Abnormal vertebral segmentation and the

Key words: abnormal vertebral segmentation; Notch signaling pathway; spondylocostal dysostosis; Alagille syndrome; DLL3; MESP2; LNFG; JAGGED1 Accepted 5 April 2007 INTRODUCTION Segmentat

SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

Abnormal Vertebral Segmentation and the

Notch Signaling Pathway in Man

Peter D Turnpenny,1* Ben Alman,2Alberto S Cornier,3Philip F Giampietro,4Amaka Offiah,5

Olivier Tassy,6Olivier Pourquie´,6Kenro Kusumi,7and Sally Dunwoodie8

Abnormal vertebral segmentation (AVS) in man is a relatively common congenital malformation but cannot

be subjected to the scientific analysis that is applied in animal models Nevertheless, some spectacular advances in the cell biology and molecular genetics of somitogenesis in animal models have proved to be directly relevant to human disease Some advances in our understanding have come through DNA linkage analysis in families demonstrating a clustering of AVS cases, as well as adopting a candidate gene approach.

Only rarely do AVS phenotypes follow clear Mendelian inheritance, but three genes—DLL3, MESP2, and LNFG— have now been identified for spondylocostal dysostosis (SCD) SCD is characterized by extensive

hemivertebrae, trunkal shortening, and abnormally aligned ribs with points of fusion In familial cases clearly following a Mendelian pattern, autosomal recessive inheritance is more common than autosomal dominant and the genes identified are functional within the Notch signaling pathway Other genes within the pathway cause diverse phenotypes such as Alagille syndrome (AGS) and CADASIL, conditions that may have their origin in defective vasculogenesis Here, we deal mainly with SCD and AGS, and present a new classification system for AVS phenotypes, for which, hitherto, the terminology has been inconsistent and

confusing Developmental Dynamics 236:1456 –1474, 2007.©2007 Wiley-Liss, Inc.

Key words: abnormal vertebral segmentation; Notch signaling pathway; spondylocostal dysostosis; Alagille syndrome;

DLL3; MESP2; LNFG; JAGGED1

Accepted 5 April 2007

INTRODUCTION

Segmentation of the vertebrae refers

to the embryonic developmental

pro-cess that results in the formation of

the spine with a series of divided,

sim-ilar anatomical units, that are the

ver-tebrae A key part of this process is

somitogenesis In vertebrate species,

somites are symmetrically aligned,

paired blocks of mesoderm formed

from the segmentation of paraxial

pre-somitic mesoderm The process begins

shortly after gastrulation and contin-ues until the preprogrammed number

of somite blocks is formed In man, 31 blocks of paired tissue are formed, but the number is specific for each species

In animal models, the formation of somite boundaries is precisely timed and takes place in a rostrocaudal di-rection, first forming the most rostral somites and progressively laying down more caudal somites In human embryonic development, this process

takes place between 20 and 35 days after conception and the formation of each somite boundary may take 4 – 6

hr Somites ultimately give rise to three substructures: sclerotome, which forms the axial skeleton and ribs; dermotome, which forms the der-mis; and myotome, which forms the axial musculature The potential number and diversity of human condi-tions due to defective somitogenesis is, therefore, large, but in this review, we

1 Clinical Genetics, Royal Devon & Exeter Hospital, and Peninsula Medical School, Exeter, United Kingdom

2 Department of Surgery, University of Toronto & Hospital for Sick Children, Toronto, Canada

3 Department of Genetics, San Juan Bautista University, Caguas, Puerto Rico

4 Department of Medical Genetics, Marshfield Clinic, Marshfield, Wisconsin

5 Department of Radiology, Hospital for Sick Children, London, United Kingdom

6 Stowers Institute for Medical Research, Kansas City, Missouri

7 Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona, Phoenix, Arizona

8 Victor Chang Cardiac Research Institute, University of New South Wales, Sydney, Australia

*Correspondence to: Peter D Turnpenny, Clinical Genetics Department, Royal Devon & Exeter Hospital, Gladstone Road, Exeter EX1 2ED, UK E-mail: peter.turnpenny@rdeft.nhs.uk

DOI 10.1002/dvdy.21182

Published online 15 May 2007 in Wiley InterScience (www.interscience.wiley.com).

2007 Wiley-Liss, Inc.

concentrate on well-defined conditions

of the axial skeleton—the spine

it-self—and their genetic basis where

this is known Abnormal vertebral segmentation (AVS), in all its various and diverse manifestations, is a

com-mon congenital abnormality, although the true incidence and prevalence are difficult to ascertain The proportion

of cases for which a cause can be con-fidently assigned is small, and progress in understanding the etiolo-gies has been slow As a consequence,

in a clinical setting, it may be difficult providing accurate genetic risk coun-seling when a single case has occurred

in a family

In man, the problems associated with abnormal spinal segmentation are of interest to a variety of disci-plines Radiologists seek to describe abnormal patterns on imaging, spinal surgeons have to make difficult deci-sions about surgery on affected chil-dren and adults, pediatricians have to care for the wider consequences such

as respiratory insufficiency, and ge-neticists try to make specific diag-noses, consider genetic testing, and of-fer recurrence risk figures when appropriate Currently, there is sub-stantial confusion over nomenclature for the various radiological pheno-types, with clinicians using terminol-ogy inconsistently In the future, it is hoped that multidisciplinary ap-proaches involving developmental bi-ologists and the various clinical disci-plines, using large data collections and candidate gene approaches, will facilitate progress in this complex field

CLINICAL HETEROGENEITY IN AVS PHENOTYPES

Axial skeletal development is very sensitive to genetic and disruptive perturbations of normal somitogen-esis and a wide range of dysmorphic syndromes, most of them rare, mani-fest AVS in various forms Table 1 lists conditions that include multiple vertebral segmentation defects (MVSD), for only a small proportion of which the pathophysiology is known This review deals mainly with non-syndromic forms of AVS, i.e., those conditions where abnormal formation

of the spine (and usually the ribs) is

an isolated anatomical anomaly with

no other body systems affected These conditions are frequently referred to

as different types of spondylocostal dysostosis (SCD), but we discuss

ter-TABLE 1 Some Syndromes and Disorders That Include Abnormal

Vertebral Segmentation a

Syndromes / disorders OMIM reference Gene(s)

Acrofacial dysostosisb 263750

Alagille 118450 JAGGED1, NOTCH2

Anhaltb 601344

Atelosteogenesis III 108721 FLNB

Campomelic dysplasia 211970 SOX9

Casamassima-Morton-Nanceb 271520

Caudal regressionb 182940

Cerebro-facio-thoracic dysplasiab 213980

CHARGE 214800 CHD7

“Chromosomal”

Currarino 176450 HLXB9

De La Chapelleb 256050

DiGeorge / Sedla´cˇkova´ 188400 Chromosomal

Dysspondylochondromatosisb

Femoral hypoplasia-unusual faciesb 134780

Fibrodysplasia ossificans

progressiva

135100 ACVR1 Fryns-Moermanb

Goldenharb 164210

Holmes-Schimkeb

Incontinentia pigmenti 308310 NEMO

Kabukib 147920

Kaufman-McKusick 236700 MKKS

KBG syndromea 148050

Klippel-Feilb 148900 ?PAX1

Larsen 150250 FLNB

Lower mesodermal agenesisb

Maternal diabetesb

MURCS associationb 601076

Multiple pterygium syndrome 265000 CHRNG

OEIS syndromeb 258040

Phaverb 261575

Rapadilino 266280 RECQL4

Robinow 180700 ROR2

Rolland-Desbuquoisb 224400

Rokitansky sequenceb 277000 ? WNT4

Silverman 224410 HSPG2

Simpson-Golabi-Behmel 312870 GPC3

Sirenomeliab 182940

Spondylocarpotarsal synostosis 269550 FLNB

Spondylocostal dysostosis 277300 DLL3, MESP2, LNFG

Spondylothoracic dysostosisb 277300

Thakker-Donnaib 227255

Toriellob

Uriosteb

VATER / VACTERLb 192350

Verloove-Vanhorickb 215850

Wildervanckb 314600

Zimmerb 301090

aVATER, vertebral defects, anal atresia, tracheoesophageal fistula, radial defects, and

renal anomalies; VACTERL, vertebral defects, anal atresia, cardiac defects,

tracheoesophageal fistula, radial defects and renal anomalies, and nonradial limb

defects

bUnderlying cause not known

minology and nomenclature later in

the review

AVS Phenotypes Following

Mendelian Inheritance

This group includes the distinctive

condition known as spondylothoracic

dysostosis (STD) and SCD types 1–3,

all of which appear to follow

autoso-mal recessive (AR) inheritance STD is

the term best reserved for the

distinc-tive condition, most commonly

re-ported in Puerto Ricans, which is

characterized by severe shortening of

the trunk and a radiological

appear-ance of the ribs fanning out from their

vertebrocostal origin in a “crab-like”

manner (Fig 1) Fusion of the ribs is

present posteriorly at their vertebral

origin, but otherwise they are usually

neatly aligned and packed tightly

to-gether This condition has been well

characterized in a recent study

(Corn-ier et al., 2004) Infant mortality is

approximately 50% due to restrictive

respiratory insufficiency, although the

availability of pediatric intensive care

greatly improves the prognosis In early life, the prominence of the ver-tebral pedicles radiologically has led

us to suggest that the term “tramline”

sign (Fig 1) neatly describes the ap-pearance In addition, in horizontal section on CT scanning, the vertebral bodies conform to a “sickle cell” shape (Cornier et al., 2004) Studies of the ultrastructure at necropsy are scarce

However, one report (Solomon et al., 1978) commented on an increased ra-tio between cartilage and bone on parasagittal sections of the vertebral bodies, with persistence of cartilage about the midline The ossification centers were disorganized and varied

in size and distribution and in the lat-eral portion of some vertebral bodies longitudinal clefts of cartilage sepa-rated anterior and posterior ossifica-tion centers Although the modeling of the vertebral bodies was severely dis-organized, microscopic bone formation and structure were not disturbed

The STD phenotype, apparently seen most frequently in Puerto

Ricans, is sometimes reported as Jar-cho-Levin syndrome (JLS) (Lavy et al., 1966; Moseley and Bonforte, 1969; Pochaczevsky et al., 1971; Pe´rez-Co-mas and Garcı´a-Castro, 1974; Gellis and Feingold, 1976; Trindade and de No´brega, 1977; Solomon et al., 1978; Tolmie et al., 1987; Schulman et al., 1993; McCall et al., 1994; Mortier et al., 1996) Jarcho and Levin (1938) de-scribed a Puerto Rican family with two affected siblings, but the radiolog-ical phenotype (Fig 2) differed slightly from STD as illustrated in Figure 1 and it is not certain that they had the same condition Use of the eponymous term JLS tends to be in-discriminate, and for that reason, we believe should be discontinued For example, there are other case reports designated as JLS, which are neither very similar to those described by Jar-cho and Levin nor consistent with STD (Poor et al., 1983; Karnes et al., 1991; Simpson et al., 1995; Aurora et al., 1996; Eliyahu et al., 1997; Rastogi

et al., 2002) Once a gene for STD in the Puerto Rican families is identified,

it will be possible to undertake geno-type–phenotype studies that will fur-ther facilitate classification

The phenotype for which we prefer to

Fig 1. X-ray of a child with spondylothoracic dysostosis Severe shortening of the spine occurs

with poorly formed vertebrae The ribs have very “crowded” origins but do not show points of fusion

along their length The prominence of the vertebral pedicles has led to the suggestion of the

“tramline” sign (Courtesy of Alberto Cornier.)

Fig 2. A reversed radiograph from Jarcho and Levin’s original paper in 1938 Abnormal seg-mentation affects all vertebrae, and the ribs are fused at their origins Today, this finding fits best into the diagnosis of spondylocostal dys-ostosis, type 2.

restrict the use of the term

spondylocos-tal dysostosis (SCD) differs from STD

by virtue of the ribs being irregularly

aligned and manifesting variable points

of fusion along their length (Fig 3) The

spine is not usually so severely

short-ened compared with STD but, as with

STD, involvement of the vertebral

bod-ies is extensive, usually contiguous, and

often affecting all spinal regions

Nev-ertheless, the thoracic spine is often

more severely affected than other

re-gions AR inheritance has been

regu-larly reported for this phenotype

(No-rum and McKusick, 1969; Cantu´ et al.,

1971; Castroveijo et al., 1973;

France-schini et al., 1974; Silengo et al., 1978;

Beighton and Horan, 1981; Turnpenny

et al., 1991; Satar et al., 1992), and we

now know that SCD can be due to

mu-tations in the Notch pathway genes

DLL3 (SCD1), MESP2 (SCD2), or

LNFG (SCD3) genes, although it is

likely that other gene(s) will be

identi-fied in due course The phenotypic fea-tures of SCD types 1–3 are described below in discussion with the molecular aspects of their respective genes Exten-sive and contiguous involvement of all spinal regions appears to be a feature that is highly suggestive of a Mendelian form of SCD and is likely to explain some isolated cases reported in the lit-erature, for example the case of Young and Moore (1984) However, the af-fected twin in a case of monozygotic twins discordant for SCD showed a sim-ilar phenotype (Van Thienen and Van der Auwera, 1994) and this finding is difficult to explain on this basis

Additional abnormalities have been reported in some families with SCD apparently demonstrating AR inheri-tance For example, urogenital anom-alies (Casamassima et al., 1981), con-genital heart disease (Delgoffe et al., 1982; Simpson et al., 1995; Aurora et al., 1996), and inguinal herniae in

males (Bonaime, 1978) Limb anoma-lies have been described in COVES-DEM syndrome (Wadia et al., 1978) but this is believed to be a case of Robinow syndrome

Families with SCD apparently fol-lowing autosomal dominant (AD) in-heritance have also been reported (Van der Sar, 1952; Ru¨ tt and Degen-hardt, 1959; Peralta et al., 1967; Rimoin et al., 1968; Kubryk and Borde, 1981; Temple et al., 1988; Lorenz and Rupprecht, 1990), but the causative genes in these cases are not known

The roles of Notch signaling path-way genes in causing SCD (types 1–3) are at least partially understood (see below) However, there are syndromes following Mendelian inheritance that include variable degrees of AVS but the functions of the genes with respect

to spinal development is poorly under-stood This group includes, for

exam-ple, ROR2 in Robinow syndrome and CHD7 in CHARGE syndrome It is not

clear what role these genes play in somitogenesis

NON-MENDELIAN FORMS

OF AVS

In clinical practice, sporadically occur-ring cases of AVS are both far more common than familial cases and more likely to be associated with additional anomalies (Martı´nez-Frı´as et al., 1994; Mortier et al., 1996) Anal and urogenital anomalies occur most fre-quently (Pochaczevsky et al., 1971; Eller and Morton, 1976; Bonaime et al., 1978; Devos et al., 1978; Solomon

et al., 1978; Poor et al., 1983; Kozlow-ski, 1984; Roberts et al., 1988; Giacoia and Say, 1991; Karnes et al., 1991; Murr et al., 1992; Lin and Harster, 1993; Mortier et al., 1996) followed by

a variety of congenital heart disease (Delgoffe et al., 1982; Kozlowski, 1984; Ohzeki et al., 1990; Aurora et al., 1996; Mortier et al., 1996) Limb ab-normalities occur but are generally of

a relatively minor nature, for exam-ple, talipes and oligodactyly or poly-dactyly (Karnes et al., 1991; Mortier

et al., 1996) Infrequently, diaphrag-matic hernia is a feature (Martı´nez-Frı´as et al., 1994) As a minor anom-aly, inguinal and abdominal herniae are frequently reported in association with MVSD As a generalization,

Fig 3. A typical case of spondylocostal dysostosis showing major abnormality of all vertebrae.

There is no “tramline” effect of the vertebral pedicles (see Fig 1), and the ribs are irregularly aligned

with variable points of fusion along their length.

these sporadically occurring cases are

more likely to show marked

asymme-try in chest shape and rib number

(Fig 4) compared with the familial

phenotypes apparently following

Mendelian inheritance

Many cases can be loosely assigned

a diagnosis of the VATER (Vertebral

defects, Anal atresia,

Tracheo-Esoph-ageal fistula, Radial defects, and

Re-nal anomalies) or VACTERL

(Verte-bral defects, Anal atresia, Cardiac

defects, Tracheo-Esophageal fistula,

Radial defects and Renal anomalies,

and nonradial Limb defects)

associa-tions (Kozlowski, 1984) associaassocia-tions, a

heterogeneous group with few clues

regarding causation (Fig 5)

A frequent association, which must

be causally linked, is neural tube

de-fect (NTD; Wynne-Davies, 1975; Eller

and Morton, 1976; McLennan, 1976;

Naik et al., 1978; Lendon et al., 1981;

Kozlowski, 1984; Giacoia and Say,

1991; Martı´nez-Frı´as et al., 1994;

Sharma and Phadke, 1994) However,

we believe this NTD-associated group should be classified separately from the SCD group, because the primary developmental pathology presumably lies in the processes determining neu-ral tube closure as distinct from somi-togenesis (Martı´nez-Frı´as, 1996)

Similarly, an association between spina bifida occulta, and/or diastema-tomyelia, and AVS has been reported (Poor et al., 1983; Ayme´ and Preus, 1986; Herold et al., 1988; Reyes et al., 1989), strongly suggesting a causal link or sequence, although the mech-anisms remain to be elucidated

AVS has been reported in maternal diabetes syndrome, which can give rise to multiple congenital anomalies

Classically, the axial skeletal malfor-mation in maternal diabetes syn-drome is caudal regression to a vary-ing degree, that is, absent sacrum or agenesis of the lower vertebral column (Bohring et al., 1999), but there are patients with hemivertebrae (Novak and Robinson, 1994) and various

forms of axial skeletal defect (Fig 6) following poorly controlled diabetes in pregnancy

AVS Associated With Chromosomal Aberrations

Some clues to genetic causes of AVS may come from patients with axial skeletal defects and chromosomal ab-normalities These cases are relatively rare, and apart from trisomy 8 mosa-icism (Riccardi, 1977), there is no clear consistency to the group Dele-tions affecting both 18q (Dowton et al., 1997) and 18p (Nakano et al., 1977) have been reported, a supernumerary dicentric 15q marker (Crow et al., 1997), and an apparently balanced translocation between chromosomes

14 and 15 (De Grouchy et al., 1963) Genomic haploinsufficiency may be unmasking a Mendelian locus for SCD such that the mechanism is autosomal recessive at the molecular level but

Fig 4. An example of a child with a severe segmentation malformation of the spine causing gross

asymmetry and almost complete absence of ribs on one side These cases are probably best not

referred to as spondylocostal dysostosis Mutations in Notch signaling pathway genes have not so

far been found in cases like this.

Fig 5. The X-ray of a child with multiple con-genital abnormalities that best fits a form of the VACTERL association She had abnormal ver-tebral segmentation affecting mainly the tho-racic region, progressive scoliosis, anal steno-sis, unilateral renal agenesteno-sis, and tricuspid regurgitation.

the paucity and inconsistency of these

cases may indicate that MVSD in

as-sociation with MCA represents a

com-mon pathway of complex pleiotropic

developmental mechanisms that are

sensitive to a range of unbalanced

karyotypes It is also possible that

chromosome mosaicism accounts for

some cases where there is marked

asymmetry in the radiological

pheno-type, which would also explain the

ap-parent sporadic occurrence, but

cur-rently there is no evidence base for

this as skin or tissue biopsy is rarely

undertaken

NOTCH SIGNALING

PATHWAY GENES AND SCD

Notch signaling is a key cascade

path-way in somitogenesis, and the

func-tion of many genes and their products,

together with their complex

interac-tions, has been partially elucidated

through the study of animal models

In man the functions of orthologous

genes and their proteins obviously

cannot be studied in the same way

Nevertheless, DNA linkage studies

and candidate gene sequencing has

led to the identification of several

genes that are important clinically

The diseases that result from

muta-tions in Notch signaling genes are

sur-prisingly diverse, the affected organ systems including the vascular and central nervous systems; the skeleton, face, and limb; hematopoiesis; the de-termination of laterality; and the liver, heart, kidney and eye Notch pathway genes and their associated diseases appear in Table 2 and several good review articles are available (Joutel and Tournier-Lasserve, 1998;

Gridley, 1997, 2003, 2006; Pourquie´

and Kusumi, 2001; Harper et al., 2003) As this review deals primarily with abnormalities of somitogenesis affecting the axial skeleton,

consider-ation is now given to the DLL3,

NOTCH2 genes The cellular

relation-ships of these genes are illustrated in Figure 7

Delta-like 3 (DLL3)

Autozygosity DNA mapping studies in

a large inbred Arab kindred with seven affected individuals was a key breakthrough in identifying the ge-netic basis of SCD (Turnpenny et al.,

1991, 1999) The locus identified, 19q13.1, is syntenic with mouse chro-mosome 7 (Giampietro et al., 1999),

which harbors the Dll3 gene The

other key breakthrough was the

iden-tification of a mutation in Dll3 as the

cause of MVSD in a

(Gru¨ neberg, 1961; Dunwoodie et al.,

1997; Kusumi et al., 1998) DLL3 was,

therefore, the obvious candidate SCD

in the large inbred Arab kindred, and other families, demonstrating linkage

to 19q13.1 Sequencing initially iden-tified mutations in three consanguin-eous families (Bulman et al., 2000)

DLL3 encodes a ligand for Notch

signaling and the gene comprises eight exons and spans approximately 9.2 kb of chromosome 19 A 1.9-kb transcript encodes a protein of 618 amino acids The protein consists of a signal sequence, a Delta–Serrate–

Lag2 (receptor interacting) domain, six epidermal growth factor (EGF) -like domains, and a transmembrane

domain (Fig 8) In animal models Dll3

shows spatially restricted patterns of expression during somite formation and is believed to have a key role in the cell signaling processes, giving rise to somite boundary formation, which proceeds in a rostrocaudal di-rection with a precise temporal

peri-odicity driven by an internal oscilla-tor, or molecular “segmentation clock” (McGrew and Pourquie´, 1998; Pour-quie´, 1999)

Mutated DLL3 in man results in

abnormal vertebral segmentation

throughout the entire spine, with all

vertebrae losing their normal form and regular three-dimensional shape The most dramatic changes, radiologically, affect the thoracic vertebrae and the ribs are mal-aligned with a variable number of points of fusion along their length (Fig 9a) There is an overall symme-try of the thoracic cage and minor, nonprogressive, scoliotic curves that

do not require corrective surgery These features define SCD In early childhood, before ossification is com-plete, the vertebrae have smooth, rounded outlines— especially in the thoracic region—an appearance for which we suggested the term “pebble beach” sign (Fig 9b) We designate this as SCD type 1 (SCD1), although

DLL3-associated SCD is an

alterna-tive term (Martı´nez-Frı´as, 2004) Additional anomalies appear to be rare, but in one case abdominal situs inversus was present, although the

link with mutated DLL3 is

uncer-tain In one family, affected siblings homozygous for the exon 8 mutation 1369delCGCTCCCGGCTACATGG (C655M660del17) manifested a form

of distal arthrogryposis in keeping with fetal akinesia sequence, and both succumbed in early childhood (C McKeown, personal communica-tion) This additional phenotype may have been a distinct AR condition segregating coincidentally to SCD, because there was multiple inbreed-ing in the family Spinal cord com-pression and associated neurological features have not been observed in SCD1, and intelligence and cognitive performance are normal This

find-ing suggests that DLL3 is not

ex-pressed in human brain, which con-trasts to findings in the mouse, where central nervous system de-fects have been found (Kusumi et al., 2001), including defects in the

neu-roventricles of the Pudgy mouse

(Ku-sumi et al., 1998; Dunwoodie et al., 2002)

To date, 24 mutations (Table 3) have been identified in SCD patients

Fig 6. The X-ray of a child with

hemiverte-brae, axial skeletal defects, and left renal

agen-esis The mother was a poorly controlled

insu-lin-dependent diabetic, and it is likely that this is

causally related to the pattern of malformations.

from 26 families (Table 4) Only five of

these families are definitely

noncon-sanguineous, and mutations have

been identified in a wide range of

eth-nic groups Eighteen mutations

trun-cate the protein and six are missense

Some missense mutations may give

rise to a slightly milder phenotype and

protein modeling studies may help

ex-plain these subtle phenotypic

differ-ences in due course With the one exception of a mutation in the trans-membrane domain, all mutations af-fect the extracellular domain of the gene and are clustered in exons 4 – 8

Three mutations have been identified

in more than one family The 945de-lAT (T315C316del2) mutation was found in two ethnic Pakistani kin-dreds originating from Kashmir, and

DNA marker analysis flanking the

DLL3 locus has identified a common

haplotype, supporting a common an-cestry Similarly, the 614insGTC-CGGGACTGCG (R205ins13) muta-tion was found in two consanguineous families, one ethnic Lebanese Arab and the other ethnic Turkish Haplo-type analysis again supports a com-mon ancestry for these two Levant kindreds The 599-603dup mutation is present in the original Arab kindred, homozygous in those affected, but in a Spanish family, the affected child is heterozygous for the same mutation Haplotype analysis on these two

ped-igrees does not support a common

an-cestry, and the 599-603dup mutation

is, therefore, believed to be recurrent, occurring as it does within a region of the gene with multiple repeat GCGGT sequences Slipped mispairing during

TABLE 2 Notch Signaling Pathway Genes and Human Disease

Gene Chromosomal locus Condition / disease System

Notch 1 9q34 T-cell ALL / lymphoma Lymphoid development / neoplasia

Aortic valve disease Angiogenesis Notch 3 19p13 CADASILa Angiogenesis

Notch 4b 6p21 ? Schizophrenia ? Neural maintenance

JAGGED 1 20p12 Alagille syndrome Hepatic / angiogenesis / ocular

NOTCH2 1p12 Alagille syndrome Hepatic / angiogenesis / ocular

DLL3 19q13 Spondylocostal dysostosis – type 1 Axial skeleton

MESP2 15q26 Spondylocostal dysostosis – type 2 Axial skeleton

LNFG 7p22 Spondylocostal dysostosis – type 3 Axial skeleton

Presenilin 1 14q24 Presenile dementia Neural maintenance

Presenilin 2 1q31 Presenile dementia Neural maintenance

NIPBL 5p13 Cornelia de Lange syndrome Upper limb / central nervous system / growth

aCADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

bAssociation studies only, which remain controversial

TABLE 3 DLL3 Mutations, Reported, and Unreporteda

Gene domain Protein truncating mutations Missense mutations

N-terminus 215del28, 395delG

DSL 602delGb, 599-603dupc, 614ins13b, 615delCd, C207Xb

EGF 1-6 712C⬎Td, 868del11b, 945delATc, 948delTGb, Q360X, C362Xb,

1256ins18, 1285-1301dup17d, 1365del17b

C309Ye, C309R, G325S, G385Dc, G404C C-terminus, pre-TM 1418delCb

aReference sequence NM 203486 DSL, delta-serrate-lag; EGF, epidermal growth factor; TM, transmembrane

bPublished (Turnpenny et al., 2003)

cPublished (Bulman et al., 2000)

dPublished (Bonafe´ et al., 2003)

ePublished (Sparrow et al., 2002)

fPublished (Whittock et al., 2004a)

TABLE 4 Origins of DLL3 Mutation Positive Cases

Ethnic origin - families N Consanguineous Nonconsanguineous

Pakistan 6 6 –

Middle-East – Arab 4 4 –

Northern Europe 9 6 3

Northern Europe – Turkey 1 – 1

Southern Europe 1 – 1

Total 26 21 5

DNA replication is the likely

explana-tion of this inserexplana-tion mutaexplana-tion

The missense mutation G504D was

found in a Northern European family

originally reported as demonstrating

autosomal dominant SCD (Floor et al.,

1989) but recently shown to be an

ex-ample of pseudodominant inheritance

(Whittock et al., 2004a) At present,

there is no confirmed case of AD SCD

due to mutated DLL3 However, in the

large Arab family reported by Turn-penny et al (1991), one female het-erozygous for the 599-603dup muta-tion had a mild thoracic scoliosis, but

no associated segmentation abnormal-ity in the thoracic region, and a very localized segmentation anomaly in the

Fig 7. The cellular relationships of Notch signaling pathway genes discussed in this review.

Fig 8. Schematic of the DLL3 gene in man, showing the mutations identified to date DSL,

delta-serrate-lag; EGF, epidermal growth factor; TM, transmembrane.

Fig 9 a: Spondylocostal dysostosis (SCD)

due to homozygous mutations in DLL3 All

ver-tebrae show abnormal segmentation, and the ribs show irregular points of fusion along their length However, there is an overall symmetry

to the thoracic cage We designate this SCD

type 1 (Courtesy of Yanick Crow.) b: Because

of the similarity to smooth, eroded pebbles on a beach, we have suggested calling the

radiolog-lower lumbar vertebrae It is possible

her scoliosis and lumbar

segmenta-tion anomaly were coincidental to her

DLL3 carrier status, as no other

obli-gate carrier in the kindred is known to

have similar features It is possible

she carried a mutation in a separate

somitogenesis gene and was,

there-fore, a manifesting “double

heterozy-gote,” an example of which is known in

an animal model (Kusumi et al., 2003)

In general, we have observed a

re-markable consistency in the

radiolog-ical phenotype in mutation-positive

cases (Turnpenny et al., 2003), and

with experience, scrutiny of the

radio-graph is usually possible to identify

those patients who will prove to have

DLL3 mutations Importantly, DLL3

mutations have not been found in the

wide variety of more common,

al-though diverse, phenotypes that

in-clude MVSD and abnormal ribs

(Maisenbacher et al., 2005;

Giampi-etro et al., 2006) Therefore, there is

remarkably little clinical

heterogene-ity for the axial skeletal malformation

due to mutated DLL3, which has

sig-nificant implications for the

applica-tion of genetic testing in the clinical

setting

In relation to defects of the axial

skeleton in man, identification of the

DLL3 gene in SCD has represented a

breakthrough in understanding the

causative basis of this group of

mal-formations, as well as highlighting

an-other example of cross-species

biolog-ical homology It has become the

paradigm for searching for the genetic

basis of other SCD phenotypes, which

led directly to the identification of

MESP2, and later LNFG, in cases of

SCD

Mesoderm Posterior 2

(MESP2)

The identification of mutated MESP2

in association with SCD arose from

the study of two small SCD families in

whom no DLL3 mutations were found

and linkage to 19q13.1 was excluded

In these families, the radiological

phe-notype was similar but subtly

differ-ent to SCD type 1 Thoracic vertebrae

were severely affected but the lumbar

vertebrae were only mildly, as shown

by magnetic resonance imaging (MRI;

Fig 10)

Genome wide homozygosity

map-ping in one family of Lebanese Arab origin revealed 84 homozygous mark-ers scattered throughout the genome,

of which 6 were concentrated in a block on 15q (D15S153 to D15S120) and 5 were concentrated in a block on 20q (D20S117 to D20S186) Subse-quent mapping excluded linkage to the 20q region but demonstrated link-age to the 15q markers D15S153, D15S131, D15S205 and D15S127

Fine mapping using additional mark-ers demonstrated a 36.6 Mb region on 15q21.3-15q26.1, between markers D15S117 and D15S1004, with a max-imum two-point lod score of 1.588, at

␪ ⫽ 0 for markers D15S131, D15S205, D15S1046, and D15S127 The region

D15S1004 contains in excess of 50 genes and is syntenic to mouse

chro-mosome 7 that contains the Mesp2 gene The Mesp2 knockout mouse

manifests altered rostrocaudal

polar-ity, resulting in axial skeletal defects (Saga et al., 1997) The predicted

hu-man gene, MESP2, comprises two

ex-ons spanning approximately 2 kb of genomic DNA at 15q26.1 Direct

se-quencing of the MESP2 gene in the

two affected siblings demonstrated a homozygous 4-bp (ACCG) duplication mutation in exon 1, termed 500-503dup (Whittock et al., 2004b) The parents were shown to be heterozy-gous and the unaffected sibling ho-mozygous normal, consistent with the duplication segregating with SCD in the family Fluorescent polymerase chain reaction excluded this mutation from 68 ethnically matched control chromosomes Analysis of the genomic

structure of the MESP2 gene

high-lighted a discrepancy between the Sanger Centre and NCBI human genomic assembly databases In the latter, there is an additional short in-tron located after base 502 of the

MESP2 coding region This finding

does not appear in the Ensembl gene prediction and, due to a lack of con-sensus splice sites within this pro-posed intronic sequence, the Exeter group concluded that the intron does not exists However, the presence or absence of such an intron did not ef-fect the conclusion concerning the 4-bp insertion on MESP2 protein pro-duction, which is predicted to inter-rupt splicing and lead to a frameshift

at the same point in the MESP2 pro-tein Having confirmed mutated

MESP2 as a cause of AR SCD, we

designated this as SCD type 2, or

MESP2-associated SCD.

In the second, nonconsanguineous family under study, haplotype data were consistent with linkage to the

MESP2 locus, but sequencing of the

gene failed to identify mutations The

MESP1 gene, which is located up-stream of MESP2 on 15q, was also

sequenced without any alteration be-ing identified It remains to be seen whether a mutation in one of the pro-moter regions, or some other position effect, might be responsible

The MESP2 gene is predicted to

produce a transcript of 1,191 bp en-coding a protein of 397 amino acids The human MESP2 protein has 58.1% identity with mouse MesP2, and

47.4% identity with human MESP1 Human MESP2 amino terminus

con-tains a basic helix–loop– helix (bHLH)

Fig 10. Magnetic resonance image of the spine in an affected individual homozygous for

a mutation in MESP2 The most striking

seg-mentation abnormality is seen in the thoracic spine with relative sparing of the lumber verte-brae We designate this as spondylocostal dys-ostosis type 2.

region encompassing 51 amino acids

divided into an 11 residue basic

do-main, a 13 residue helix I dodo-main, an

11 residue loop domain, and a 16

res-idue helix II domain The loop region

is slightly longer than that found in

homologues such as paraxis The

length of the loop region is conserved

between mouse and human MESP1,

MESP2, Thylacine 1 and 2, and chick

mesogenin In addition, both MESP1

and MESP2 contain a unique CPXCP

motif immediately carboxy-terminal

to the bHLH domain (Fig 11) The

amino- and carboxy-terminal domains

are separated in human MESP2 by a

GQ repeat region also found in human

MESP1 (2 repeats) but expanded in

human MESP2 (13 repeats) Mouse

MesP1 and MesP2 do not contain GQ

repeats but they do contain two QX

repeats in the same region: mouse

MesP1 QSQS, mouse MesP2 QAQM

Hydrophobicity plots indicate that

MesP1 and MesP2 share a

carboxy-terminal region that is predicted to

adopt a similar fold, although MesP2

sequences do contain a unique region

at the carboxy-terminus

Sequence analysis of 20 ethnically

matched and 10 nonmatched

individ-uals revealed the presence of a

vari-able length polymorphism in the GQ

region of human MESP2, beginning at

nucleotide 535 This region contains a

series of 12-bp repeat units The

smallest GQ region detected contains

two type A units (GGG CAG GGG

CAA, encoding the amino acids

GQGQ), followed by two type B units

GQGQ) and one type C unit (GGG CAG GGG CGC, encoding GQGR)

Analysis of this polymorphism in the matched and nonmatched controls re-vealed allele frequencies that were not significantly different, statistically, between the two groups

MESP2 is a member of the bHLH

family of transcriptional regulatory proteins essential to a vast array of developmental processes (Massari

and Murre, 2000) Murine Mesp1 and Mesp2, located on chromosome 7, are

separated by approximately 23 kb

They are positioned head to head and transcribed from the interlocus region (Saga et al., 1996) At least two en-hancers are involved in the expression

of these genes in mouse (Haraguchi et al., 2001): one in early mesoderm ex-pression and the other in presomitic mesoderm (PSM) expression In addi-tion, a suppressor responsible for the rostrally restricted expression in the PSM has been identified (Haraguchi

et al., 2001) These enhancers are es-sential to the specific and coordinated expression of the MesP proteins The

expression of MesP1 and Mesp2 is first

detected in the mouse embryo at the onset of gastrulation (⬃6.5 days post coitum [dpc]) and is restricted to early nascent mesoderm (Saga et al., 1996;

Kitajima et al., 2000) At this stage,

the expression domain of Mesp1 is broader than that of Mesp2, and lin-eage analysis of Mesp2-expressing

cells shows that they contribute to cranial, cardiac, and extraembryonic mesoderm (Saga et al., 1999)

Expres-sion is then down-regulated as Mesp

transcripts are not detected later in

development A second site of Mesp

expression is detected at 8.0 dpc im-mediately before somitogenesis A pair of MesP-expressing bands appear

on each side of the embryonic midline,

at the anterior part of the PSM where the somites are anticipated to form (Saga et al., 1997, 1999) During

somi-togenesis MesP1 and MesP2 continue

to be expressed in single bilateral bands in the anterior PSM MesP2 ex-pression within the PSM continues until approximately the time when somite formation ceases (⬃13.5 dpc;

Saga et al., 1999), after which Mesp

expression is rapidly down-regulated

as transcripts are not detected in newly formed somites

Alignment of MesP2 homologues

from human, mouse, Xenopus, and

chick demonstrates a highly divergent carboxy-terminus between species (Fig 11), and it is unknown whether functional domains are similarly ar-ranged in all Mesp2 orthologues, though this is well characterized for

the Xenopus Mesp2 orthologue,

Thyl-acine (Sparrow et al., 1998) If the car-boxy-terminus in human MesP2 is re-quired for transcriptional activation, then the mutant form of the protein described here, lacking the carboxy-terminus, would lose this function Mouse pups lacking MesP2 die within

20 minutes of birth, presenting with short tapered trunks and abnormal segmentation, affecting all but a few caudal (tail) vertebrae (Saga et al.,

1997) This finding resembles

Dll3-null mice, where the rib and vertebral architecture is disturbed along the en-tire axis

Unlike the mouse null MesP2 allele,

the human mutant protein retains its bHLH region and, although trun-cated, could still dimerize, bind DNA, and act in a dominant-negative man-ner However, in the affected family the heterozygous parents demon-strated no axial skeletal defects, from

which we deduce that this MESP2

mutation is recessive Similarly, het-erozygous mice are normal and fertile (Saga et al., 1997) It is possible that a single functional carboxy-terminus is sufficient to activate transcription but

Fig 11. Schematic showing the MesP2 and MesP1 homologues from human, mouse, and chick.

The carboxy-terminus is highly divergent between species.