Carrier detection in families affected by duchenne muscular dystrophy using multiplex ligation dependent probe amplification (2)

According to an analysis ofprevious studies, two-thirds of cases the defective gene is passed on to a son through the mother’s faulty X chromosome; the diagnosis offemale carriers mother

Duchenne muscular dystrophy (DMD) is a recessive disorderassociated with the chromosome X caused by mutations in thedystrophin gene It affects mainly boys According to an analysis ofprevious studies, two-thirds of cases the defective gene is passed on

to a son through the mother’s faulty X chromosome; the diagnosis offemale carriers (mother, aunt, sister) to detect mutations which wouldenable medical staff to provide prenatal genetic counseling for them

is the most effective solution to reduce incidence With manyadvantages such as rapid, sensitive, cost effective, reliable so MLPA

is used as the first option and is a useful quantitative method fordetecting mutation for the analysis of both affected males and femalecarriers

In this study, we have succeeded in the application of theMLPA method to identify female carriers Using the MLPA method,

we detected 7 out of 10 female carriers in 5 affected DMD families.Four of them show heterozygous deletion exons 45-52, 8-43, 3-47and 48-53, in the DMD gene, respectively The remaining threefemale carriers have heterozygous duplication exons 11-20 and 51-60

in the DMD gene Most of these mutations are located in the ‘hotspot’ regions In addition, we also detected one patient withduplication exons 11-20 and 51-60 in the gene MLPA assays areperformed according to manufacturer recommendations

TÓM TẮT

Bệnh loạn dưỡng cơ Duchenne (Duchenne MuscularDystrophy- DMD) là bệnh di truyền lặn liên kết với nhiễm sắc thể Xgây ra bởi đột biến ở gen dystrophin.Theo một số nghiên cứu, khoảnghai phần ba số trường hợp gen khiếm khuyết được truyền sang contrai từ người mẹ có nhiễm sắc thể X bị lỗi; chẩn đoán người nữ dịhợp tử (mẹ, cô, dì, chị gái) để phát hiện các đột biến sẽ cho phépnhân viên y tế tư vấn di truyền trước khi sinh và là giải pháp hiệu quảnhất để giảm tỷ lệ mắc căn bệnh này Với nhiều ưu điểm như nhanh,nhạy, chi phí hiệu quả, độ tin cậy cao nên MLPA được sử dụng như

là sự lựa chọn đầu tiên và là một phương pháp định lượng hữu íchcho việc phát hiện đột biến trong phân tích của cả nam giới bị bệnh

và người nữ dị hợp tử

Trong nghiên cứu này, chúng tôi đã thành công trong việc ápdụng phương pháp MLPA để xác định người nữ dị hợp tử Sử dụngphương pháp MLPA, chúng tôi phát hiện được 7 phụ nữ có mang gen

dị hợp tử trong số 10 phụ nữ trong 5 gia đình bị ảnh hưởng bởi bệnhDMD Bốn người trong số họ có đột biến xóa đoạn dị hợp tử ở cácexon 45-52, 8-43, 3-47 và 48-53 Ba người nữ còn lại có đột biến dịhợp tử lặp đoạn ở các exon 11-20 và 51-60 trong gen DMD Hầu hếtnhững đột biến này được phát hiện tại các vùng “hot spot” Ngoài ra,chúng tôi cũng phát hiện một bệnh nhân có đột biến lặp đoạn ở cácexon 11-20 và 51-60 trong gen dystrophy Kỹ thuật MLPA được thựchiện theo khuyến nghị của nhà sản xuất

Duchenne muscular dystrophy (DMD) is one of the most mon fatal genetic disorders affecting children around the world Thecauses of DMD are mutations in the dystrophin gene on chromosome X;hence, it is diagnosed mostly in males Although DMD is the most com-mon fatal genetic disorder to affect children, at the moment no cureshave been found Researchers are still looking for treatments to alterthe course of the disease and improve the quality of life for patients.Previous studies have shown that two thirds of patients receive themutation from their mothers and the other one third has new mutation[14], [19], [47], [54] Thus, detection of the heterozygous status ofmother as well as other female members of family with suitable con-sequent antenatal screening of the fetus at risk is highly appreciatedactive prevention (reduces new incidence of the disease) In recentyears, many studies show that Multiplex Ligation- dependent ProbeAmplification (MLPA) is a rapid and accurate technique, which al-lows high-throughput screening mutations, especial deletions and du-plications, in DMD and other genetic diseases This is a molecularbiology method based on the basic principle of PCR; nonetheless, ituses one pair of primer, two reactions to detection mutation in all 79exons Thus, within 1 week, MLPA can screen all mutations in thedystrophin gene This is a particular advantage of the MLPAcompared with other methods In order to carry out the diagnosis,prognosis and genetic counseling for female carriers, we carry out the

com-study: "Carrier detection in families affected by Duchenne muscular dystrophy using Multiplex Ligation- dependent Probe Amplification”.

CHAPTER 1 INTRODUCTION 1.1 Duchenne muscular dystrophy

1.1.1 Characteristics of DMD

DMD is an X-linked recessive disease caused by mutations

in the DMD gene [56] Therefore, it was found to be rather morecommon in males than females DMD is a progressive disease inwhich the patient's muscle injury is due to a lack of dystrophinprotein The dystrophin gene can be passed on from the carrierwoman to her child (complies with the rules of the genetic X- linkedinheritance) According to Mendelian inheritance, there is 50%chance a mother who carries the DMD gene can pass the Xchromosome carrying DMD mutated gene to the sons and they willdevelop disease and 50% chance that her daughters will carry thegene A carrier mother may or may not pass on the gene with themutation Normally, the majority of female carriers usually have nosymptom However, 2-20% of carriers have symptoms of muscleweakness or clinical signs of disease

1.1.2 Treatment and management of DMD

It is nearly 30 years since the discovery of the genetic defectcausing DMD, but the disease has yet to be cured To date only onetreatment, the use of corticosteroids, has been shown to be effective

in DMD patients [5], [55] At present, some areas in which research

is being focused include: gene therapy, read-through stop codonstrategies, stem cell therapy, vival vectors and utrophin [59] How-ever, these methods are only of partial support for patients and theyare not the complete cures Therefore, diagnosis of women with ahigh risk and genetic counseling for female carriers in patients’ fami-

lies are still the best options to aid in the prevention of this disease[32], [39]

1.2 The dystrophin gene

The dystrophin gene is the largest known human gene It islocated on short arm of the X- chromosome at position Xp21.2spaning approximately 2400 kb, consists of 79 exons and produces a14.6 kb mRNA [6], [8], [17], [64] It is also composed of at least 7 al-ternative promoters: brain (B) promoter, muscle (M) promoter,cerebellum promoter, promoter Dp 260, Dp 140, Dp 116, Dp 71,leading to a number of different isoforms (Figure 3) [35]

1.3 Protein dystrophin

The product of the dystrophin gene is dystrophin protein Dystrophin is a hydrophobic, rod-shaped protein that is found typically in muscles and is used for muscle movement It

is encoded by the DMD gene and it has a molecular weight of 427kDa, and contains about 3685 amino acids [17], [23] This protein islocated in the plasma membrane of muscle cells and is divided intofour domains [23]: the amino-terminal domain, the central- rod-domain, the cystein- rich domain, he carboxy- terminal domain.Dystrophin plays an important structural role as part of a largecomplex in muscle fiber membranes It provides a structural linkbetween the muscle cytoskeleton and extracellular matrix to maintainmuscle integrity [7], [42]

1.4 Mutations in the dystrophin gene

1.4.1 Deletion mutations

Deletion one or several exons is common mutations inpatients with DMD, accounting for 60-65% of DMD disease-causingmutations [9] Deletion mutations in DMD genes are the mostcommonly found intragenic deletions and concentrated in two known

"hot spot" regions

1.4.2 Point mutations

Point mutation, accounting for 25-30%, is the second largestmutation in the dystrophin gene after deletion mutations [43] Most pointmutations in the DMD gene created stop codon and caused seriouslydisease Point mutations are located along the entire gene and this is amajor obstacle in identifying these mutations

1.4.3 Duplication mutations

Duplication mutations are the cause of DMD in most of the

remaining cases (approximately 5%-15%) Prior (2005) suggested that

80% of mutations occur in the 5 'end and 20% in the center In addition,

a small percentage of DMD patients have small mutations scatteredalong the length of the entire gene making them difficult to detect [39]

1.5 The methods to detection mutations in the dystrophin gene

1.5.1 PCR method

PCR (polymerase chain reaction) is based on the synthesis of a target DNA segment under the catalysis of the enzyme DNA-polymerase (taq polymerase), and occurs in repeated cycles Components in the PCR reaction include DNA sample, deoxynucleotide triphosphate (dATP, dGTP, dCTP, dTTP), MgCl2, primers, DNA polymerase and PCR buffer

solution [33] PCR is a three-step process that is carried out in repeated cycles The number of cycles per PCR reaction depends on the initial number of DNA template, usually, does not exceed 40 cycles In the case of DMD, PCR plays an especiallyimportant role in the process of mutation detection For example,multiplex PCR is appreciated in the diagnosis of deletion and about98% of deletions are easily detectable using multiplex PCR inaffected males [3]

1.5.2 Southern blot method

Southern blot is a method used in molecular biology fordetection of a specific DNA sequence in DNA samples The principle

in this method combines agarose gel electrophoresis for sizeseparation of DNA with methods to transfer the size- separated DNA

to a filter membrane for probe hybridization Prior (2005) used thismethod to detect mutations in DMD patients as well as femalecarriers [39]

1.5.3 Fluorescence in situ hybridization - FISH

FISH (fluorescence in situ hypridization) is a cytogenetic technique that permits DNA sequences to be detected onmetaphase chromosomes, in interphase nuclei, in a tissue section, or

molecular-in blastomeres and gametes [40] This method uses fluorescentprobes that bind to only those parts of the chromosome with whichthey show a high degree of sequence complementarity Many studiesindicate that FISH is an efficient, sensitive method that bringsconfident results to detection, identification and to screen DMDfemale carriers [27], [51], [57]

1.5.4 Sequencing method

Today, with advancement of technology, both of Sangerdideoxy method and Maxam-Gilbert chemical cleavage method arereplaced by modern sequencing equipment (automatic sequencing).The new technology is based on the same principles of Sanger'smethod but four different fluorescent dye-labelled ddNTPs are used.Thus each fluorescent label can be detected by its characteristicspectrum The products are separated by automated electrophoresisand the bands detected by fluorescence spectroscopy For DMD,sequence analysis of the dystrophin gene is a rapid way to detectsmall mutation that nearly entire of the 79 exons but this method isvery costly and time consuming

1.5.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method

In the MLPA technique, genomic DNA is hybridized insolution to probe sets, each of which consists of twooligonucleotides: one short synthetic oligonucleotide and one longprobe oligonucleotide The short synthetic oligonucleotide contains atarget-specific sequence (20–30 nucleotides) at the 3’ end and acommon sequence (19 nucleotides) that is the primer binding sites, atthe 5’ end The long MLPA probe contains the 25-43 nucleotidestarget-specific sequence at the 5’ end, a 36 nucleotides sequence thatcontains primer binding sites and is common to all probes, at the 3’end and a suffer sequence (19–370 nucleotides)- a variable lengthrandom fragment in between to generate the length differences Thedifferent lengths of the products allow separation on an automatedcapillary sequencer, and the peak areas are quantified [46] MLPA

assay has become a widely used technique in laboratories performinggenetic testing for the molecular diagnosis in general and DMD inparticular Compared to other techniques, MLPA is rapid, sensitive,reliable and very simple to perform Therefore, MLPA is highlyrecommended by scientists for detecting DMD and others geneticdeseases.

1.6 The aim of the study

DMD is an X- linked disease which has a 100% fatality rate

Up to now, DMD still has no effective method of treatment Severalprevious studies show that two-third of patients receives DMD genes

from heterozygous mothers and one-third of patients are new (de novo) mutations [19], [54] Hence, diagnosis of carriers is the most

effective option to restrict the development of this disease In nam, MLPA has been used commonly because MLPA is rapid, sensi-tive and accurate The long term objective of this research is to applyMLPA to detect DMD mutated gene in carriers and from which, weaim to further develop the project into early diagnosis prenatal pro-gram To achieve the goal, we propose to pursue the followingspecifics aim: Apply MLPA to detect the carriers in families affected

Viet-by DMD

CHAPTER 2 MATERIALS AND METHODS

2.1 Patient, female carrier and normal control

1 male patient; 10 female relatives from 5 different familiescharacterized by DMD patients with exons 45-50, 11-20, 51-60, 3-

47, 8-43 and 48-50 dystrophin deletions and duplications; 2 healthyfemale and 1 healthy male without family history ofdystrophinopathies were analyzed as control

2.2 Reagents and equipment

2.2.1 Reagents

a Reagents for DNA extraction from blood

Lysis buffer solution; K solution; proteinase K solution (20mg/ml); SDS 10% solution; Phenol: chloroform: isoamyl (25:24:1),Chloroform: isoamyl (24:1); ethanol 100% (cold); ethanol 70%(cold); sodium acetate 3M (pH 5.2), TE (Tris- EDTA)

b Reagents for MLPA: SALSA MLPA probe mix P034 (DMD

exons 1-10, 21-30, 41-50, 61-70) and P035 (DMD exons 11-20,

31-40, 51-60, 71-79) DMD/Becker kit is purchased by MRC Holland,Amsterdam, The Netherlands

2.2.2 Equipment

Thermal cycler (Eppendorf branch); GenomeLabTM GeXP Genetic

Analysis System (Applied Biosystems); Genemarker software v.1.95

(Softgenetic, State College, PA, USA); Automatic pipettes (range:0.5-1000µl with tips matched together); Centrifuge suitable for 1.5

ml eppendorf tubes; Micro centrifuge; Thermomixer R (Eppendorfbranch); Nano drop spectrophotometer; Vortex machine; Timer,Freezer (2-8oC, -20oC); 0.5, 0.2 ml PCR tubes

2.3 Methods

2.3.1 Sampling process

Blood samples were collected in EDTA vacutainer tube (3 mlquantities) from female relative, patients and matched control Theprocess ensures absolute sterility

2.3.2 DNA extraction from blood

The DNA was extracted from peripheral blood byphenol/chloroform method Then, the DNA will be tested bymeasurement of optical density at wavelength of 260nm and 280 nm.DNA samples, which has to concentrations > 20ng/μll and purity(A260/A280) from 1.8 ÷ 2.0, will be used to carry out the next steps

of this study

2.3.3 Calculation of DNA concentration

Calculation of DNA concentration depends onSpectrophotometric method A ratio of OD260nm/OD280nm in therange of 1.8 – 2 indicates highest -quality DNA whereas lower valuesindicate protein contamination Higher values indicate RNA contami-nation [45]

2.3.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method

The MLPA DMD test kit (SALSA P34/P035) was purchasedfrom MRC Holland, Amsterdam, the Netherlands The procedurewas performed according to the protocol provided by the manufac-turer’s recommendation [6] Before carry out of MLPA, prepare

DNA sample: 20 ng/ml (DNA working stock) and thermocycler pendorf branch) The process is described as follows:

(Ep-1 DNA denaturation

Add 5 μll of DNA working stock to each PCR tube Denature sampleDNA for 5 minutes at 98°C and cool the samples to 25°C

2 Hybridization reaction

- Prepare hybridisation master mix for hybridization reaction

- Add 3 μll of the hybridisation master mix to each PCR sampletube above Continue incubate for 1 minute at 95 °C, then for 16 – 20hours at 60 °C following the thermocycler program

3 Ligation reaction

- Prepare ligase mix for ligation reaction

- Continue the thermocycler program: pause at 54°C When thesamples are at 54°C, add 32 µl of ligase mix and incubate 15 minutes

at 54oC for ligation;

- Heat to inactivate the ligase for 5 minutes at 98oC and thenpause at 15 oC

4 PCR reaction

- Label new tubes for the PCR reaction

- Prepare PCR buffer mix (well mix)

- Add 30µl of PCR buffer mix to each new tube and well mix

by pipetting

- At room temperature, transfer 10 μll of each ligation product

to its corresponding PCR tube Spin

- Prepare PCR master mix (in icebox)