role

PHenotyPic HeteroGeneity Phenotypic heterogeneity occurs when more

than one phenotype is caused by allelic mutations (e.g., different tions in the same gene) (Table 82-3) For example, laminopathies are monogenic multisystem disorders that result from mutations in the

muta-LMNA gene, which encodes the nuclear lamins A and C Twelve

auto-somal dominant and four autoauto-somal recessive disorders are caused by

mutations in the LMNA gene They include several forms of

lipodys-trophies, Emery-Dreifuss muscular dystrophy, progeria syndromes,

a form of neuronal Charcot-Marie-Tooth disease (type 2B1), and a group of overlapping syndromes Remarkably, hierarchical cluster analysis has revealed that the phenotypes vary depending on the

position of the mutation ( genotype-phenotype correlation) Similarly, identical mutations in the FGFR2 gene can result in very distinct

phenotypes: Crouzon’s syndrome (craniofacial synostosis) or Pfeiffer’s syndrome (acrocephalopolysyndactyly)

locus or nonallelic HeteroGeneity anD PHenocoPies Nonallelic or locus heterogeneity refers to the situation in which a similar disease phe-

notype results from mutations at different genetic loci (Table 82-3)

This often occurs when more than one gene product produces ferent subunits of an interacting complex or when different genes are involved in the same genetic cascade or physiologic pathway

dif-For example, osteogenesis imperfecta can arise from mutations

in two different procollagen genes (COL1A1 or COL1A2) that are

located on different chromosomes, and at least eight other genes

(Chap 427) The effects of inactivating mutations in these two genes are similar because the protein products comprise different subunits

of the helical collagen fiber Similarly, muscular dystrophy syndromes

can be caused by mutations in various genes, consistent with the

fact that it can be transmitted in an X-linked (Duchenne or Becker),

autosomal dominant (limb-girdle muscular dystrophy type 1), or

autosomal recessive (limb-girdle muscular dystrophy type 2) manner

(Chap 462e) Mutations in the X-linked DMD gene, which encodes

dystrophin, are the most common cause of muscular dystrophy This

feature reflects the large size of the gene as well as the fact that the

phenotype is expressed in hemizygous males because they have only

a single copy of the X chromosome Dystrophin is associated with a

large protein complex linked to the membrane-associated cytoskeleton

in muscle Mutations in several different components of this protein

complex can also cause muscular dystrophy syndromes Although the

phenotypic features of some of these disorders are distinct, the

pheno-typic spectrum caused by mutations in different genes overlaps, thereby

leading to nonallelic heterogeneity It should be noted that mutations in

dystrophin also cause allelic heterogeneity For example, mutations in

the DMD gene can cause either Duchenne’s or the less severe Becker’s

muscular dystrophy, depending on the severity of the protein defect

Recognition of nonallelic heterogeneity is important for several

rea-sons: (1) the ability to identify disease loci in linkage studies is reduced

by including patients with similar phenotypes but different genetic disorders; (2) genetic testing is more complex because several differ-ent genes need to be considered along with the possibility of different mutations in each of the candidate genes; and (3) novel information is gained about how genes or proteins interact, providing unique insights into molecular physiology

Phenocopies refer to circumstances in which nongenetic

condi-tions mimic a genetic disorder For example, features of toxin-

or drug-induced neurologic syndromes can resemble those seen

in Huntington’s disease, and vascular causes of dementia share phenotypic features with familial forms of Alzheimer’s dementia

(Chap 448) As in nonallelic heterogeneity, the presence of ies has the potential to confound linkage studies and genetic testing Patient history and subtle differences in phenotype can often provide clues that distinguish these disorders from related genetic conditions

phenocop-variable exPressivity anD incomPlete Penetrance The same genetic mutation may be associated with a phenotypic spectrum in different affected individuals, thereby illustrating the phe-

nomenon of variable expressivity This may include different

manifestations of a disorder variably involving different organs

Phenotypic Heterogeneity

Familial partial lipodystrophy Dunnigan AD 151660

Emery-Dreifuss muscular dystrophy (AR) AR 604929Limb-girdle muscular dystrophy type 1B AR 159001

Locus Heterogeneity

Myosin-binding

dystrophy)

(e.g., multiple endocrine neoplasia [MEN]), the severity of the

disor-der (e.g., cystic fibrosis), or the age of disease onset (e.g., Alzheimer’s

dementia) MEN 1 illustrates several of these features In this

autoso-mal dominant tumor syndrome, affected individuals carry an

inacti-vating germline mutation that is inherited in an autosomal dominant

fashion After somatic inactivation of the alternate allele, they can

develop tumors of the parathyroid gland, endocrine pancreas, and

the different glands, the age at which tumors develop, and the types

of hormones produced vary among affected individuals, even within

a given family In this example, the phenotypic variability arises, in

part, because of the requirement for a second somatic mutation in the

normal copy of the MEN1 gene, as well as the large array of different

cell types that are susceptible to the effects of MEN1 gene mutations

In part, variable expression reflects the influence of modifier genes,

or genetic background, on the effects of a particular mutation Even

in identical twins, in whom the genetic constitution is essentially the

same, one can occasionally see variable expression of a genetic disease

Interactions with the environment can also influence the course of

a disease For example, the manifestations and severity of

of phenylketonuria is affected by exposure to phenylalanine in the diet

(Chap 434e) Other metabolic disorders, such as hyperlipidemias and

porphyria, also fall into this category Many mechanisms, including

genetic effects and environmental influences, can therefore lead to

variable expressivity In genetic counseling, it is particularly important

to recognize this variability, because one cannot always predict the

course of disease, even when the mutation is known

Penetrance refers to the proportion of individuals with a mutant

genotype that express the phenotype If all carriers of a mutant

express the phenotype, penetrance is complete, whereas it is said to

be incomplete or reduced if some individuals do not exhibit features

of the phenotype Dominant conditions with incomplete penetrance

are characterized by skipping of generations with unaffected carriers

transmitting the mutant gene For example, hypertrophic obstructive

cardiomyopathy (HCM) caused by mutations in the myosin-binding

protein C gene is a dominant disorder with clinical features in only a

have the mutation but no evidence of the disease can still transmit the

disorder to subsequent generations In many conditions with postnatal

onset, the proportion of gene carriers who are affected varies with age

Thus, when describing penetrance, one has to specify age For

exam-ple, for disorders such as Huntington’s disease or familial amyotrophic

lateral sclerosis, which present later in life, the rate of penetrance is

influenced by the age at which the clinical assessment is performed

Imprinting can also modify the penetrance of a disease For example,

in patients with Albright’s hereditary osteodystrophy, mutations in the

Gsα subunit (GNAS1 gene) are expressed clinically only in individuals

sex-influenceD PHenotyPes Certain mutations affect males and females

quite differently In some instances, this is because the gene resides on

the X or Y sex chromosomes (X-linked disorders and Y-linked

dis-orders) As a result, the phenotype of mutated X-linked genes will be

expressed fully in males but variably in heterozygous females,

depend-ing on the degree of X-inactivation and the function of the gene For

example, most heterozygous female carriers of factor VIII deficiency

(hemophilia A) are asymptomatic because sufficient factor VIII is

hand, some females heterozygous for the X-linked lipid storage defect

caused by α-galactosidase A deficiency (Fabry’s disease) experience

mild manifestations of painful neuropathy, as well as other features of

mutations in genes such as SRY, which causes male-to-female sex

reversal, or DAZ (deleted in azoospermia), which causes abnormalities

Other diseases are expressed in a sex-limited manner because of

the differential function of the gene product in males and females

Activating mutations in the luteinizing hormone receptor cause

The phenotype is unique to males because activation of the receptor induces testosterone production in the testis, whereas it is function-ally silent in the immature ovary Biallelic inactivating mutations

of the follicle-stimulating hormone (FSH) receptor cause primary ovarian failure in females because the follicles do not develop in the absence of FSH action In contrast, affected males have a more subtle phenotype, because testosterone production is preserved (allowing

sexual maturation) and spermatogenesis is only partially impaired

(Chap 411) In congenital adrenal hyperplasia, most commonly caused by 21-hydroxylase deficiency, cortisol production is impaired and ACTH stimulation of the adrenal gland leads to increased produc-

androgen level causes ambiguous genitalia, which can be recognized

at the time of birth In males, the diagnosis may be made on the basis

of adrenal insufficiency at birth, because the increased adrenal gen level does not alter sexual differentiation, or later in childhood, because of the development of precocious puberty Hemochromatosis

andro-is more common in males than in females, presumably because of ferences in dietary iron intake and losses associated with menstruation

Chromosomal Disorders Chromosomal or cytogenetic disorders are caused by numerical or structural aberrations in chromosomes For

a detailed discussion of disorders of chromosome number and

causes of abortions, developmental disorders, and malformations

Contiguous gene syndromes (e.g., large deletions affecting several

genes) have been useful for identifying the location of new causing genes Because of the variable size of gene deletions in differ-ent patients, a systematic comparison of phenotypes and locations of deletion breakpoints allows positions of particular genes to be mapped within the critical genomic region

disease-Monogenic Mendelian Disorders Monogenic human diseases are

fre-quently referred to as Mendelian disorders because they obey the

prin-ciples of genetic transmission originally set forth in Gregor Mendel’s classic work The continuously updated OMIM catalogue lists several thousand of these disorders and provides information about the clini-cal phenotype, molecular basis, allelic variants, and pertinent animal models (Table 82-1) The mode of inheritance for a given phenotypic trait or disease is determined by pedigree analysis All affected and unaffected individuals in the family are recorded in a pedigree using

and the transmission of alleles from parents to children, are illustrated

in Fig 82-12 One dominant (A) allele and one recessive (a) allele can display three Mendelian modes of inheritance: autosomal dominant, autosomal recessive, and X-linked About 65% of human monogenic disorders are autosomal dominant, 25% are autosomal recessive, and 5% are X-linked Genetic testing is now available for many of these disorders and plays an increasingly important role in clinical medicine

(Chap 84)

autosomal Dominant DisorDers These disorders assume particular evance because mutations in a single allele are sufficient to cause the disease In contrast to recessive disorders, in which disease pathogen-esis is relatively straightforward because there is loss of gene function, dominant disorders can be caused by various disease mechanisms, many of which are unique to the function of the genetic pathway involved

rel-In autosomal dominant disorders, individuals are affected in cessive generations; the disease does not occur in the offspring of unaffected individuals Males and females are affected with equal fre-quency because the defective gene resides on one of the 22 autosomes

suc-(Fig 82-13A) Autosomal dominant mutations alter one of the two alleles at a given locus Because the alleles segregate randomly at meiosis, the probability that an offspring will be affected is 50% Unless there is a new germline mutation, an affected individual has an affected parent Children with a normal genotype do not transmit the disor-der Due to differences in penetrance or expressivity (see above), the

clinical manifestations of autosomal dominant disorders may be

vari-able Because of these variations, it is sometimes challenging to

deter-mine the pattern of inheritance

It should be recognized, however, that some individuals acquire a

mutated gene from an unaffected parent De novo germline mutations

occur more frequently during later cell divisions in gametogenesis,

which explains why siblings are rarely affected As noted before, new

germline mutations occur more frequently in fathers of advanced age

For example, the average age of fathers with new germline mutations

that cause Marfan’s syndrome is ~37 years, whereas fathers who

trans-mit the disease by inheritance have an average age of ~30 years

autosomal recessive DisorDers In recessive disorders, the mutated

alleles result in a complete or partial loss of function They frequently

involve enzymes in metabolic pathways, receptors, or proteins in

signaling cascades In an autosomal recessive disease, the affected

individual, who can be of either sex, is a homozygote or compound

heterozygote for a single-gene defect With a few important

excep-tions, autosomal recessive diseases are rare and often occur in the

context of parental consanguinity The relatively high frequency of

cer-tain recessive disorders such as sickle cell anemia, cystic fibrosis, and

thalassemia, is partially explained by a selective biologic advantage for

the heterozygous state (see below) Although heterozygous carriers of

a defective allele are usually clinically normal, they may display subtle

differences in phenotype that only become apparent with more precise

testing or in the context of certain environmental influences In sickle

cell anemia, for example, heterozygotes are normally asymptomatic However, in situations of dehydration or diminished oxygen pressure,

In most instances, an affected individual is the offspring of erozygous parents In this situation, there is a 25% chance that the offspring will have a normal genotype, a 50% probability of a hetero-zygous state, and a 25% risk of homozygosity for the recessive alleles

het-(Figs 82-10, 82-13B) In the case of one unaffected heterozygous and one affected homozygous parent, the probability of disease increases

to 50% for each child In this instance, the pedigree analysis mimics

an autosomal dominant mode of inheritance (pseudodominance) In

contrast to autosomal dominant disorders, new mutations in recessive alleles are rarely manifest because they usually result in an asymptom-atic carrier state

x-linkeD DisorDers Males have only one X chromosome; quently, a daughter always inherits her father’s X chromosome in addi-tion to one of her mother’s two X chromosomes A son inherits the

conse-Y chromosome from his father and one maternal X chromosome Thus, the characteristic features of X-linked inheritance are (1) the absence

of father-to-son transmission, and (2) the fact that all daughters of an

The risk of developing disease due to a mutant X-chromosomal gene differs in the two sexes Because males have only one X chromosome, they are hemizygous for the mutant allele; thus, they are more likely

Male

Mating

Monozygotic twins Dizygotic twins

Consanguineous union

Multiple siblings Spontaneousabortion

FIGURE 82-12 Segregation of alleles Segregation of genotypes in

the offspring of parents with one dominant (A) and one recessive

(a) allele The distribution of the parental alleles to their offspring

depends on the combination present in the parents Filled symbols =

Autosomal recessive with pseudodominanceAutosomal recessive

FIGURE 82-13 (A) Dominant, (B) recessive, (C) X-linked, and (D) mitochondrial (matrilinear) inheritance.

to develop the mutant phenotype, regardless of whether the mutation

is dominant or recessive A female may be either heterozygous or

homozygous for the mutant allele, which may be dominant or

reces-sive The terms X-linked dominant or X-linked recessive are therefore

only applicable to expression of the mutant phenotype in women In

addition, the expression of X-chromosomal genes is influenced by

X chromosome inactivation

y-linkeD DisorDers The Y chromosome has a relatively small number

of genes One such gene, the sex-region determining Y factor (SRY),

which encodes the testis-determining factor (TDF), is crucial for

normal male development Normally there is infrequent exchange of

sequences on the Y chromosome with the X chromosome The SRY

region is adjacent to the pseudoautosomal region, a chromosomal

segment on the X and Y chromosomes with a high degree of

homol-ogy A crossing-over event occasionally involves the SRY region

with the distal tip of the X chromosome during meiosis in the male

Translocations can result in XY females with the Y chromosome

lack-ing the SRY gene or XX males harborlack-ing the SRY gene on one of the

also result in individuals with an XY genotype and an incomplete

female phenotype Most of these mutations occur de novo Men with

oligospermia/azoospermia frequently have microdeletions on the long

arm of the Y chromosome that involve one or more of the azoospermia

factor (AZF) genes.

Exceptions to Simple Mendelian Inheritance Patterns • mitocHonDrial

DisorDers Mendelian inheritance refers to the transmission of genes

encoded by DNA contained in the nuclear chromosomes In addition,

each mitochondrion contains several copies of a small circular

and encodes transfer and ribosomal RNAs and 13 core proteins that

are components of the respiratory chain involved in oxidative

phos-phorylation and ATP generation The mitochondrial genome does not

recombine and is inherited through the maternal line because sperm

does not contribute significant cytoplasmic components to the zygote

A noncoding region of the mitochondrial chromosome, referred to

as D-loop, is highly polymorphic This property, together with the

absence of mtDNA recombination, makes it a valuable tool for studies

tracing human migration and evolution, and it is also used for specific

forensic applications

Inherited mitochondrial disorders are transmitted in a matrilineal

fashion; all children from an affected mother will inherit the disease,

but it will not be transmitted from an affected father to his children

(Fig 82-13D) Alterations in the mtDNA that involves enzymes

required for oxidative phosphorylation lead to reduction of ATP

sup-ply, generation of free radicals, and induction of apoptosis Several

syn-dromic disorders arising from mutations in the mitochondrial genome

are known in humans and they affect both protein-coding and tRNA

myopathies and encephalopathies because of the high dependence of

these tissues on oxidative phosphorylation The age of onset and the

clinical course are highly variable because of the unusual mechanisms

of mtDNA transmission, which replicates independently from nuclear

DNA During cell replication, the proportion of wild-type and mutant

mitochondria can drift among different cells and tissues The resulting

heterogeneity in the proportion of mitochondria with and without a

mutation is referred to as heteroplasmia and underlies the phenotypic

variability that is characteristic of mitochondrial diseases

Acquired somatic mutations in mitochondria are thought to be

involved in several age-dependent degenerative disorders affecting

predominantly muscle and the peripheral and central nervous

sys-tem (e.g., Alzheimer’s and Parkinson’s diseases) Establishing that

an mtDNA alteration is causal for a clinical phenotype is challenging

because of the high degree of polymorphism in mtDNA and the

phe-notypic variability characteristic of these disorders Certain

pharma-cologic treatments may have an impact on mitochondria and/or their

function For example, treatment with the antiretroviral compound

azidothymidine (AZT) causes an acquired mitochondrial myopathy

through depletion of muscular mtDNA

mosaicism Mosaicism refers to the presence of two or more genetically distinct cell lines in the tissues of an individual It results from a muta-tion that occurs during embryonic, fetal, or extrauterine development

The developmental stage at which the mutation arises will determine whether germ cells and/or somatic cells are involved Chromosomal mosaicism results from nondisjunction at an early embryonic mitotic division, leading to the persistence of more than one cell line, as exem-

mosaicism is characterized by a patchy distribution of genetically altered somatic cells The McCune-Albright syndrome, for example,

is caused by activating mutations in the stimulatory G protein α

phe-notype varies depending on the tissue distribution of the mutation;

manifestations include ovarian cysts that secrete sex steroids and cause precocious puberty, polyostotic fibrous dysplasia, café-au-lait skin pigmentation, growth hormone–secreting pituitary adenomas, and

x-inactivation, imPrintinG, anD uniParental Disomy According to ditional Mendelian principles, the parental origin of a mutant gene

tra-is irrelevant for the expression of the phenotype There are, however,

important exceptions to this rule X-inactivation prevents the

expres-sion of most genes on one of the two X chromosomes in every cell

of a female Gene inactivation through genomic imprinting occurs

on selected chromosomal regions of autosomes and leads to itable preferential expression of one of the parental alleles It is of pathophysiologic importance in disorders where the transmission of disease is dependent on the sex of the transmitting parent and, thus, plays an important role in the expression of certain genetic disorders

inher-Two classic examples are the Prader-Willi syndrome and Angelman’s

diminished fetal activity, obesity, hypotonia, mental retardation, short stature, and hypogonadotropic hypogonadism Deletions of the paternal copy of the Prader-Willi locus located on the short arm

of chromosome 15 result in a contiguous gene syndrome involving

missing paternal copies of the necdin and SNRPN genes, among

oth-ers In contrast, patients with Angelman’s syndrome, characterized

by mental retardation, seizures, ataxia, and hypotonia, have deletions involving the maternal copy of this region on chromosome 15 These

two syndromes may also result from uniparental disomy In this case,

the syndromes are not caused by deletions on chromosome 15 but by the inheritance of either two maternal chromosomes (Prader-Willi syndrome) or two paternal chromosomes (Angelman’s syndrome)

Lastly, the two distinct phenotypes can also be caused by an imprinting defect that impairs the resetting of the imprint during zygote develop-ment (defect in the father leads to Prader-Willi syndrome; defect in the mother leads to Angelman’s syndrome)

Imprinting and the related phenomenon of allelic exclusion may

be more common than currently documented, because it is difficult to examine levels of mRNA expression from the maternal and paternal alleles in specific tissues or in individual cells Genomic imprinting,

or uniparental disomy, is involved in the pathogenesis of several other

moles contain a normal number of diploid chromosomes, but they are all of paternal origin The opposite situation occurs in ovarian teratomata, with 46 chromosomes of maternal origin Expression

of the imprinted gene for insulin-like growth factor II (IGF-II) is involved in the pathogenesis of the cancer-predisposing Beckwith-

somatic overgrowth with organomegalies and hemihypertrophy, and they have an increased risk of embryonal malignancies such as Wilms’

tumor Normally, only the paternally derived copy of the IGF-II gene

is active and the maternal copy is inactive Imprinting of the IGF-II gene is regulated by H19, which encodes an RNA transcript that is not translated into protein Disruption or lack of H19 methylation leads

to a relaxation of IGF-II imprinting and expression of both alleles

Alterations of the epigenome through gain and loss of DNA tion, as well as altered histone modifications, play an important role in the pathogenesis of malignancies

somatic mutations Cancer can be considered a genetic disease at

indicating that they have arisen from a single precursor cell with one

or several mutations in genes controlling growth (proliferation or

apoptosis) and/or differentiation These acquired somatic mutations

are restricted to the tumor and its metastases and are not found in

the surrounding normal tissue The molecular alterations include

dominant gain-of-function mutations in oncogenes, recessive

loss-of-function mutations in tumor-suppressor genes and DNA repair genes,

gene amplification, and chromosome rearrangements Rarely, a single

mutation in certain genes may be sufficient to transform a normal cell

into a malignant cell In most cancers, however, the development of a

malignant phenotype requires several genetic alterations for the

grad-ual progression from a normal cell to a cancerous cell, a phenomenon

Genome-wide analyses of cancers using deep sequencing often reveal somatic

rearrangements resulting in fusion genes and mutations in multiple

genes Comprehensive sequence analyses provide further insight into

genetic heterogeneity within malignancies; these include intratumoral

heterogeneity among the cells of the primary tumor, intermetastatic

and intrametastatic heterogeneity, and interpatient differences These

analyses further support the notion of cancer as an ongoing process

of clonal evolution, in which successive rounds of clonal selection

within the primary tumor and metastatic lesions result in diverse

genetic and epigenetic alterations that require targeted (personalized)

therapies The heterogeneity of mutations within a tumor can also

lead to resistance to target therapies because cells with mutations that

are resistant to the therapy, even if they are a minor part of the tumor

population, will be selected as the more sensitive cells are killed Most

human tumors express telomerase, an enzyme formed of a protein and

an RNA component, which adds telomere repeats at the ends of

chro-mosomes during replication This mechanism impedes shortening

of the telomeres, which is associated with senescence in normal cells

and is associated with enhanced replicative capacity in cancer cells

Telomerase inhibitors provide a novel strategy for treating advanced

human cancers

In many cancer syndromes, there is an inherited predisposition to

tumor formation In these instances, a germline mutation is inherited

in an autosomal dominant fashion inactivating one allele of an

auto-somal tumor-suppressor gene If the second allele is inactivated by a

somatic mutation or by epigenetic silencing in a given cell, this will

lead to neoplastic growth (Knudson two-hit model) Thus, the

defec-tive allele in the germline is transmitted in a dominant mode, although

tumorigenesis results from a biallelic loss of the tumor-suppressor gene

in an affected tissue The classic example to illustrate this phenomenon

is retinoblastoma, which can occur as a sporadic or hereditary tumor

In sporadic retinoblastoma, both copies of the retinoblastoma (RB)

gene are inactivated through two somatic events In hereditary

retino-blastoma, one mutated or deleted RB allele is inherited in an autosomal

dominant manner and the second allele is inactivated by a subsequent somatic mutation This two-hit model applies to other inherited cancer

(Chap 118)

nucleotiDe rePeat exPansion DisorDers Several diseases are associated with an increase in the number of nucleotide repeats above a certain

coding region of the genes, as in Huntington’s disease or the X-linked form of spinal and bulbar muscular atrophy (SBMA; Kennedy’s syn-drome) In other instances, the repeats probably alter gene regulatory sequences If an expansion is present, the DNA fragment is unstable and tends to expand further during cell division The length of the nucleotide repeat often correlates with the severity of the disease When repeat length increases from one generation to the next, dis-ease manifestations may worsen or be observed at an earlier age; this

phenomenon is referred to as anticipation In Huntington’s disease,

for example, there is a correlation between age of onset and length

been documented in other diseases caused by dynamic mutations in trinucleotide repeats (Table 82-4) The repeat number may also vary in

a tissue-specific manner In myotonic dystrophy, the CTG repeat may

Complex Genetic Disorders The expression of many common diseases such as cardiovascular disease, hypertension, diabetes, asthma, psy-chiatric disorders, and certain cancers is determined by a combination

of genetic background, environmental factors, and lifestyle A trait is

called polygenic if multiple genes contribute to the phenotype or

mul-tifactorial if multiple genes are assumed to interact with environmental

factors Genetic models for these complex traits need to account for genetic heterogeneity and interactions with other genes and the envi-ronment Complex genetic traits may be influenced by modifier genes that are not linked to the main gene involved in the pathogenesis of the

trait This type of gene-gene interaction, or epistasis, plays an

impor-tant role in polygenic traits that require the simultaneous presence of variations in multiple genes to result in a pathologic phenotype

Type 2 diabetes mellitus provides a paradigm for considering a tifactorial disorder, because genetic, nutritional, and lifestyle factors are

Disease Locus Repeat Triplet Length (Normal/Disease) Inheritance Gene Product

X-chromosomal spinobulbar

Dystrophia myotonica (DM) 19q13.2-q13.3 CTG 5–30/200–1000 AD, variable penetrance Myotonin protein kinase

Spinocerebellar ataxia type 3 (SCA3);

Spinocerebellar ataxia type 6 (SCA6,

Spinocerebellar ataxia type 7 (SCA7) 3p21.1-p12 CAG 4–19/37 to >300 AD Ataxin 7

Spinocerebellar ataxia type 12

Dentatorubral pallidoluysian

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; XR, X-linked recessive.

Disorder Genes or Susceptibility Locus Chromosomal Location Other Factors

Monogenic permanent neonatal

diabetes mellitus KCNJ11 (inwardly rectifying potassium channel Kir6.2) 11p15.1 AD

ABCC8 (ATP-binding cassette, subfamily c, member 8;

GLIS3 (GLIS family zinc finger protein 3) 9p24.2 AR, diabetes, congenital

hypothyroidismMaturity-onset diabetes of the young

(MODY): Monogenic forms of diabetes

mellitus

MODY 5 (renal cysts, diabetes) HNF1β (hepatocyte nuclear factor 1β) 17cen-q21.3

Diabetes mellitus type 2; loci and

genes linked and/or associated with

susceptibility for diabetes mellitus

type 2

Genes and loci identified by linkage/association studies Heavily influenced by diet,

energy expenditure, obesity

PPARG, KCNJ11/ABCC8, TCF7L2, IGF2BP2, CDKAL1, SLC30A8, CDKN2A/B, HHEX, FTO, HNF1B, NOTCH2, THADA, ADAMSTS9, JAZF1, CDC122/CAMK1D, KCNQ1, TSPAN8/LGR5, IRS1, DUSP9, PROX1, BCK11A, G6PC2, GCKR, ADCY5, SLC2A2, WFS1, ZBED3, DGKB/TMEM195, GCK, KLF14, TP53INP1, GLIS3, TLE4, ADRA2A, CENTD2, CRY2, FADS1, MADD, MTNR1B, HMGA1, HNF1A, IGF1A, IGF1, C2CD4B, PRC1, VPS13C, ZFAND6, GIPR

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; MODY, maturity onset diabetes of the young.

The identification of genetic variations and environmental factors that

either predispose to or protect against disease is essential for

predict-ing disease risk, designpredict-ing preventive strategies, and developpredict-ing novel

therapeutic approaches The study of rare monogenic diseases may

provide insight into some of the genetic and molecular mechanisms

important in the pathogenesis of complex diseases For example, the

identification of the genes causing monogenic forms of permanent

neonatal diabetes mellitus or maturity-onset diabetes defined them

as candidate genes in the pathogenesis of diabetes mellitus type 2

(Tables 82-2 and 82-5) Genome scans have identified numerous genes

and loci that may be associated with susceptibility to development of

diabetes mellitus in certain populations Efforts to identify

suscepti-bility genes require very large sample sizes, and positive results may

depend on ethnicity, ascertainment criteria, and statistical analysis

Association studies analyzing the potential influence of (biologically

functional) SNPs and SNP haplotypes on a particular phenotype are

providing new insights into the genes involved in the pathogenesis of

these common disorders Large variants ([micro]deletions,

duplica-tions, and inversions) present in the human population also

contrib-ute to the pathogenesis of complex disorders, but their contributions

remain poorly understood

Linkage and Association Studies There are two primary strategies for

mapping genes that cause or increase susceptibility to human disease:

(1) classic linkage can be performed based on a known genetic model

or, when the model is unknown, by studying pairs of affected relatives;

or (2) disease genes can be mapped using allelic association studies

(Table 82-6)

Genetic linkaGe Genetic linkage refers to the fact that genes are

physi-cally connected, or linked, to one another along the chromosomes

Two fundamental principles are essential for understanding the cept of linkage: (1) when two genes are close together on a chromo-some, they are usually transmitted together, unless a recombination event separates them (Figs 82-6); and (2) the odds of a crossover, or recombination event, between two linked genes is proportional to the distance that separates them Thus, genes that are farther apart are more likely to undergo a recombination event than genes that are very close together The detection of chromosomal loci that segregate with

con-a disecon-ase by linkcon-age ccon-an be used to identify the gene responsible for

the disease (positional cloning) and to predict the odds of disease gene

transmission in genetic counseling

Polymorphisms are essential for linkage studies because they vide a means to distinguish the maternal and paternal chromosomes

pro-in an pro-individual On average, 1 out of every 1000 bp varies from one person to the next Although this degree of variation seems low (99.9%

identical), it means that >3 million sequence differences exist between any two unrelated individuals and the probability that the sequence

at such loci will differ on the two homologous chromosomes is high (often >70–90%) These sequence variations include variable number

of tandem repeats (VNTRs), short tandem repeats (STRs), and SNPs

Most STRs, also called polymorphic microsatellite markers, consist of

di-, tri-, or tetranucleotide repeats that can be characterized readily using the polymerase chain reaction (PCR) Characterization of SNPs, using DNA chips or beads, permits comprehensive analyses of genetic variation, linkage, and association studies Although these sequence variations often have no apparent functional consequences, they pro-vide much of the basis for variation in genetic traits

In order to identify a chromosomal locus that segregates with a disease, it is necessary to characterize polymorphic DNA markers from affected and unaffected individuals of one or several pedigrees One can

then assess whether certain marker alleles cosegregate with the disease

Markers that are closest to the disease gene are less likely to undergo

recombination events and therefore receive a higher linkage score

Linkage is expressed as a lod (logarithm of odds) score—the ratio of

the probability that the disease and marker loci are linked rather than

unlinked Lod scores of +3 (1000:1) are generally accepted as supporting

linkage, whereas a score of –2 is consistent with the absence of linkage

allelic association, linkaGe Disequilibrium, anD HaPlotyPes Allelic

asso-ciation refers to a situation in which the frequency of an allele is

sig-nificantly increased or decreased in individuals affected by a particular

disease in comparison to controls Linkage and association differ in

several aspects Genetic linkage is demonstrable in families or

sib-ships Association studies, on the other hand, compare a population

of affected individuals with a control population Association

stud-ies can be performed as case-control studstud-ies that include unrelated

affected individuals and matched controls or as family-based studies

that compare the frequencies of alleles transmitted or not transmitted

to affected children

Allelic association studies are particularly useful for identifying

susceptibility genes in complex diseases When alleles at two loci occur

more frequently in combination than would be predicted (based on

known allele frequencies and recombination fractions), they are said

to be in linkage disequilibrium Evidence for linkage disequilibrium

can be helpful in mapping disease genes because it suggests that the

two loci are tightly linked

Detecting the genetic factors contributing to the pathogenesis

of common complex disorders remains a great challenge In many

instances, these are low-penetrance alleles (e.g., variations that

indi-vidually have a subtle effect on disease development, and they can

only be identified by unbiased GWAS) (Catalog of Published

occur in noncoding or regulatory sequences but do not alter protein

structure The analysis of complex disorders is further complicated

by ethnic differences in disease prevalence, differences in allele

fre-quencies in known susceptibility genes among different populations,

locus and allelic heterogeneity, gene-gene and gene-environment

interactions, and the possibility of phenocopies The data generated

by the HapMap Project are greatly facilitating GWAS for the

charac-terization of complex disorders Adjacent SNPs are inherited together

as blocks, and these blocks can be identified by genotyping selected

marker SNPs, so-called Tag SNPs, thereby reducing cost and workload

(Fig 82-4) The availability of this information permits the ization of a limited number of SNPs to identify the set of haplotypes present in an individual (e.g., in cases and controls) This, in turn, permits performing GWAS by searching for associations of certain haplotypes with a disease phenotype of interest, an essential step for unraveling the genetic factors contributing to complex disorders

character-PoPulation Genetics In population genetics, the focus changes from alterations in an individual’s genome to the distribution pattern of dif-ferent genotypes in the population In a case where there are only two

of the genotype can be calculated Alternatively, one can determine an allele frequency if the genotype frequency has been determined

Allele frequencies vary among ethnic groups and geographic

regions For example, heterozygous mutations in the CFTR gene

are relatively common in populations of European origin but are rare in the African population Allele frequencies may vary because certain allelic variants confer a selective advantage For example, heterozygotes for the sickle cell mutation, which is particularly com-mon in West Africa, are more resistant to malarial infection because the erythrocytes of heterozygotes provide a less favorable environ-

ment for Plasmodium parasites Although homozygosity for the

sickle cell mutation is associated with severe anemia and sickle crises

(Chap 127), heterozygotes have a higher probability of survival because of the reduced morbidity and mortality from malaria; this phenomenon has led to an increased frequency of the mutant allele Recessive conditions are more prevalent in geographically isolated populations because of the more restricted gene pool

APPROACH TO THE PATIENT:

inherited disorders

For the practicing clinician, the family history remains an essential step in recognizing the possibility of a hereditary predisposition

to disease When taking the history, it is useful to draw a detailed

Linkage Studies

Classical linkage analysis (parametric

Suitable for genome scan Difficult to obtain sufficient statistical power for

complex traitsControl population not required

Useful for multifactorial disorders in isolated populationsAllele-sharing methods (nonparametric

methods) Suitable for identification of susceptibility genes in poly-genic and multifactorial disorders Difficult to collect sufficient number of subjects

Affected sib and relative pair analyses Suitable for genome scan Difficult to obtain sufficient statistical power for

complex traitsSib pair analysis Control population not required if allele frequencies are

known Reduced power compared to classical linkage, but not sensitive to specification of genetic modeStatistical power can be increased by including parents and

relatives

Association Studies

Case-control studies Suitable for identification of susceptibility genes in

polygenic and multifactorial disorders Requires large sample size and matched control populationLinkage disequilibrium Suitable for testing specific allelic variants of known

candidate loci False-positive results in the absence of suitable control populationTransmission disequilibrium test (TDT) Facilitated by HapMap data, making GWAS more feasible Candidate gene approach does not permit

detection of novel genes and pathwaysWhole-genome association studies Does not necessarily need relatives Susceptibility genes can vary among different

populations

Abbreviation: GWAS, genome-wide association study.

DNA testing is performed by mutational analysis or linkage studies in individuals at risk for a genetic disorder known to be present in a family Mass screening programs require tests of high sensitivity and specificity to be cost-effective Prerequisites for the success of genetic screening programs include the following: that the disorder is potentially serious; that it can be influenced at a presymptomatic stage by changes in behavior, diet, and/or phar-maceutical manipulations; and that the screening does not result

in any harm or discrimination Screening in Jewish populations for the autosomal recessive neurodegenerative storage disease Tay-Sachs has reduced the number of affected individuals In contrast, screening for sickle cell trait/disease in African Americans has led

to unanticipated problems of discrimination by health insurers and employers Mass screening programs harbor additional potential problems For example, screening for the most common genetic alteration in cystic fibrosis, the ΔF508 mutation with a frequency

of ~70% in northern Europe, is feasible and seems to be tive One has to keep in mind, however, that there is pronounced allelic heterogeneity and that the disease can be caused by about

effec-2000 other mutations The search for these less common mutations would substantially increase costs but not the effectiveness of the screening program as a whole Next-generation genome sequenc-ing permits comprehensive and cost-effective mutational analyses after selective enrichment of candidate genes For example, tests that sequence all the common genes causing hereditary deaf-ness are already commercially available Occupational screen-ing programs aim to detect individuals with increased risk for

smoke or dust exposure) Integrating genomic data into electronic medical records is evolving and may provide significant decision support at the point of care, for example, by providing the clini-cian with genomic data and decision algorithms for the prescrip-tion of drugs that are subject to pharmacogenetic influences

Mutational Analyses DNA sequence analysis is now widely used

as a diagnostic tool and has significantly enhanced diagnostic accuracy It is used for determining carrier status and for prenatal

pedigree of the first-degree relatives (e.g., parents, siblings, and

chil-dren), because they share 50% of genes with the patient Standard

symbols for pedigrees are depicted in Fig 82-11 The family history

should include information about ethnic background, age, health

status, and deaths, including infants Next, the physician should

explore whether there is a family history of the same or related

ill-nesses to the current problem An inquiry focused on commonly

occurring disorders such as cancers, heart disease, and diabetes

mellitus should follow Because of the possibility of age-dependent

expressivity and penetrance, the family history will need

intermit-tent updating If the findings suggest a genetic disorder, the

clini-cian should assess whether some of the patient’s relatives may be at

risk of carrying or transmitting the disease In this circumstance, it

is useful to confirm and extend the pedigree based on input from

several family members This information may form the basis for

genetic counseling, carrier detection, early intervention, and

In instances where a diagnosis at the molecular level may be

relevant, it is important to identify an appropriate laboratory that

can perform the appropriate test Genetic testing is available for

a rapidly growing number of monogenic disorders through

com-mercial laboratories For uncommon disorders, the test may only

be performed in a specialized research laboratory Approved

labo-ratories offering testing for inherited disorders can be identified

in continuously updated online resources (e.g., GeneTests; Table

82-1) If genetic testing is considered, the patient and the family

should be counseled about the potential implications of positive

results, including psychological distress and the possibility of

dis-crimination The patient or caretakers should be informed about

the meaning of a negative result, technical limitations, and the

pos-sibility of false-negative and inconclusive results For these reasons,

genetic testing should only be performed after obtaining informed

consent Published ethical guidelines address the specific aspects

that should be considered when testing children and adolescents

Genetic testing should usually be limited to situations in which the

results may have an impact on medical management

IDENTIFYING THE DISEASE-CAUSING GENE

Genomic medicine aims to enhance the quality of medical care

through the use of genotypic analysis (DNA testing) to identify genetic

predisposition to disease, to select more specific

pharmacother-apy, and to design individualized medical care based on genotype

Rare alleles Mendelian disease

Low frequency variants with intermediate effect

Typical:

Common variants with low effect on complex disease

Rare:

Common variants with high effect on complex disease

Rare variants with small effect:

difficult to identify

Low frequency

Allele frequency

Common Very rare

FIGURE 82-14 Relationship between allele frequency and effect size in monogenic and polygenic disorders In classic Mendelian

disor-ders, the allele frequency is typically low but has a high impact (single gene disorder) This contrasts with polygenic disorders that require the

combination of multiple low impact alleles that are frequently quite common in the general population

discussed in previous versions of this chapter, are available for the

detection of mutations In a very broad sense, one can distinguish

between techniques that allow for screening of known mutations

(screening mode) or techniques that definitively characterize

muta-tions Analyses of large alterations in the genome are possible using

classic methods such as cytogenetics, fluorescent in situ

sensitive novel techniques that search for multiple single exon

dele-tions or duplicadele-tions

More discrete sequence alterations rely heavily on the use of

PCR, which allows rapid gene amplification and analysis Moreover,

PCR makes it possible to perform genetic testing and mutational

analysis with small amounts of DNA extracted from leukocytes or

even from single cells, buccal cells, or hair roots DNA sequencing

can be performed directly on PCR products or on fragments cloned

into plasmid vectors amplified in bacterial host cells Sequencing of

all exons of the genome or selected chromosomes, or sequencing

of numerous candidate genes in a single run, is now possible with

next-generation sequencing platforms

The majority of traditional diagnostic methods were gel-based

Novel technologies for the analysis of mutations, genotyping,

large-scale sequencing, and mRNA expression profiles are undergoing rapid

evolution DNA chip technologies allow hybridization of DNA or

RNA to hundreds of thousands of probes simultaneously Microarrays

are being used clinically for mutational analysis of several human

dis-ease genes, as well as for the identification of viral or bacterial sequence

variations With advances in high-throughput DNA sequencing

tech-nology, complete sequencing of the genome or an exome has entered

the clinical realm Although comprehensive sequencing of large

genomic regions or multiple genes is already a reality, the subsequent

bioinformatics analysis, assembly of sequence fragments, and

compar-ative alignments remains a significant and commonly underestimated

challenge The discovery of incidental (or secondary) findings that are

unrelated to the indication for the sequencing analysis but indicators

of other disorders of potential relevance for patient care can pose a

difficult ethical dilemma It can lead to the detection of undiagnosed

medically actionable genetic conditions, but can also reveal

deleteri-ous mutations that cannot be influenced, as numerdeleteri-ous sequence

vari-ants are of unknown significance

A general algorithm for the approach to mutational analysis is

pheno-type cannot be overemphasized This is the step where one should

also consider the possibility of genetic heterogeneity and

phenocop-ies If obvious candidate genes are suggested by the phenotype, they

can be analyzed directly After identification of a mutation, it is essential to demonstrate that it segregates with the phenotype The functional characterization of novel mutations is labor intensive and may require analyses in vitro or in transgenic models in order

to document the relevance of the genetic alteration

Prenatal diagnosis of numerous genetic diseases in instances

with a high risk for certain disorders is now possible by direct DNA

analysis Amniocentesis involves the removal of a small amount of

amniotic fluid, usually at 16 weeks of gestation Cells can be lected and submitted for karyotype analyses, FISH, and mutational analysis of selected genes The main indications for amniocentesis include advanced maternal age (>35 years), an abnormal serum triple marker test (α-fetoprotein, β human chorionic gonadotropin, pregnancy-associated plasma protein A, or unconjugated estriol),

col-a fcol-amily history of chromosomcol-al col-abnormcol-alities, or col-a Mendelicol-an disorder amenable to genetic testing Prenatal diagnosis can also

be performed by chorionic villus sampling (CVS), in which a small

amount of the chorion is removed by a transcervical or dominal biopsy Chromosomes and DNA obtained from these cells can be submitted for cytogenetic and mutational analyses CVS can

transab-be performed earlier in gestation (weeks 9–12) than amniocentesis,

an aspect that may be of relevance when termination of pregnancy

is a consideration Later in pregnancy, beginning at about 18 weeks

of gestation, percutaneous umbilical blood sampling (PUBS) mits collection of fetal blood for lymphocyte culture and analysis

per-Recently, the entire fetal genome has been determined prenatally from cells taken from the mother’s plasma through deep sequencing and the counting of parental haplotypes, or by inferring it from DNA sequences obtained from blood samples from the mother, father, and umbilical cord These approaches enable screening for clinically relevant and deleterious alleles inherited from the parents, as well

as for de novo germline mutations, and they may have the potential

to change the diagnosis of genetic disorders in the prenatal setting

In combination with in vitro fertilization (IVF) techniques, it is even possible to perform genetic diagnoses in a single cell removed from the four- to eight-cell embryo or to analyze the first polar body from an oocyte Preconceptual diagnosis thereby avoids therapeutic abortions but is costly and labor intensive It should be emphasized that excluding a specific disorder by any of these approaches is never equivalent to the assurance of having a normal child

Mutations in certain cancer susceptibility genes such as BRCA1 and BRCA2 may identify individuals with an increased risk for the

development of malignancies and result in risk-reducing tions The detection of mutations is an important diagnostic and

interven-Determine functional properties

of identified mutations

in vitro and in vivo

Genetic counselingTesting of other family members

Mutational analysis

Treatment based

on pathophysiology

Characterization of phenotypeFamilial or sporadic genetic disorder

Pedigree analysis

Gene known

or candidate genesGene unknown

Linkage analysisEnrichment of linked regionDeep-sequencing

Population-basedgenetic screening

Susceptibility genes

or loci

FIGURE 82-15 Approach to genetic disease.

prognostic tool in leukemias and lymphomas The demonstration

of the presence or absence of mutations and polymorphisms is

also relevant for the rapidly evolving field of

pharmacogenom-ics, including the identification of differences in drug treatment

response or metabolism as a function of genetic background For

example, the thiopurine drugs 6-mercaptopurine and azathioprine

are commonly used cytotoxic and immunosuppressive agents

They are metabolized by thiopurine methyltransferase (TPMT),

an enzyme with variable activity associated with genetic

polymor-phisms in 10% of whites and complete deficiency in about 1 in 300

individuals Patients with intermediate or deficient TPMT activity

are at risk for excessive toxicity, including fatal myelosuppression

Characterization of these polymorphisms allows mercaptopurine

doses to be modified based on TPMT genotype Pharmacogenomics

may increasingly permit individualized drug therapy, improve drug

effectiveness, reduce adverse side effects, and provide cost-effective

ETHICAL ISSUES

Determination of the association of genetic defects with disease,

comprehensive data of an individual’s genome, and studies of genetic

variation raise many ethical and legal issues Genetic information is

generally regarded as sensitive information that should not be readily

accessible without explicit consent (genetic privacy) The disclosure

of genetic information may risk possible discrimination by insurers

or employers The scientific components of the Human Genome

Project have been paralleled by efforts to examine ethical, social,

and legal implications An important milestone emerging from these

endeavors consists in the Genetic Information Nondiscrimination

Act (GINA), signed into law in 2008, which aims to protect

asymp-tomatic individuals against the misuse of genetic information for

health insurance and employment It does not, however, protect the

symptomatic individual Provisions of the U.S Patient Protection

and Affordable Care Act, effective in 2014, will fill this gap and

prohibit exclusion from, or termination of, health insurance based

on personal health status Potential threats to the maintenance of

genetic privacy consist in the emerging integration of genomic data

into electronic medical records, compelled disclosures of health

records, and direct-to-consumer genetic testing

It is widely accepted that identifying disease-causing genes can

lead to improvements in diagnosis, treatment, and prevention

However, the information gleaned from genotypic results can have

quite different impacts, depending on the availability of

identification of mutations that cause MEN 2 or hemochromatosis

allows specific interventions for affected family members On

the other hand, at present, the identification of an Alzheimer’s or

Huntington’s disease gene does not currently alter therapy and

outcomes Most genetic disorders are likely to fall into an

interme-diate category where the opportunity for prevention or treatment

area is unpredictable, as underscored by the finding that

angioten-sin II receptor blockers may slow disease progression in Marfan’s

syndrome Genetic test results can generate anxiety in affected

indi-viduals and family members Comprehensive sequence analyses are

particularly challenging because most individuals can be expected

to harbor several serious recessive gene mutations

The impact of genetic testing on health care costs is currently

unclear It is likely to vary among disorders and depend on the

availability of effective therapeutic modalities A significant

prob-lem arises from the marketing of genetic testing directly to

consum-ers by commercial companies The validity of these tests has not

been defined, and there are numerous concerns about the lack of

appropriate regulatory oversight, the accuracy and confidentiality

of genetic information, the availability of counseling, and the

han-dling of these results

Many issues raised by the genome project are familiar, in

prin-ciple, to medical practitioners For example, an asymptomatic

patient with increased low-density lipoprotein (LDL) cholesterol, high blood pressure, or a strong family history of early myocardial infarction is known to be at increased risk of coronary heart disease

In such cases, it is clear that the identification of risk factors and an appropriate intervention are beneficial Likewise, patients with phe-nylketonuria, cystic fibrosis, or sickle cell anemia are often identi-fied as having a genetic disease early in life These precedents can be helpful for adapting policies that relate to genetic information We can anticipate similar efforts, whether based on genotypes or other markers of genetic predisposition, to be applied to many disorders

One confounding aspect of the rapid expansion of information is that our ability to make clinical decisions often lags behind initial insights into genetic mechanisms of disease For example, when

genes that predispose to breast cancer such as BRCA1 are described,

they generate tremendous public interest in the potential to predict disease, but many years of clinical research are still required to rig-orously establish genotype and phenotype correlations

Genomics may contribute to improvements in global health by providing a better understanding of pathogens and diagnostics, and through contributions to drug development There is, however, concern about the development of a “genomics divide” because of the costs associated with these developments and uncertainty as

to whether these advances will be accessible to the populations of developing countries The World Health Organization has sum-marized the current issues and inequities surrounding genomic medicine in a detailed report titled “Genomics and World Health.”

Whether related to informed consent, participation in research,

or the management of a genetic disorder that affects an individual

or his or her family, there is a great need for more information about fundamental principles of genetics The pervasive nature of the role

of genetics in medicine makes it important for physicians and other health care professionals to become more informed about genetics and to provide advice and counseling in conjunction with trained

prevention strategies will therefore require intensive patient and physician education, changes in health care financing, and legisla-tion to protect patient’s rights

Alterations of the chromosomes (numerical and structural) occur in

about 1% of the general population, in 8% of stillbirths, and in close

encode the human genome are packaged into 23 pairs of

chromo-somes, which consist of discrete portions of DNA, bound to several

classes of regulatory proteins Technical advances that led to the

abil-ity to analyze human chromosomes immediately translated into the

revelation that human disorders can be caused by an abnormality of

chromosome number In 1959, the clinically recognizable disorder,

Down syndrome, was demonstrated to result from having three copies

of chromosome 21 (trisomy 21) Very soon thereafter, in 1960, a small,

structurally abnormal chromosome was recognized in the cells of some

patients with chronic myelogenous leukemia (CML), and this

abnor-mal chromosome is now known as the Philadelphia chromosome

Since these early discoveries, the techniques for analysis of human

chromosomes, and DNA in general, have gone through several

revolu-tions, and with each technical advancement, our understanding of the

role of chromosomal abnormalities in human disease has expanded

While early studies in the 1950s and 1960s easily identified

abnormali-ties of chromosome number (aneuploidy) and large structural

altera-tions such as delealtera-tions (chromosomes with missing regions),

duplica-tions (extra copies of chromosome regions), or translocaduplica-tions (where

portions of the chromosomes are rearranged), many other types of

structural alterations could only be identified as techniques improved

The first important technical advance was the introduction of

chro-mosome banding in the late 1960s, a technique that allowed for the

staining of the chromosomes, so that each chromosome could be

rec-ognized by its pattern of alternating dark and light (or fluorescent and

nonfluorescent) bands Other technical innovations ranged from the

introduction of fluorescence in situ hybridization in the 1980s to use of

array-based and sequencing technologies in the early 2000s Currently,

we can appreciate that many types of chromosome abnormalities

con-tribute to human disease including aneuploidy; structural alterations

such as deletions and duplications, translocations, or inversions;

uni-parental disomy, where two copies of one chromosome (or a portion

of a chromosome) are inherited from one parent; complex alterations

such as isochromosomes, markers, and rings; and mosaicism for all of

the aforementioned abnormalities The first chromosome disorders

identified had very striking and generally severe phenotypes, because

the abnormalities involved large regions of the genome, but as

meth-ods have become more sensitive, it is now possible to recognize many

more subtle phenotypes, often involving smaller genomic regions

METHODS FOR CHROMOSOME ANALYSIS

STANDARD CYTOGENETIC ANALYSIS

Standard cytogenetic analysis refers to the examination of banded

human chromosomes Banded chromosome analysis allows for both

the determination of the number and identity of chromosomes in the

cell and recognition of abnormal banding patterns associated with a

structural rearrangement A stained band is defined as the part of a

chromosome that is clearly distinguishable from its adjacent segments

by appearing darker or lighter with one or more banding techniques

Cytogenetic analysis is most commonly carried out on cells in mitosis,

requiring dividing cells Actively growing cells are most often obtained

from peripheral blood; however, it is only a small subset of the blood

cells that are actually used for cytogenetic analysis Often, chemicals,

like phytohemagglutinin (PHA), are used to specially stimulate growth

of T cells in a blood sample Other sources of dividing cells include

skin-derived fibroblasts, amniotic fluid or placental tissue (for

prena-tal diagnosis), or tumor tissue (for cancer diagnosis) After culturing,

cells are treated with a mitotic spindle inhibitor, which prevents

the separation of the chromatids during metaphase Halting mitosis

in metaphase is essential, because chromosomes are at their most condensed state during this stage of mitosis The banding pattern of a metaphase chromosome is easily recognizable and is ideal for karyo-typing There are several different types of chromosome staining tech-niques, including R-banding, C-banding, and quinacrine staining, but the most commonly used is G-banding G-banding is accomplished

by treatment of the chromosomes with a proteolytic enzyme, such as trypsin, which digests some of the proteins holding DNA in a three-dimensional structure, followed by staining with a dye (Giemsa) that binds DNA The resulting patterns have both dark and light bands; in general, the light bands occur in regions on the chromosome in which genes are actively being transcribed, and dark bands are in regions of less active transcription

The banded human karyotype has now been standardized based

on an internationally agreed upon system for designating not only individual chromosomes but also chromosome regions, providing a way in which structural rearrangements and variants can be described

in terms of their composition The normal human female karyotype

is referred to as 46,XX (46 chromosomes, with 22 pairs of autosomes and two of the same type of sex chromosomes [two Xs], indicating this is a female); and the normal human male karyotype is referred

to as 46,XY (46 chromosomes, with 22 pairs of autosomes and one of each type of sex chromosome [one X and one Y], indicating this is a male) The anatomy of a chromosome includes the central constric-tion, known as the centromere, which is critical for movement of the chromosomes during mitosis and meiosis; the two chromosome arms (p for the smaller or petite arm, and q for the longer arm); and the chromosome ends, which contain the telomeres The telomeres are made up of a hexanucleotide repeat (TTAGGG)n, and unlike the centromere, they are not visible at the light microscope level Telomeres are functionally important because they confer stability

to the end of the chromosome Broken chromosomes tend to fuse end to end, whereas a normal chromosome with an intact telomere structure is stable To create the standard chromosome-banding map, each chromosome is divided into segments that are numbered, and then further subdivided The precise band names are recorded in

an international document so that each band has a distinct number

Figure 83e-1 shows an ideogram (chromosome map with bands)

of the X chromosome and a G-banded X chromosome This system provides a way for a chromosome abnormality to be written, with an indication of which band is deleted, duplicated, or rearranged

83e

p2

p1q1

q2

p arm

q arm

p22.3p21.3p11.2

q21.1q23

q28

centromeretelomere

num-Numbering begins at the centromere and moves out along each arm toward the telomeres

Molecular cytogenetics provides a link between chromosome and

molecular analysis and overcomes some of the limitations of standard

cytogenetics Deletions smaller than several million base pairs are not

routinely detectable by standard G-banding techniques, and

chromo-somal abnormalities with indistinct or novel banding patterns can be

difficult or impossible to interpret To carry out cytogenetic analysis,

cells must be dividing, which is not always possible to obtain (e.g., in

autopsy or tumor material that has already been fixed) Finally, growth

selection or bias may occasionally cause the results of cytogenetic

studies to be misleading because cells that proliferate in vitro may not

be representative of the original population, as is often the case with

tumor specimens

Fluorescence in situ hybridization (FISH) is a combined

cytoge-netic-molecular technique that solves many of the aforementioned

problems FISH permits determination of the number and location

of specific DNA sequences in human cells FISH can be performed

on metaphase chromosomes, as with G-banding, but can also be

performed on cells not actively progressing through mitosis FISH

performed on nondividing cells is referred to as interphase or nuclear

between the two strands of the DNA double helix and uses a molecular

probe, which can be a pool of sequences across an entire chromosome,

a DNA sequence for a repetitive part of the genome (e.g., centromeres

or telomeres), or a specific DNA sequence found only once in the

genome (e.g., a disease-associated gene) The choice of probes for FISH

studies is important and will vary with the information needed for the

diagnosis of a particular disorder The most common type of probes

are locus-specific probes, which are used to determine if a critical gene

or region is absent (indicating a deletion), or present in the normal

number of copies, or if an additional copy of the region is present FISH on metaphase chromosomes will give the additional information

of the location of the additional copy, which is necessary information

to determine whether a structural rearrangement, such as a tion, is present FISH can also be performed with probes that bind to repeated sequences, such as DNA found in centromeres or telomeres,

transloca-or with probes that bind to an entire chromosome (“painting” probes),

to determine the chromosome composition of an abnormal some Interphase FISH studies can also help to identify structural alterations when probes are used that map to both sides of a transloca-tion breakpoint Each side of the breakpoint is labeled in a different color, and when no translocation is present the two probes appear

chromo-to be overlapping When a translocation is present, the two probes appear separate from one another These set of probes, called “break-apart” probes, are commonly used to detect recurrent translocations

in cancer cells

ARRAY-BASED METHODOLOGIES (CYTOGENOMICS)

Array-based methods were introduced into the clinical lab beginning

in 2003 and quickly revolutionized the field of cytogenetics These techniques used arrays (collections of DNA segments from the entire genome) which could be interrogated with respect to copy number With standard cytogenetics, the missing or extra pieces of DNA have

to be big enough to see in the microscope on banded chromosomes (usually larger than 5 Mb) FISH requires a preselection of an infor-mative molecular probe prior to analysis In contrast, array-based techniques permit analysis of many regions of the genome in a single analysis, with greatly increased resolution over standard cytogenetics Array-based techniques allow for scanning of the genome for small deletions or duplications quickly and accurately The resolution of the

0.50 0.75 1.00

FIGuRE 83e-2 G-banding, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) array demonstrate an

abnormal chromosome 15 A G-banding shows an abnormal chromosome 15, with unrecognizable material in place of the p arm in the

chromosome on the right (top arrow) B Metaphase FISH (only chromosome 15s are shown) using a probe from the 15q telomere region (red) and a control probe that maps outside of the duplicated region (green) C Interphase FISH demonstrates three copies of the 15q tel probe in

red, and two copies of the 15q control probe (green) D Genome-wide SNP array demonstrates the increased copy number for a portion of 15q

Note that the G-banding alone indicates the abnormal chromosome 15, but the origin of the extra material can only be demonstrated by FISH

or array The FISH analysis requires additional information about possible genetic causes to select the correct probe The array can exactly tify the origin of the extra material, but by itself would not provide positional information

test is a function of the number of probes or DNA sequences present

on the array Arrays may use probes of different sizes (ranging from

50 to 200,000 base pairs of DNA) and different probe densities

depend-ing on the requirements of the application Low-resolution platforms

can have hundreds of probes, targeted to known disease regions,

whereas high-resolution platforms can have millions of probes spread

across the entire genome Depending on the size of the probes and the

probe placement across the genome, array-based testing may be able

to detect single exon deletions or duplications

Comparative Genomic Hybridization (CGH) and Single Nucleotide

Polymorphism (SNP) Analysis CGH and SNP-based genotyping arrays

can both be used for the analysis of genomic deletions and

duplica-tions For both techniques, oligonucleotide probes are placed onto a

slide or chip in a grid format Each of these probes is specific for a

particular genomic region In array CGH, the amount of DNA from

a patient is compared to that in a clinically normal control, or pool

of controls, for each of the probes present on the array DNA from

a patient is fluorescently labeled with a dye of one color, and DNA

from a control individual is labeled with another color These DNA

samples are then hybridized at the same time to the array The

result-ing fluorescent signal will vary dependresult-ing on whether both the control

and patient DNA are present in equal amounts or if one has a different

copy number than the other SNP platforms use arrays targeting SNPs

that are distributed across the genome SNP arrays vary in density of

markers and in the technology used for genotyping, depending on the

manufacturer of the array SNP arrays were initially designed to

deter-mine genotypes at a biallelic, polymorphic base (e.g., CC, CT, or TT)

and have been increasingly used in genome-wide association studies

to identify disease susceptibility genes SNP arrays were subsequently

adapted to identify genomic deletions and duplications (Fig 83e-2)

SNP arrays, in addition to identifying copy number changes, can

also detect regions of the genome that have an excess of homozygous

genotypes and absence of heterozygous genotypes (e.g., CC and TT

genotypes only, with no CT genotypes) Absence of heterozygosity is

sometimes associated with uniparental disomy (discussed later in this

chapter) but is also observed when an individual’s parents are related

to one another (identity by descent) Regions of homozygosity have

been used to help identify genes in which homozygous mutations

result in disease phenotypes in families with known consanguinity

Array-based techniques (which we will now refer to as cytogenomic

analysis) have proven superior to chromosome analysis in the

identifi-cation of clinically significant deletions or dupliidentifi-cations It is estimated

that for a deletion or duplication to be visualized by standard

cyto-genetics it must be minimally between 5 and 10 million base pairs in

size In almost all cases, deletions and duplications of this size contain

multiple genes, and these deletions and duplications are disease

caus-ing However, utilization of array-based cytogenomic testing, which

can routinely identify deletions and duplications smaller than 50,000

base pairs, reveals that clinically normal individuals all have some

deletions and duplications This presents a dilemma for the analyst

to discern which smaller copy number variations (CNVs) are disease

causing (pathogenic) and which are likely benign polymorphisms

Although initially burdensome, the cytogenomics community has

been curating these CNVs for almost a decade, and databases have

been created reporting CNVs routinely seen in clinically normal

indi-viduals and those routinely seen in indiindi-viduals with clinical

abnormali-ties Nevertheless, each copy number variant that is identified in an

individual undergoing genomic testing must be evaluated for gene content and overlap with CNVs in other patients and in controls

Array technologies are DNA based, unlike cytogenetic gies, which are cell based Although resolution of gains and losses are greatly increased with array technology, this technique cannot identify structural changes When DNA is extracted for array stud-ies, chromosomal structure is lost because the DNA is fragmented for better hybridization to the slides As an example, the array may be able to detect a duplication of a small region of a chromosome, but no informa-tion on the location of this extra material can be determined from this test The location of this extra copy in the genome may be critical, as the chromosomal material may be involved in a translocation, insertion, marker, or other complex rearrangement Depending on the chromo-somal position of this extra material, the patient may have different clinical outcomes, and recurrence risks for the family can be significantly different Often, combinations of array-based and cytogenetic-based techniques are required to fully characterize chromosomal abnormali-

NEXT-GENERATION SEQuENCING—BASED METHODOLOGIES

Recent advances in genomic sequencing, known as next-generation sequencing (NGS), have vastly increased the speed and throughput of DNA sequence analysis NGS is rapidly finding its way into the diag-nostic lab for detection of clinically relevant intragenic mutations, and new bioinformatic tools for analysis of genomic deletions and duplica-tions are being developed It is anticipated that NGS will soon allow the complete analysis of a patient’s genome, with identification of intragenic mutations as well as chromosome abnormalities resulting in gain or loss of genetic material Identification of completely balanced translocations is the most challenging for NGS, but recent reports of successes in this area suggest that in a matter of time, sequencing will

be used for all types of genomic analysis

INDICATIONS FOR CHROMOSOME/CYTOGENOMIC ANALYSIS

Cytogenetic analysis is most commonly used for (1) examination of the fetal chromosomes or genome during pregnancy (prenatal diagnosis)

or in the event of a spontaneous miscarriage; (2) examination of mosomes in the neonatal or pediatric population to look for an under-lying diagnosis in the case of congenital or developmental anomalies, including short stature and abnormalities of sexual differentiation or progression; (3) chromosome analysis in adults who are facing fertil-ity problems; or (4) examination of cancer cells to look for alterations that aid in establishing a diagnosis or contributing to the prognosis of

PRENATAL DIAGNOSIS

Prenatal diagnosis is carried out by analysis of samples obtained by four techniques: amniocentesis, chorionic villous sampling, fetal blood sampling, and analysis of cell free DNA from maternal serum Amniocentesis, which has been the most commonly used test to date,

is usually performed between 15 and 17 weeks of gestational age and carries a small but significant risk for miscarriage Amniocentesis can

be performed as early as 12 weeks, but because there is a lower volume

of fluid, the risks for fetal injury or miscarriage are greater Chorionic villous sampling (CVS) or placental biopsy is routinely carried out earlier than amniocentesis, between 10 and 12 weeks, but a reported increase in limb defects when the procedure is carried out earlier than

Method Requires Growing Cells Detects Deletions and Duplications

Detects Balanced Structural Rearrangements Detects Uniparental Disomy Detection Limits (Lower Limit)

10 weeks has resulted in reduced use of this test in some centers Fetal

blood sampling (percutaneous umbilical blood sampling [PUBS]) is a

riskier procedure that is carried out in the second or third trimester of

pregnancy, usually to follow up on an unclear finding from an

amnio-centesis (such as mosaicism) or an ultrasound abnormality that was

detected later in pregnancy One of the far-reaching recent advances

in prenatal diagnosis of chromosome and other genetic disorders is

the utilization of cell free fetal DNA that can be identified in maternal

serum The obvious advantages of using fetal DNA obtained from

maternal serum is that the DNA can be obtained at minimal risk to the

pregnancy, because it requires a maternal blood sample, rather than

amniotic fluid which is obtained by puncturing the uterine membranes

and carries a risk of miscarriage or infection Although cell free fetal

DNA screening, also called noninvasive prenatal screening, has started

to be offered clinically, it requires further confirmation of fetal tissues

when an abnormal result is identified Furthermore, ethical concerns

have been raised, because it is feared that the ease of doing this test

may encourage testing for individuals who are not truly prepared to

deal with the choices that accompany diagnosis of a genetic disease

and this testing may change the ethical implications of prenatal testing

Nevertheless, this is an active of area of research, both in terms of the

technology and the utilization and implications

Common Indications Common indications for prenatal diagnosis by

cytogenetic or cytogenomic analysis are (1) advanced maternal age,

(2) presence of an abnormality of the fetus on ultrasound

examina-tion, and (3) abnormalities in maternal serum screening that reveal an

increased risk for chromosome abnormality

Maternal age is well known to be an important risk factor for

hav-ing a fetus with trisomy At a maternal age less than 25 years, 2% of all

clinically recognized pregnancies are trisomic, but by a maternal age

of 36 years, this figure increases to 10%, and by the maternal age of

42 years, the figure increases to >33% Based on the risk of having

a chromosomally abnormal fetus in comparison to the risk for an

adverse event from amniocentesis or CVS, the recommendation is

that women over the age of 35 consider prenatal testing if they want

to know the chromosomal status of their fetus The precise

mecha-nism for the maternal age effect is not known, but it is believed that

it involves a breakdown in the process of chromosome segregation A

similar effect is not seen for trisomy and paternal age This difference

may reflect the fact that oocytes are generated early in ovary

develop-ment in the female, whereas spermatogonia are generated

continu-ously after puberty in the male

Abnormalities on prenatal ultrasound are the second most frequent

indication for prenatal genetic screening Ultrasound screening can

reveal structural or functional anomalies in the fetus, which might be associated with chromosome or genomic disorders Follow-up chro-mosome studies may therefore be recommended

Maternal serum screening results are the third most frequent

indica-tion for prenatal chromosome analysis There have been several sions of maternal serum screening offered over the past few decades Currently, the “quad” screen analyzes levels of α fetoprotein (AFP), human chorionic gonadotropin (hCG), estriol, and inhibin-A The values of these analytes are used to adjust the maternal age–predicted risk of a trisomy 21 or trisomy 18 fetus

ver-POSTNATAL INDICATIONS

Postnatal indications for cytogenetic or cytogenomic analysis in neonates or children are varied, and the list has been growing with the increasing ability to diagnose smaller genomic alterations via array-based techniques Common indications include multiple con-genital anomalies, suspicion of a known cytogenetic or cytogenomic syndrome, intellectual disability or developmental delay both with and without accompanying dysmorphic features, autism, failure to thrive

in infancy or short stature during childhood, and disorders of sexual development The ability to detect smaller genomic alterations with involvement of fewer genes, sometimes as few as a single gene, suggests that a wider range of phenotypes could be investigated by cytogenomic analysis Reasons for chromosome testing in adults include recurrent miscarriages or infertility, where balanced chromosome rearrange-ments such as reciprocal translocations may occur Additionally, some adults with anomalies who were not diagnosed when they were chil-dren are referred for cytogenetic analysis, often when other members

of their family want to understand any potential genetic implications,

as they plan their own families

TYPES OF CHROMOSOME ABNORMALITIESNuMERICAL CHROMOSOME ABNORMALITIES

Aneuploidy (extra or missing chromosomes) is the most common

type of abnormality, occurring in 3/1000 newborns and at much higher frequency (about 35%) in spontaneously aborted fetuses The only autosomal trisomies that are compatible with being live born in humans are trisomies 13, 18, and 21, although there are several chro-mosomes that can be trisomic in mosaic form Trisomy 21 is associ-ated with the relatively common disorder Down syndrome Down syndrome has characteristic features including recognizable facial fea-tures, along with intellectual disability and abnormalities of multiple other organ systems including the heart Both trisomy 13 and trisomy

18 are much more severe disorders than Down syndrome, with low frequency of patients surviving past 1 year of age Trisomy 13 is char-acterized by low birth weight, postaxial polydactyly, microcephaly, ocular malformations such as anophthalmia or microphthalmia, cleft lip and palate, cardiac defects, and renal malformations Trisomy 18 neonates have distinct facial characteristics at birth accompanied by

an abnormal neurologic exam, underdeveloped genitalia, general lack

of responsiveness, and structural birth defects such as congenital heart disease, esophageal atresia, and omphalocele

Mosaicism refers to the presence of two or more populations of

cells with distinct chromosome constitutions: for example, an vidual with a normal female karyotype in some cells (46,XX) and trisomy 21 in other cells (47,XX,+21) In general, individuals who are mosaic for a chromosomal abnormality have less severe phenotypes than individuals with that same finding in every cell The severity and presentation of phenotypes are related to the mosaic levels and the tissue distribution of the abnormal cells There are a number of trisomies that have been reported in mosaic form including mosaic trisomies for chromosomes 8, 9, 14, 17, and 22 A number of trisomies have also been reported in spontaneous abortions (SABs) that have not been seen in live-born individuals, including trisomy 16, which is the most common trisomy in SABs Monosomy for human chromo-somes is very rare, with the single exception being monosomy for the

indi-X chromosome, associated with Turner syndrome (45,indi-X) Monosomy for the X chromosome occurs in 1% of all conceptions, yet 98% of these conceptions do not go to term and result in SABs Trisomies for

aCross the lifespan Timing of Testing Indications for Testing

Abnormalities on ultrasoundIncreased risk for genetic disorder on maternal serum screen

Neonatal and Childhood Multiple congenital anomalies

Intellectual disabilityAutism

Developmental delayFailure to thriveShort statureDisorders of sexual developmentHistory of familial chromosomal alterationCancer

Recurrent miscarriageCancer

the sex chromosomes also occur, with 47,XXX (trisomy X or triple X

syndrome), 47,XXY (Klinefelter syndrome), and 47,XYY all reported

syndrome is the most common clinically recognized sex chromosome

abnormality, and clinical features include gynecomastia, azoospermia,

small testes, and hypogonadism The 47,XYY karyotype is most often

found in boys with developmental delay and or behavioral

difficul-ties, but population-based studies have shown that intelligence for

individuals with this karyotype is generally within the normal range,

although slightly lower than that found in siblings

STRuCTuRAL CHROMOSOME ABNORMALITIES

Structural chromosome abnormalities include deletions, duplications,

translocations, inversions, as well as other types of abnormalities,

each relatively rare, but nonetheless contributing to clinical disease

resulting from chromosome anomalies These rare alterations include

isochromosomes, ring chromosomes, dicentric chromosomes, and

marker chromosomes (structurally abnormal chromosomes that

can-not be identified based on cytogenetics alone) Both translocations and

inversions can be completely balanced in some cases, such that there

is no disruption of coding regions of the genome, with a completely

normal clinical phenotype; however, carriers are at risk for unbalanced

forms of these rearrangements in their offspring

Reciprocal translocations are found in approximately 1/500–1/600

individuals in the general population and result from the exchange

of chromosomal segments between at least two chromosomes These

usually occur between nonhomologous chromosomes and can be

identified based on an altered banding pattern on G-banding Balanced

translocation carriers are at risk for abnormal chromosome

segrega-tion during meiosis and therefore have a higher risk for infertility,

SAB, and live-born offspring with multiple congenital

malforma-tions These phenotypes are observed when only one of the pairs of

chromosomes involved in a translocation is inherited from a parent,

exchanged segments are so small that they cannot be appreciated by

banding (cryptic translocation), and these are sometimes recognized when a phenotypically affected child with an unbalanced form is born Parental chromosomes can then be studied by FISH to determine if the rearrangement is inherited from a parent with a balanced form

of the translocation The majority of reciprocal, apparently balanced translocations occur in phenotypically normal individuals The risk for a clinical abnormality when a new reciprocal translocation is identified (usually during prenatal diagnostic studies) is about 7% Analysis of cytogenetically reciprocal translocations using arrays has demonstrated that translocations in clinically normal individuals are more likely to show no deletions or duplications at the breakpoint, whereas translocations in clinically affected individuals are more likely

to have breakpoint-associated deletions or duplications Most rocal translocations occur uniquely, at apparently random positions throughout the genome; however, there are a few exceptions with multiple cases of recurrent translocations occurring These recurrent translocations include t(11;22), which results in Emanuel syndrome

recip-in the unbalanced form, and several translocations recip-involvrecip-ing a region

on 4p, 8p, and 12p These recurrent translocations occur in regions

of the genome that contain specific types of AT-rich repeats, or other repeat sequences, that are prone to rearrangement A special category

of translocations is the Robertsonian translocations, which involve the acrocentric chromosomes An acrocentric chromosome has unique genetic material only on the long arm of the chromosomes, whereas the short arm contains repetitive DNA The acrocentric chromosomes are 13, 14, 15, 21, and 22 Robertsonian translocations occur when an entire long arm of an acrocentric chromosome is translocated onto the short arm of another acrocentric chromosome Balanced carriers of a Robertsonian translocation contain only 45 chromosomes, with one chromosome consisting of two long arms of an acrocentric chromo-some Technically, this is an unbalanced translocation, as two short arms of the acrocentric chromosomes are missing; however, because the short arms are repetitive, there is no phenotypic consequence Unbalanced Robertsonian carriers have 46 chromosomes, but have three copies of the long arm of an acrocentric chromosome The most

FIGuRE 83e-3 Segregation of a balanced translocation in a mother, with inheritance of an unbalanced form in her child Note that the

mother has two rearranged chromosomes, but her child only received one of these, resulting in extra copies of a region of the blue

chromo-some, with loss of some material from the red chromosome

common Robertsonian translocation involves chromosomes 13 and

14 Unbalanced Robertsonian translocations involving chromosomes

13 and 21 result in trisomy 13 and Down syndrome, respectively

Approximately 4% of patients with Down syndrome have a

transloca-tion, and because recurrence risks are different for families of these

individuals, all patients with clinically identified Down syndrome

should have a karyotype to look for translocations

Inversions are another type of chromosome abnormality involving

rearranged segments, where there are two breaks within a

chromo-some, with the intervening chromosomal material inserted in an

inverted orientation As with reciprocal translocations, if a break

occurs within a gene or control region for a gene, a clinical phenotype

may result, but often there are no consequences for the inversion

car-rier; however, there is risk for abnormalities in the offspring of carriers,

as recombinant chromosomes may result after crossing over between

a normal chromosome and an inverted chromosome during meiosis

Deletion refers to the loss of a chromosomal segment, which results

in the presence of only a single copy of that region in an individual’s

genome A deletion can be at the end of a chromosome (terminal), or it

can be within the chromosome (interstitial) Deletions that are visible

at the microscopic level in standard cytogenetic analysis are generally

greater than 5 Mb in size Smaller deletions have been identified by

FISH and by chromosomal microarray The clinical consequences of

a deletion depend on the number and function of genes in the deleted

region Genes that cause a phenotype when a single copy is deleted are

known as haploinsufficient genes (one copy is not sufficient), and it

is estimated that less than 10% of genes are haploinsufficient Genes

associated with disease that are not haploinsufficient include genes for

known recessive disorders, such as cystic fibrosis or Tay-Sachs disease

The first chromosome deletion syndromes were diagnosed clinically

and were subsequently demonstrated to be caused by a chromosome

deletion on cytogenetic analysis Examples of these disorders include

the Wolf-Hirschhorn syndrome, which is associated with deletions of

a small region of the short arm of chromosome 4 (4p); the cri-du-chat

syndrome, associated with deletion of a small region of the short arm

of chromosome 5 (5p); Williams syndrome, which is associated with

interstitial deletions of the long arm of chromosome 7 (7q11.23); and

the DiGeorge/velocardiofacial syndromes, associated with

intersti-tial deletions of the long arm of chromosome 22 (22q11.2) Iniintersti-tial

cytogenetic studies were able to provide a rough localization of the

deletions in different patients, but with the increased usage of arrays,

precise mapping of the extent and gene content of these deletions has

become much easier In many cases, one or two genes that are critical

for the phenotype associated with these deletions have been

identi-fied In other cases, the phenotype stems from the deletion of multiple

genes The increased utilization of genomic testing by array, which can

identify deletions that are much smaller than those detectable by

stan-dard cytogenetic analysis, has resulted in the discovery of several new

cytogenomic disorders These include the 1q21.1, 15q13.3, 16p11.2,

and 17q21.31 microdeletion syndromes

Duplication of genomic regions is better tolerated than deletion,

as evidenced by the viability of several autosomal trisomies (whole

chromosome duplications) but no autosomal monosomies (whole

chromosome deletions) There are several duplication syndromes

where the duplicated region of the genome is present as a

supernu-merary chromosome Utilization of chromosome microarray analysis

has made analysis of the origins of duplicated chromosome material

straightforward (Fig 83e-2) Recurrent syndromes associated with

supernumerary chromosomes include the inverted duplication 15 (inv

dup 15) syndrome, caused by the presence of a marker chromosome

derived from chromosome 15, with two copies of proximal 15q

result-ing in tetrasomy (four copies) of this region The inv dup 15 syndrome

has a distinct phenotype and is associated with hypotonia,

develop-mental delay, intellectual disability, epilepsy, and autistic behavior

Another syndrome is the cat eye syndrome, named for the

“cat-eye-like” appearance of the pupil, resulting from a coloboma of the iris

This syndrome results from a supernumerary chromosome derived

from a portion of chromosome 22, and the marker chromosomes can

vary in size and are often mosaic Consistent with expectations of a

mosaic disorder, the phenotype of this syndrome is highly variable and includes renal malformations, urinary tract anomalies, congenital heart defects, anal atresia with fistula, imperforate anus, and mild to moderate intellectual disability Another rare duplication syndrome

is the Pallister-Killian syndrome (PKS), which illustrates the principle

of tissue-specific mosaicism Individuals with PKS have coarse facial features with pigmentary skin anomalies, localized alopecia, pro-found intellectual disability, and seizures The disorder is caused by a supernumerary isochromosome for the short arm of chromosome 12 (isochromosome 12p) Isochromosomes consist of two copies of one chromosome arm (p or q), rather than one copy of each arm This isochromosome is not generally seen in peripheral blood lymphocytes when they are analyzed by G-banding, but it is detected in fibroblasts Array technology has been reported to detect the isochromosome in uncultured peripheral blood in some patients, and it has been hypothe-sized that a growth bias against cells with the isochromosome prevents their identification in cytogenetic studies

Numerical abnormalities, translocations, and deletions are the most common chromosome alterations observed in the diagnostic labora-tory, but in addition to inversions and duplications, several other types of abnormal chromosomes have been reported, including ring chromosomes, where the two ends of the chromosome fuse to form

a circle, and insertions, where a piece of one chromosome is inserted into another chromosome or elsewhere into the same chromosome

Uniparental disomy (UPD) is the inheritance of a pair of

chro-mosomes (or part of a chromosome) from only one parent This usually occurs as a result of nondisjunction during meiosis, with a gamete missing or having an extra copy of a chromosome A result-ing fertilized egg would then have only one parental contribution for a given chromosome pair, or a trisomy for a given chromosome

If the monosomy or trisomy is not compatible with life, the embryo may undergo a “rescue” to normal copy number If a monosomy is rescued, the single chromosome may be duplicated, resulting in a cell

the case of trisomies, a subsequent nondisjunction can result in cells where one of the extra chromosomes is lost (trisomy rescue) (Fig 83e-4) For trisomy rescue, there is a one in three chance that the lost

FIGuRE 83e-4 Mechanisms of formation of uniparental disomy

Panel A demonstrates nondisjunction in one parent (mother,

repre-sented in red), with trisomy in the zygote A subsequent tion, with loss of the paternal chromosome (represented in blue),

nondisjunc-restores the diploid karyotype but leaves two copies of the maternal

chromosome (maternal uniparental disomy [UPD]) Panel B

dem-onstrates nondisjunction in one parent (mother, indicated by red

oval), resulting in only one copy of this chromosome in the zygote

Subsequent nondisjunction duplicates the single chromosome, ing the monosomy, but resulting in two copies of the paternal chro-

rescu-mosome (represented in blue; paternal UPD).

chromosome will be the sole chromosome from one parent, resulting

in a cell with two chromosomes from the same parent UPD is

some-times associated with clinical abnormalities, and this can occur by two

mechanisms UPD can cause disease when there is an imprinted gene

on the involved chromosome, resulting in altered gene expression

Imprinting is the chemical marking of the parental origin of a

chromo-some, and genes that are imprinted are only expressed from either the

results in the differential expression of affected genes, based on parent

of origin Imprinting usually occurs through differential modification

of the chromosome from one of the parents, and methylation is one

of several epigenetic mechanisms (others include histone acetylation,

ubiquitylation, and phosphorylation) Imprinted chromosomes that

are associated with phenotypes include paternal UPD6 (associated

with neonatal diabetes), maternal UPD7 and UPD11 (associated

with Russell-Silver syndrome), paternal UPD11 (associated with

Beckwith-Wiedemann syndrome), paternal UPD14, maternal UPD15

(Angelman syndrome), and paternal UPD15 (Prader-Willi syndrome)

UPD can also result in disease if the two copies from the same parent

are the same chromosome (uniparental isodisomy), and the

chromo-some contains an allele involving a pathogenic mutation associated

with a recessive disorder Two copies of the deleterious allele would

result in the associated disease, even though only one parent is a

dis-ease carrier

ACQuIRED CHROMOSOME ABNORMALITIES IN CANCER

Chromosome changes can occur during meiosis or mitosis and can

occur at any point across the lifespan Mosaicism for a developmental

disorder is one consequence of mitotic chromosome abnormalities,

and another consequence is cancer, when the chromosome change confers a growth or proliferation advantage on the cell The types of chromosome abnormalities seen in cancer are similar to those seen in the developmental disorders (e.g., aneuploidy, deletion, duplication, translocation, isochromosomes, rings, inversion) Tumor cells often have multiple chromosome changes, some of which happen early

in the development of a tumor, and may contribute to its selective advantage, whereas others are secondary effects of the deregulation that characterizes many tumors Chromosome changes in cancer have been studied extensively and have been shown to provide important diagnostic, classification, and prognostic information The identifica-tion of cancer type–specific translocation breakpoints has led to the identification of a number of cancer-associated genes.�For example, the small abnormal chromosome found to be associated with chronic myelogenous leukemia (CML) in 1960 was shown to be the result of translocation between chromosomes 9 and 22 once techniques for analysis of banded chromosomes were introduced, and subsequently,

the translocation breakpoint was cloned to reveal the c-abl oncogene

on chromosome 9 This translocation produces a fusion protein, which

cancer genetics, see Chap 101e.

Susan M Domchek, J Larry Jameson, Susan Miesfeldt

APPLICATIONS OF MOLECULAR GENETICS IN CLINICAL MEDICINE

Genetic testing for inherited abnormalities associated with disease

risk is increasingly used in the practice of clinical medicine Germline

gene mutations with autosomal dominant or recessive patterns of

small relative risks associated with disease Germline alterations are

responsible for disorders beyond classic Mendelian conditions with

genetic susceptibility to common adult-onset diseases such as asthma,

hypertension, diabetes mellitus, macular degeneration, and many

forms of cancer For many of these diseases, there is a complex

inter-play of genes (often multiple) and environmental factors that affect

lifetime risk, age of onset, disease severity, and treatment options

The expansion of knowledge related to genetics is changing our

understanding of pathophysiology and influencing our classification

of diseases Awareness of genetic etiology can have an impact on

clini-cal management, including prevention and screening for or treatment

of a range of diseases Primary care physicians are relied upon to help

patients navigate testing and treatment options Consequently, they

must understand the genetic basis for a large number of genetically

influenced diseases, incorporate personal and family history to

deter-mine the risk for a specific mutation, and be positioned to provide

counseling Even if patients are seen by genetic specialists who assess

genetic risk and coordinate testing, primary care providers should

provide information to their patients regarding the indications,

limita-tions, risks, and benefits of genetic counseling and testing They must

also be prepared to offer risk-based management following genetic risk

assessment Given the pace of genetics, this is an increasingly difficult

task The field of clinical genetics is rapidly moving from single gene

testing to multigene panel testing, with techniques such as

whole-exome and -genome sequencing on the horizon, increasing the

com-plexity of test selection and interpretation, as well as patient education

and medical decision making

COMMON ADULT-ONSET GENETIC DISORDERS

INHERITANCE PATTERNS

Adult-onset hereditary diseases follow multiple patterns of

inheri-tance Some are autosomal dominant conditions These include many

common cancer susceptibility syndromes such as hereditary breast

and ovarian cancer (due to germline BRCA1 and BRCA2 mutations)

and Lynch syndrome (caused by germline mutations in the mismatch

repair genes MLH1, MSH2, MSH6, and PMS2) In both of these

examples, inherited mutations are associated with a high penetrance

(lifetime risk) of cancer, although risk is not 100% In other

condi-tions, although there is autosomal dominant transmission, there is

lower penetrance, thereby making the disorders more difficult to

recognize For example, germline mutations in CHEK2 increase the

risk of breast cancer, but with a moderate lifetime risk in the range of

20–40%, as opposed to 50–70% for mutations in BRCA1 or BRCA2

Other adult-onset hereditary diseases are transmitted in an autosomal

recessive fashion where two mutant alleles are necessary to cause

dis-ease Examples include hemochromatosis and MYH-associated colon

cancer There are more pediatric-onset autosomal recessive disorders,

such as lysosomal storage diseases and cystic fibrosis

The genetic risk for many adult-onset disorders is multifactorial

Risk can be conferred by genetic factors at a number of loci, which

individually have very small effects (usually with relative risks of <1.5)

These risk loci (generally single nucleotide polymorphisms [SNPs])

combine with other genes and environmental factors in ways that are

not well understood SNP panels are available to assess risk of disease,

but the optimal way of using this information in the clinical setting remains uncertain

Many diseases have multiple patterns of inheritance, adding to the complexity of evaluating patients and families for these conditions For example, colon cancer can be associated with a single germline muta-tion in a mismatch repair gene (Lynch syndrome, autosomal domi-

nant), biallelic mutations in MYH (autosomal recessive), or multiple

SNPs (polygenic) Many more individuals will have SNP risk alleles than germline mutations in high-penetrance genes, but cumulative lifetime risk of colon cancer related to the former is modest, whereas the risk related to the latter is significant Personal and family histories provide important insights into the possible mode of inheritance

FAMILY HISTORY

When two or more first-degree relatives are affected with asthma, cardiovascular disease, type 2 diabetes, breast cancer, colon cancer, or melanoma, the relative risk for disease among close relatives ranges from two- to fivefold, underscoring the importance of family history for these prevalent disorders In most situations, the key to assessing the inherited risk for common adult-onset diseases is the collection and interpretation of a detailed personal and family medical history in conjunction with a directed physical examination

Family history should be recorded in the form of a pedigree Pedigrees should convey health-related data on first- and second-degree relatives When such pedigrees suggest inherited disease, they should be expanded to include additional family members The deter-mination of risk for an asymptomatic individual will vary depend-ing on the size of the pedigree, the number of unaffected relatives, the types of diagnoses, and the ages of disease onset For example, a woman with two first-degree relatives with breast cancer is at greater risk for a specific Mendelian disorder if she has a total of 3 female first-degree relatives (with only 1 unaffected) than if she has a total

of 10 female first-degree relatives (with 7 unaffected) Factors such as adoption and limited family structure (few women in a family) should

to be taken into consideration in the interpretation of a pedigree Additional considerations include young age of disease onset (e.g., a 30-year nonsmoking woman with a myocardial infarction), unusual diseases (e.g., male breast cancer or medullary thyroid cancer), and the finding of multiple potentially related diseases in an individual (e.g., a woman with a history of both colon and endometrial cancer) Some adult-onset diseases are more prevalent in certain ethnic groups For instance, 2.5% of individuals of Ashkenazi Jewish ancestry carry one

of three founder mutation in BRCA1 and BRCA2 Factor V Leiden

mutations are much more common in Caucasians than in Africans

or Asians

Additional variables that should be documented are nonhereditary risk factors among those with disease (such as cigarette smoking and myocardial infarction; asbestos exposure and lung disease; and mantle radiation and breast cancer) Significant associated environmental exposures or lifestyle factors decrease the likelihood of a specific genetic disorder In contrast, the absence of nonhereditary risk fac-tors typically associated with a disease raises concern about a genetic association A personal or family history of deep vein thrombosis in the absence of known environmental or medical risk factors suggests

a hereditary thrombotic disorder The physical examination may also provide important clues about the risk for a specific inherited disorder

A patient presenting with xanthomas at a young age should prompt consideration of familial hypercholesterolemia The presence of trichi-lemmomas in a woman with breast cancer raises concern for Cowden

syndrome, associated with PTEN mutations.

Recall of family history is often inaccurate This is especially so when the history is remote and families lose contact or separate geo-graphically It can be helpful to ask patients to fill out family history forms before or after their visits, because this provides them with an opportunity to contact relatives Ideally, this information should be embedded in electronic health records and updated intermittently Attempts should be made to confirm the illnesses reported in the family history before making important and, in certain circum-stances, irreversible management decisions This process is often labor

84

intensive and ideally involves interviews of additional family members

or reviewing medical records, autopsy reports, and death certificates

Although many inherited disorders will be suggested by the

cluster-ing of relatives with the same or related conditions, it is important to

note that disease penetrance is incomplete for most genetic disorders

As a result, the pedigree obtained in such families may not exhibit a

clear Mendelian inheritance pattern, because not all family members

carrying the disease-associated alleles will manifest clinical evidence

of the condition Furthermore, genes associated with some of these

disorders often exhibit variable disease expression For example,

the breast cancer–associated gene BRCA2 can predispose to several

different malignancies in the same family, including cancers of the

breast, ovary, pancreas, skin, and prostate For common diseases such

as breast cancer, some family members without the susceptibility allele

(or genotype) may develop breast cancer (or phenotype) sporadically

Such phenocopies represent another confounding variable in the

pedi-gree analysis

Some of the aforementioned features of the family history are

(IV-1), has a strong history of breast and ovarian cancer on the

pater-nal side of her family The early age of onset and the co-occurrence of

breast and ovarian cancer in this family suggest the possibility of an

inherited mutation in BRCA1 or BRCA2 It is unclear however,

with-out genetic testing, whether her father harbors such a mutation and

transmitted it to her After appropriate genetic counseling of the

pro-band and her family, the most informative and cost-effective approach

to DNA analysis in this family is to test the cancer-affected 42-year-old

living cousin for the presence of a BRCA1 or BRCA2 mutation If a

mutation is found, then it is possible to test for this particular

altera-tion in other family members, if they so desire In the example shown,

if the proband’s father has a BRCA1 mutation, there is a 50:50

prob-ability that the mutation was transmitted to her, and genetic testing

can be used to establish the absence or presence of this alteration In

this same example, if a mutation is not detected in the cancer-affected

cousin, testing would not be indicated for cancer-unaffected relatives

GENETIC TESTING FOR ADULT-ONSET DISORDERS

A critical first step before initiating genetic testing is to ensure that the

correct clinical diagnosis has been made, whether it is based on

fam-ily history, characteristic physical findings, pathology, or biochemical

testing Such careful clinical assessment can define the phenotype In

the traditional model of genetic testing, testing is directed initially

FIGURE 84-1 A 36-year-old woman (arrow) seeks consultation

because of her family history of cancer The patient expresses

concern that the multiple cancers in her relatives imply an inherited

predisposition to develop cancer The family history is recorded, and

records of the patient’s relatives confirm the reported diagnoses

ca 44

46 Ovarian

ca 43

Ovarian cancer 2

40 Ovarian

ca 38

42 Breast

ca 38

24 Pneumonia

56

36

62

69 Breast

ca 44

55 Ovarian

ca 54

62 10

mutations in a number of distinct genes The patterns of disease mission, disease risk, clinical course, and treatment may differ signifi-cantly depending on the specific gene affected Historically, the choice

trans-of which gene to test has been determined by unique clinical and family history features and the relative prevalence of candidate genetic disorders However, rapid changes in genetic testing techniques, as discussed below, may impact this paradigm It is now technically and financially feasible to sequence many genes (or even the whole exome)

at one time The incorporation of multiplex testing for germline tions is rapidly evolving

muta-METHODOLOGIC APPROACHES TO GENETIC TESTING

Genetic testing is regulated and performed in much the same way as other specialized laboratory tests In the United States, genetic test-ing laboratories are Clinical Laboratory Improvement Amendments (CLIA) approved to ensure that they meet quality and proficiency standards A useful information source for various genetic tests is

www.genetests.org It should be noted that many tests need to be

ordered through specialized laboratories

Genetic testing is performed largely by DNA sequence analysis

for mutations, although genotype can also be deduced through the study of RNA or protein (e.g., apolipoprotein E, hemoglobin S, and immunohistochemistry) For example, universal screening for Lynch syndrome via immunohistochemical analysis of colorectal cancers for absence of expression of mismatch repair proteins is under way at multiple hospitals throughout the United States The determination

of DNA sequence alterations relies heavily on the use of polymerase chain reaction (PCR), which allows rapid amplification and analysis of the gene of interest In addition, PCR enables genetic testing on mini-mal amounts of DNA extracted from a wide range of tissue sources including leukocytes, mucosal epithelial cells (obtained via saliva or buccal swabs), and archival tissues Amplified DNA can be analyzed directly by DNA sequencing, or it can be hybridized to DNA chips

or blots to detect the presence of normal and altered DNA sequences

Direct DNA sequencing is frequently used for determination of itary disease susceptibility and prenatal diagnosis Analyses of large alterations of the genome are possible using cytogenetics, fluorescent

hered-in situ hybridization (FISH), Southern blotthered-ing, or multiplex

Massively parallel sequencing (also called next-generation sequencing)

is significantly altering the approach to genetic testing for adult-onset hereditary susceptibility disorder This technology encompasses several high-throughput approaches to DNA sequencing, all of which can reliably sequence many genes at one time Technically, this involves the use of amplified DNA templates in a flow cell, a very different process than traditional Sanger sequencing which is time-consuming and expensive

Multiplex panels for inherited susceptibility are commercially

avail-able and include testing of a number of genes that have been ated with the condition of interest For example, panels are available for Brugada syndrome, hypertrophic cardiomyopathy, and Charcot-Marie-Tooth neuropathy For many syndromes, this type of panel test-ing may make sense However, in other situations, the utility of panel testing is less certain Currently available breast cancer susceptibility panels contain six genes or more Many of the genes included in the larger panels are associated with only a modest risk of breast cancer, and the clinical application is uncertain An additional problem of sequencing many genes (rather than the genes for which there is most suspicion) is the identification of one or more variants of uncertain significance (VUS), discussed below

associ-Whole-exome sequencing (WES) is also now commercially available,

although largely used in individuals with syndromes unexplained by

VUS are another limitation to genetic testing A VUS (also termed

unclassified variant) is a sequence variation in a gene where the effect

of the alteration on the function of the protein is not known Many of these variants are single nucleotide substitutions (also called missense mutations) that result in a single amino acid change Although many VUSs will ultimately be reclassified as benign polymorphisms, some will prove to be functionally important As more genes are sequenced (for example, in a multiplex panel or through WES), the percentage of individuals found to have a VUS increases significantly The finding

of a VUS is difficult for patients and providers alike and complicates decisions regarding medical management

Clinical utility is an important consideration because genetic testing for susceptibility to chronic diseases is increasingly integrated into the practice of medicine In some situations, there is clear clinical utility

to genetic testing with significant evidence-based changes in medical management decisions based on results However, in many cases, the discovery of disease-associated genes has outpaced studies that assess how such information should be used in the clinical management of the patient and family This is particularly true for moderate- and low-penetrance gene mutations Therefore, predictive genetic testing should be approached with caution and only offered to patients who have been adequately counseled and have provided informed consent.Predictive genetic testing falls into two distinct categories Presymptomatic testing applies to diseases where a specific genetic alteration is associated with a near 100% likelihood of developing disease In contrast, predisposition testing predicts a risk for disease that is less than 100% For example, presymptomatic testing is avail-able for those at risk for Huntington’s disease; whereas, predisposition testing is considered for those at risk for hereditary colon cancer It is important to note that for the majority of adult-onset disorders, testing

is only predictive Test results cannot reveal with confidence whether,

when, or how the disease will manifest itself For example, not everyone with the apolipoprotein E4 allele will develop Alzheimer’s disease, and individuals without this genetic marker can still develop the disorder

The optimal testing strategy for a family is

to initiate testing in an affected family member first Identification of a mutation can direct the testing of other at-risk family members (whether symptomatic or not) In the absence of addi-tional familial or environmental risk factors, individuals who test negative for the mutation found in the affected family member can be informed that they are at general population risk for that particular disease Furthermore, they can be reassured that they are not at risk for passing the mutation on to their children On the other hand, asymptomatic family members who test positive for the known mutation must

be informed that they are at increased risk for disease development and for transmitting the alteration to their children

Pretest counseling and education are tant, as is an assessment of the patient’s ability

impor-to understand and cope with test results Genetic testing has implications for entire families, and thus individuals interested in pursuing genetic testing must consider how test results might impact their relationships with relatives, part-ners, spouses, and children In families with a known genetic mutation, those who test posi-tive must consider the impact of their carrier

traditional genetic testing As cost declines, WES may be more widely

used Whole-genome sequencing is also commercially available

Although it may be quite feasible to sequence the entire genome, there

are many issues in doing so, including the daunting task of

analyz-ing the vast amount of data generated Other issues include: (1) the

optimal way in which to obtain informed consent, (2) interpretation

of frequent sequence variation of uncertain significance, (3)

interpreta-tion of alterainterpreta-tions in genes with unclear relevance to specific human

pathology, and (4) management of unexpected but clinically

signifi-cant genetic findings

Testing strategies are evolving as a result of these new genetic

testing platforms As the cost of multiple gene panels and WES

con-tinue to fall, and as interpretation of such test results improve, there

may be a shift from sequential single-gene (or a few genes) testing

to multigene testing For example, presently, a 30-year-old woman

with breast cancer but no family history of cancer and no syndromic

features would undergo BRCA1/2 testing If negative, she would

sub-sequently be offered TP53 testing Notably, a reasonable number of

individuals offered TP53 testing for Li-Fraumeni syndrome decline

because mutations are associated with extremely high cancer risks

(including childhood cancers) in multiple organs and there are no

proven interventions to mitigate risk Without features consistent

with Cowden syndrome, the woman would not be routinely offered

PTEN testing or testing for CHEK2, ATM, BRIP, BARD, NBN, and

PALB2 However, it is now possible to synchronously analyze all of

the aforementioned genes, for a nominally higher cost than BRCA1/2

testing alone Concerns about such panels include appropriate consent

strategies related to unexpected findings, VUS, and unclear clinical

utility of testing moderate-penetrance genes Thus, changes from the

traditional model of single-gene genetic testing should be done with

Limitations to the accuracy and interpretation of genetic testing

exist In addition to technical errors, genetics tests are sometimes

designed to detect only the most common mutations In addition,

genetic testing has evolved over time For example, it was not

pos-sible to obtain commercially available comprehensive large genomic

rearrangement testing for BRCA1 and BRCA2 until 2006 Therefore, a

FIGURE 84-2 Approach to genetic testing.

Traditional approach to genetic testing Genetic testing in the era of

next-generation sequencing?

Patient identified by clinicalhistory of familial disorder

Pretest counseling for risks and

benefits of analysis of specific genes and informed consent

Mutationalanalysis

Posttest counseling andtreatment implications forpatient and family members

Pretest counseling for risks andbenefits of analysis of multiple genes (or whole exome) and

informed consent

Mutational analysis

Interpretation of the findings by

a physician in the context of theindividual’s personal and familymedical history

Posttest counseling andtreatment implications forpatient and family members

Return to primary carephysician for follow-up Return to primary carephysician for follow-up

If negativeconsider nextmost likely genes