Carrier detection in families affected by duchenne muscular dystrophy using multiplex ligation dependent probe amplification (1)

University of Liege, Belgium Vietnam National University, Hanoi JOINT MASTER PROGRAM IN BIOTECHNOLOGY ------Nguyen Thi Tuong An CARRIER DETECTION IN FAMILIES AFFECTED BY DUCHENNE M

University of Liege, Belgium Vietnam National University, Hanoi

JOINT MASTER PROGRAM IN BIOTECHNOLOGY

- -Nguyen Thi Tuong An

CARRIER DETECTION IN FAMILIES AFFECTED BY DUCHENNE MUSCULAR DYSTROPHY USING MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION

Major: Biotechnology Code: 60 42 80

MASTER THESIS

SUPERVISOR PHD., MD TRAN VAN KHANH

Hanoi, 2013

This work was supported by The Center for Gene and Protein research

First of all, I would like to express my greatest appreciation to my supervisor PhD., MD Tran Van Khanh, who has offered her continuous advice and encouragement throughout the course of this thesis

I am so thankful to committee members their helpful suggestions and comments for my thesis

I would like to thank the lecturers of the Vietnam National University (Viet Nam) and University of Liege (Belgium) for their scientific lectures

I would also like to thank the leaders and colleagues of the National Institute for Control of Vaccine and Biologicals (NICVB) for giving me opportunity to take part in this master program.

I would also like to thank staff members of the Institute of Microbiology and Biotechnology and The Center for Gene and Protein research, Hanoi Medical University for making me feel welcome, and providing guidance whenever I needed it

To my classmates, a “big” thank you for their friendship and the many hours of discussions and bouncing of ideas This is a very special time in my life and I have learnt much from their sharing

The most special thanks goes to my family and all of my friends who always help and support me silently and without their help, I would not have been able completed this project.

And the last word, I would like to say thank you to the patients and their families for their cooperation.

Hanoi, November 2013

Nguyen Thi Tuong An

CK Creatine Kinase

DGC Dystrophin-Glycoprotein Complex

DMD Duchenne Muscular Dystrophy

DNA Deoxyribonucleic Acid

dNTP Deoxynucleoside triphosphate

FISH Fluorescence In Situ Hybridization

MLPA Multiplex Ligation-dependent Probe AmplificationmRNA RNA messenger

OD Optical Density

PCR Polymerase Chain Reaction

SDS Sodium Dodecyl Sulfate

LIST OF FIGURES

Figure 1: Typical progression of clinical symptoms with age of DMD patients 8

Figure 2: Genetic of DMD 9

Figure 3: A: location of DMD gene B: protein products of the DMD gene 11

Figure 4: The dystrophin- associated glycoprotein complex (DGC) in skeletal muscular 12

Figure 5: The steps in PCR 15

Figure 6: Test hybridization on a metaphase from the Duchenne muscular dystrophy carrier 18

Figure 7: Sequencing of the dystrophin gene 19

Figure 8: Outline of the MLPA reaction 20

Figure 9: MLPA results of control and DMD male patient 21

Figure 10: Family pedigree of patient A 37

Figure 11: MLPA results of DMD female control (C2) and mother (D1) in using DMD Probe set P034 the dystrophin gene 38

Figure 12: MLPA results of DMD female control (C2) and second aunt (D2) in using DMD Probe set P034 the dystrophin gene 39

Figure 13: MLPA results of DMD female control (C2) and first aunt’s daughter (D3) in using DMD Probe set P034 the dystrophin gene 40 Figure 14: Family pedigree of patient B 42

Figure 15: MLPA results of DMD male control (C1) and patient B (D8) in using DMD Probe set P035 the dystrophin gene 43

Figure 16: MLPA results of DMD female control (C2) and first aunt (D4) in using DMD Probe set P035 the dystrophin gene 44

Figure 17: MLPA results of DMD female control (C2) and second aunt (D5) in using DMD Probe set P035 the dystrophin gene 45

Figure 18: MLPA results of DMD female control (C2) and first aunt’s daughter (D6) in using DMD Probe set P035 the dystrophin gene 46

Figure 19: MLPA results of DMD female control (C2) and second aunt’s daughter (D7) in using DMD Probe set P035 of the dystrophin gene47 Figure 20: MLPA results of DMD female control (C3) and patient E’s mother (D9) in using DMD Probe set P035 the dystrophin gene 50

Figure 21: MLPA results of DMD female control (C3) and patient F’s mother (D10) in using DMD Probe set P035 the dystrophin gene 51

Figure 22: MLPA results of DMD female control (C3) and patient G’s mother (D11) in using DMD Probe set P034 the dystrophin gene 52

LIST OF TABLE

Table 1.1 Genotype of parent and rate of affected in offspring 7Table 1.2 Comparision between Multiplex Ligation dependent Probe Amlification

(MLPA) and other methods 22Table 2.1 PCR program for the MLPA reaction 34Table 3.1 Results of DNA extraction 36Table 3.2 Results of multiplex ligation-dependent probe amplification (MLPA) 54

ACKNOWLEDGEMENTS i

ABBREVIATIONS ii

LIST OF FIGURES iii

LIST OF TABLE iv

ABSTRACT 1

TÓM TẮT 3

PREFACE 5

CHAPTER 1 INTRODUCTION 7

1.1 Duchenne muscular dystrophy 7

1.1.1 Characteristics of DMD 7

1.1.2 Treatment and management of DMD 9

1.2 The dystrophin gene 10

1.3 Protein dystrophin 11

1.4 Mutations in the dystrophin gene 13

1.4.1 Deletion mutations 13

1.4.2 Point mutations 13

1.4.3 Duplication mutations 14

1.5 The methods to detection mutations in the dystrophin gene 14

1.5.1 PCR method 14

1.5.2 Southern blot method 16

1.5.3 Fluorescence in situ hybridization - FISH 17

1.5.4 Sequencing method 18

1.5.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method 19

1.6 The aim of the study 23

CHAPTER 2 MATERIALS AND METHODS 24

2.1 Samples 24

2.2 Reagents and equipment 24

2.2.1 Reagents 24

2.2.2 Equipment 27

2.3 Methods 27

2.3.1 Sampling process 27

2.3.2 DNA extraction from blood 27

2.3.3 Calculation of DNA concentration 30

2.3.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method 31

CHAPTER 3 RESULTS AND DISCUSSION 36

3.1 DNA extraction 36

3.2 MLPA results 37

3.2.1 MLPA results of patient A’s family 37

3.2.2 MLPA results of patient B’s family 42

3.2.3 MLPA result of patient E’s mother (D9) 50

3.2.4 MLPA result of patient F’mother (D10) 51

3.2.5 MLPA result of patient G’s mother (D11) 52

CONCLUSION AND SUGGESTION 55 BIBLIOGRAPHY

Duchenne muscular dystrophy (DMD) is a recessive disorderassociated with the chromosome X caused by mutations in the dystrophingene It mostly affects boys, and is characterized by a rapidly progressivemuscle weakness that almost always results in death, usually by 20 years ofage When a family member has DMD, all member of the family are affected

by caregiving demands and emotional reactions In Vietnam the economic burden of disease is very high, as the parents have to cover the cost

socio-of treatment themselves, without much help from the government Therefore,prenatal diagnosis is greatly in demand

According to an analysis of previous studies, two-thirds of cases thedefective gene is passed on to a son through the mother’s faulty Xchromosome; the diagnosis of female carriers (mother, aunt, sister) to detectmutations which would enable medical staff to provide prenatal geneticcounseling for them is the most effective solution to reduce incidence Manymethods have been used to help the detection of heterozygous females, such

as PCR, Southern blot, FISH, etc, in which, MLPA is increasingly becomewidely used in laboratories worldwide With many advantages such as rapid,sensitive, cost effective and reliable so MLPA is the first option and is auseful quantitative method for detecting mutation for the analysis of bothaffected males and female carriers

In this study, we have succeeded in the application of the MLPAmethod to identify female carriers We detected 7 out of 10 female carriers in

5 affected DMD families Four of them show heterozygous deletion exons

45-52, 8-43, 3-47 and 48-53, in the DMD gene, respectively The remaining three

female carriers have heterozygous duplication exons 11-20 and 51-60 in theDMD gene In addition, we also detected one patient with duplication exons11-20 and 51-60 in the gene The normal control samples show no deletions

in any of the exons tested MLPA assays are performed according tomanufacturer recommendations

TÓM TẮT

Tên luận văn: "Ứng dụng kỹ thuật Multiplex Liagation-dependent Probe

Amplification để xác định người lành mang gen bệnh trong các gia đình bịảnh hưởng bởi bệnh loạn dưỡng cơ Duchenne"

Người hướng dẫn: TS BS Trần Vân Khánh

Trung tâm nghiên cứu Gen- Protein, Trường đại học Y Hà Nội

Ngành: Công nghệ Sinh học Chuyên ngành: Công nghệ Sinh học

Mã số: 604280

Bệnh loạn dưỡng cơ Duchenne (Duchenne Muscular DMD) là bệnh di truyền lặn liên kết với nhiễm sắc thể X gây ra bởi đột biến ởgen dystrophin Căn bệnh này chủ yếu ảnh hưởng đến trẻ em trai, nó đặctrưng bởi yếu cơ tiến triển nhanh và phần lớn bệnh nhân chết ở tuổi 20 Khi

Dystrophy-có một thành viên trong gia đình mắc bệnh DMD, các thành viên khác của giađình đó cũng bị ảnh hưởng bởi nhu cầu chăm sóc và những phản ứng tâm lý

Ở Việt Nam, gánh nặng về mặt kinh tế, xã hội của bệnh là rất cao do đó cácbậc cha mẹ phải tự trang trải các chi phí điều trị cho con em mình mà không

có sự giúp đỡ từ chính phủ Chính vì vậy, việc chẩn đoán trước khi sinh là yêucầu cấp thiết

Theo một số nghiên cứu, khoảng hai phần ba số trường hợp gen khiếmkhuyết được truyền sang con trai từ người mẹ có nhiễm sắc thể X bị lỗi; chẩnđoán người nữ dị hợp tử (mẹ, cô, dì, chị em gái) để phát hiện các đột biến sẽcho phép nhân viên y tế tư vấn di truyền trước khi sinh là giải pháp hiệu quảnhất để giảm tỷ lệ mắc căn bệnh này Có nhiều phương pháp được sử dụng đểgiúp phát hiện người nữ dị hợp tử, chẳng hạn như PCR, MLPA, Southernblot, FISH, … trong đó MLPA là kỹ thuật ngày càng được sử dụng rộng rãitrong các phòng thí nghiệm trên toàn thế giới Với nhiều ưu điểm như nhanh

chóng, chính xác, độ nhạy cao và chi phí hiệu quả nên MLPA là sự lựa chọnđầu tiên và là phương pháp định lượng hữu ích để phát hiện đột biến ở cả namgiới bị bệnh và người nữ mang gen bệnh.

Trong nghiên cứu này, chúng tôi đã thành công trong việc áp dụngphương pháp MLPA để xác định người nữ dị hợp tử Chúng tôi phát hiệnđược 7 phụ nữ có mang gen dị hợp tử trong số 10 phụ nữ ở 5 gia đình bị ảnhhưởng bởi bệnh DMD Bốn người trong số họ có đột biến xóa đoạn dị hợp tử

ở các exon 45-52, 8-43, 3-47 và 48-53; ba người còn lại có đột biến lặp đoạn

dị hợp tử ở các exon 11-20 và 51-60 trong gen dystrophin Ngoài ra, chúng tôicũng phát hiện một bệnh nhân có đột biến lặp đoạn ở các exon 11-20 và 51-60trong gen dystrophin Các mẫu chứng hiển thị đầy đủ các exon Kỹ thuậtMLPA được thực hiện theo khuyến nghị của nhà sản xuất

Duchenne muscular dystrophy (DMD) is one of the most common fatal netic disorders affecting children around the world (every ethnicity and geo-graphic location) It affects approximately 1 in every 3,500 boys, or 20,000 livemale births each year worldwide [13], [52] The causes of DMD are mutations inthe dystrophin gene on chromosome X; hence, it is diagnosed mostly in males InVietnam, the frequency of occurrence of this disease does not have officialstatistics

ge-All the DMD patients are males since females are heterozygous ers; they lose the ability to walk because of inability produce dystrophin, aprotein necessary for muscle strength and function As a result, every skeletalmuscle in the body deteriorates Although DMD is the most common fatal ge-netic disorder to affect children, at the moment no cures have been found.This disease causes serious health problems and significant economic bur-dens Researchers are still looking for treatments to alter the course of the dis-ease and improve the quality of life for patients Previous studies have shownthat two thirds of patients receive the mutation from their mothers and theother one third has new mutation [14], [19], [47], [54] Thus, detection of theheterozygous status of mother as well as other female members of family withsuitable consequent antenatal screening of the fetus at risk is highly appreci-ated active prevention (reduces new incidence of the disease) In Vietnam,routine molecular genetic testing used to detect mutations includes: Poly-merase Chain Reaction- PCR (Multiplex PCR), Southern blots, etc However,these methods require a long time to detect mutations on the entire 79 exons;

carri-it takes about 3-4 weeks, normally In recent years, many studies show thatMultiplex Ligation- dependent Probe Amplification (MLPA) is a rapid andaccurate technique, which allows high-throughput screening mutations, espe-

cial deletions and duplications, in DMD and other genetic diseases This is amolecular biology method based on the basic principle of PCR; nonetheless,

it uses one pair of primer to amplify all probe ligation products Thistechnique was performed with 2 reactions: MLPA-P034 (DMD exon 1-10,21-30, 41-50 and 61-70) and MLPA-P035 (DMD exon 11-20, 31-40, 51-60and 71-79) Each reaction amplifies of 50% total exons so that 79 exons of thedystrophin gene will be amplified in 2 reactions Thus, within 1 week, MLPAcan screen all mutations in the dystrophin gene This is a particular advantage

of the MLPA compared with other methods In order to carry out thediagnosis, prognosis and genetic counseling for female carriers, we carry out

the 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

Duchenne muscular dystrophy (DMD) was first described by Duchenne

de Boulogne, the French neurologist, in the 1860s Although the disease wasdiscovered a long time ago, as recently as the early 1980s, people still did notunderstand the cause of any form of muscular dystrophy In 1986, researchersdiscovered the gene mutation causing DMD and the protein associated withthis gene was identified and named dystrophinby Hoffman in 1987 [17]

DMD is an X-linked recessive disease caused by mutations in the DMDgene [56] Genetically, it complies with the rules of the genetic chromosomeX-linked recessive inheritance so that it mostly affects boys Females havetwo X chromosomes: one X chromosome has the faulty DMD gene whileother contains a normal gene, which compensates for the faulty one Incontrast, males have only one X chromosome so that they always showsymptoms of the disease Therefore, the DMD was found to be rather morecommon in males than females

Table 1.1 Genotype of parent and rate of affected in offspring

boys begin to show signs of weakness when they are 3-5 years old, forexample they are slow runners and have difficulty jumping From 7- 9 yearsold, they have trouble climbing stairs, followed by a complete loss ofambulation by age 11 [11] At the onset of their teenage years or even sooner,they are affected by diseases relating to the heart, respiratory system, nervoussystem and this eventually leads to death, typically in their twenties [15], [39].

Figure 1: Typical progression of clinical symptoms with age of DMD patients.

Resouce: http://prosensa.eu/hc-professionals/duchenne-muscular-dystrophThe dystrophin gene can be passed on from the carrier woman to herchild (complies with the rules of the genetic X- linked inheritance) According

to Mendelian inheritance, there is 50% chance a mother who carries the DMDgene can pass the X chromosome carrying DMD mutated gene to the sons andthey will develop disease and 50% chance that her daughters will carry thegene A carrier mother may or may not pass on the gene with the mutation(figure 2)

1.1.2 Treatment and management of DMD

It is nearly 30 years since the discovery of the genetic defect causingDMD, but the disease has yet to be cured To date, corticosteroids are the only

medication that has been shown to be effective in DMD patients [5], [55].

However, the specific mechanisms by which corticosteroids improve strength

in DMD are not known [59] Accordingly, before using, the potential risksand benefits of this medical intervention need to be adequately investigated

In addition, researchers have made great advances in their knowledge ofDMD and continue to search for a cure At present, some areas in which re-search is being focused include: gene therapy, read-through stop codon strate-gies, stem cell therapy, vival vectors and utrophin [59] Nonetheless, thesemethods are only of partial support for patients and they are not the completecures Therefore, diagnosis of women with a high risk and genetic counselingfor female carriers in patients’ families are still the best options to aid in theprevention of this disease [32], [39] In addition, a healthy lifestyle, exerciseand medication can contribute to a better quality of life for those with the dis-ease

1.2 The dystrophin gene

The dystrophin gene is the largest known human gene It is located

on short arm of the X- chromosome at position Xp21.2 spaning approximately 2400 kb, consists of 79 exons and produces a 14.6 kb mRNA [6], [8], [17], [64] It is also composed of at least 7 alternative pro- moters: brain (B) promoter, muscle (M) promoter, cerebellum promoter, promoter Dp 260, Dp 140, Dp 116, Dp 71, leading to a number of differ- ent isoforms (Figure 3) [35].

Figure 3: A: location of DMD gene B: protein products of the DMD gene [35] 1.3 Protein dystrophin

The product of the dystrophin gene is dystrophin protein A completeunderstanding about the functions of protein dystrophin is important for thediagnosis of dystrophinopathies Dystrophin is a hydrophobic, rod-shapedprotein 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 427 kDa, andcontains about 3685 amino acids [17], [23] This protein is located in theplasma membrane of muscle cells and is divided into four domains [23]:

 The amino-terminal domain

 The central- rod- domain

 The cystein- rich domain

 The carboxy- terminal domain

The first domain is the amino-terminal domain It has homology with actin and contains between 232 and 240 amino acid residues depending on theisoform The larget domain of dystrophin is the central-rod- domain and it is asuccession of 25 triple-helical repeats similar to spectrin and contains about

α-3000 residues The third domain is a cysteine- rich domain of 280 residues.The finally- carboxy-terminal domain comprises 420 residues [35]

Figure 4: The dystrophin- associated glycoprotein complex (DGC) in

skeletal muscular [4]

Dystrophin plays an important structural role as part of a large complex

in muscle fiber membranes It provides a structural link between the musclecytoskeleton and extracellular matrix to maintain muscle integrity [7], [42].Many membrane proteins associated with dystrophin protein complex aremade up of DGC (dystrophin-glycoprotein complex) One of the other mainroles of the DGC is to stabilise the sarcolemma and to protect muscle fibres

from long-term contraction-induced damage and necrosis [35] Moreover, theDGC may also play a role in cell signaling by interacting with proteins thatsend and receive chemical signals The signal loss can also contribute tocausing the disease [17], [41] When dystrophin is absent, the DGC isdestabilized leads to muscle wasting which characterizes Duchenne musculardystrophy.

1.4 Mutations in the dystrophin gene

Mutations in the large dystrophin gene, which consists of 79 exonsgenerally causes a disruption of the open reading frame in the dystrophinprotein production process which leads to progressive muscle degeneration[1], [34] There are 3 main types of mutation in the dystrophin gene:

1.4.2 Point mutations

Point mutation, accounting for 25-30%, is the second largest mutation inthe dystrophin gene after deletion mutations [43] Most point mutations in theDMD gene created stop codon and caused seriously disease Point mutations are

located along the entire gene and this is a major obstacle in identifying thesemutations Currently, over 200 point mutations of the dystrophin gene have beenidentified [38], [43].

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 ofDMD patients have small mutations scattered along the length of the entire genemaking them difficult to detect [39]

1.5 The methods to detection mutations in the dystrophin gene

As mentioned above, there is currently no cure for DMD Thus prenataldiagnosis and detection of the carrier is the best approach to reduce theburden of disease for the family in particular and society in general Severalmolecular diagnostic techniques have been applied to detect these mutationsall over the world and Vietnam Some of them are listed below:

PCR is a three-step process that is carried out in repeated cycles

Step1 (initial or denaturation stage): The initial step is the denaturation

or separation of the two strands of the DNA molecule This is accomplished

by heating the starting material to temperatures of about 94°- 95° C Eachstrand is a template on which a new strand is built This step takes place in therange of 30-60 seconds.

Step 2 (annealing stage): In the second step the temperature is reduced

to about 55° C so that the primers can anneal to the template Annealingtemperature is lower and depends on the length of the primers This step takesplace in the range of 30-60 seconds

Step 3 (synthesis or extended stage): In the third step the temperature israised to about 72° C, and the DNA polymerase begins adding nucleotidesonto the ends of the annealed primers This step takes place in the range of 30seconds to several minutes

Figure 5: The steps in PCR

Resource: Andy Vierstraete (1999)

The number of cycles per PCR reaction depends on the initial number ofDNA template, usually, does not exceed 40 cycles Normally, 25 to 30 cyclesproduces a sufficient amount of DNA The number of copies doubles after eachcycle So, after n cycles, a target DNA is cloned into 2n copies [33].

PCR technique is a key contributor in the development of molecularbiology techniques Simple, economy (personal, time, material) and a highdegree of sensitivity are the advantages of this technique With its enormouspotential, PCR techniques are widely used in many different fields in bothscience and in social life In medicine, PCR can deliver a fast and accuratediagnosis of genetic diseases and genetic factors involved Particularly in thecase of DMD, PCR plays an especially important role in the process ofmutation detection Several techniques such as multiplex PCR, Nested PCR,RT-PCR have been used to detect mutations are based on the principle ofPCR Among these methods, multiplex PCR is appreciated in the diagnosis ofdeletion and about 98% of deletions are easily detectable using multiplexPCR in affected males [3] However, for detection of mutations in thedystrophin gene by PCR at the DNA level, we must amplify the entire 79exons To design 79 primer pairs at both ends of each specific exon is bothcostly and time consuming This is a limitation of this method

1.5.2 Southern blot method

Southern blot is a method used in molecular biology for detection of aspecific DNA sequence in DNA samples The principle in this methodcombines agarose gel electrophoresis for size separation of DNA withmethods to transfer the size- separated DNA to a filter membrane for probehybridization The method is named after its inventor, the British biologistEdwin Southern Southern blot is not only applied to detect deletion

mutations, but it also allows identification of the patient’s duplication mutations.

In addition, Southern blot is used to detect the mother and sister of the patient inthe heterozygous form Prior (2005) used this method to detect mutations inDMD patients as well as female carriers [39] The drawback of this technique isthat time consuming and that it is difficult to exactly determine the duplicationboundaries and to detect duplications in females [63]

1.5.3 Fluorescence in situ hybridization - FISH

FISH (fluorescence in situ hypridization) is a molecular- cytogenetictechnique developed by biomedical researchers in the early 1980s This is atechnique that permits DNA sequences to be detected on metaphasechromosomes, in interphase nuclei, in a tissue section, or in blastomeres andgametes [40] This method uses fluorescent probes that bind to only thoseparts of the chromosome with which they show a high degree of sequencecomplementarity Fluorescence microscopy can be used to find out where thefluorescent probe is bound to the chromosomes Besides, FISH is often usedfor finding specific features in DNA for use in genetic counseling, medicine,and species identification Many studies indicate that FISH is an efficient,sensitive method that brings confident results to detection, identification and

to screen DMD female carriers [27], [51], [57] However, the method hassome disadvantages such as being costly and has a limited number of targetsand throughput

Figure 6: Test hybridization on a metaphase from the Duchenne muscular dystrophy carrier [29]

1.5.4 Sequencing method

In the past, two main methods of DNA sequencing: Sanger dideoxymethod and Maxam-Gilbert chemical cleavage method were used Today,with advancement of technology, both of methods are replaced by modernsequencing equipment (automatic sequencing) The new technology is based

on the same principles of Sanger's method but four different fluorescent labelled ddNTPs are used Thus each fluorescent label can be detected by itscharacteristic spectrum The products are separated by automatedelectrophoresis and the bands detected by fluorescence spectroscopy As inSanger's method, the DNA is separated on a gel, but they are all run on thesame lane as opposed to four different ones For DMD, sequence analysis ofthe dystrophin gene is a rapid way to detect small mutation that nearly entire

dye-of the 79 exons but this method is very costly and time consuming

Figure 7: Sequencing of the dystrophin gene

Resource: Center for Gene and Protein research- Hanoi medical University

1.5.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method

In recent years, among the different approaches used for the detection

of gene deletions/duplications, particular interest has been devoted to theMultiplex Ligation-dependent Probe Amplification (MLPA) method In theMLPA technique, genomic DNA is hybridized in solution to probe sets, each

of which consists of two oligonucleotides: one short synthetic oligonucleotideand one long probe oligonucleotide The short synthetic oligonucleotidecontains a target-specific sequence (20–30 nucleotides) at the 3’ end and acommon sequence (19 nucleotides) that is the primer binding sites, at the 5’end The long MLPA probe contains the 25-43 nucleotides target-specificsequence at the 5’ end, a 36 nucleotides sequence that contains primer bindingsites and is common to all probes, at the 3’ end and a suffer sequence (19–370nucleotides)- a variable length random fragment in between to generate thelength differences The different lengths of the products allow separation on

an automated capillary sequencer, and the peak areas are quantified [46]

Figure 8: Outline of the MLPA reaction [46]

MLPA assay has become a widely used technique in laboratoriesperforming genetic testing for the molecular diagnosis in general and DMD inparticular This method is considered to be a simple, rapid and reliable tool in

the screening of deletions and duplications of the DMD gene [50] Currently,

MLPA is one of the most powerful tools used in the diagnosis of effectedmales and female carriers with dystrophin gene mutations in the Gen-ProteinResearch Center of Hanoi Medical University

Figure 9: MLPA results of control and DMD male patient

Resource: Center for Gene and Protein research- Hanoi medical University MLPA electropherograms of DMD male patient showing absence of peaks in DMD Probe- P034 representing deletion of the exons 21-30 of the Dystrophin gen Each peak represents one exon of the Dystrophin gene.

160.32 168.34 177.91

192.97

203.06 209.20

234.42

242.08

249.89 265.63

273.41 282.18 289.56 305.94 314.36 337.60 353.88

360.80

378.59 386.99 394.97

410.09 418.60

425.72 433.79 449.91 460.14 466.50 483.80

Exon 21

Exon22

Exon 2 3 Exon24

Exon25 Exon26

Exon 27 Exon 28 Exon 29

160.34

161.42

168.22

177.69 184.86 192.89 202.79 209.15 216.07

224.02 234.25 241.91

248.46

249.67 257.13 265.36

273.66 281.98 289.56 298.54 314.13 327.55 337.47 353.77

356.53 360.56 369.80 378.30 386.64 394.80 401.39

402.78 409.84 418.33

425.56 433.83 442.25 449.69 459.72 466.17 476.75 483.49 490.68

Control

Table 1.2 Comparision between Multiplex Ligation dependent Probe

Amlification (MLPA) and other methods

PCR - Simple to perform - Limited number of targets

- Time consuming

Southern

blot - Detects small rearrangements

- Laborious and time consuming

- Limited number of targets and throughput

FISH - Fast results

1.6 The aim of the study

DMD is an X- linked disease which has a 100% fatality rate It is cally diagnosed in boys between the ages of 3-7 Boys with DMD generallylose their ability to walk between the ages of 8 and 12 Up to now, DMD stillhas no effective method of treatment In addition, with the serious clinicalmanifestations of mental disorders and premature mortality, DMD is actually

typi-a fintypi-ancitypi-al typi-and menttypi-al burden for ptypi-atients, their ftypi-amilies typi-and communities.Several previous studies show that two-third of patients receives DMD genes

from heterozygous mothers and one-third of patients are new (de novo)

muta-tions [14], [19], [47], [54] Hence, diagnosis of carriers is the most effectiveoption to restrict the development of this disease Amongst the molecular ge-netic testing used to detect female carriers in Vietnam, MLPA has been usedcommonly because MLPA is rapid, sensitive and accurate The long term ob-jective of this research is to apply MLPA to detect DMD mutated gene in car-riers and from which, we aim to further develop the project into early diagno-sis prenatal program To achieve the goal, we propose to pursue the followingspecifics aim: Apply MLPA to detect the carriers in families affected byDMD

CHAPTER 2 MATERIALS AND METHODS

2.1 Samples

Patient: 1 male patient

Female: 10 female relatives of DMD patients from 5 different familiescharacterized by DMD patients with exons 45-50, 11-20, 51-60, 3-47, 8-43and 48-50 dystrophin deletions and duplications

Control: 2 healthy female and 1 healthy male without family history ofdystrophinopathies were analyzed as control

Vietnamese DMD male patients and female relatives from North ofVietnam were referred to the Center for Gene and Protein research – HanoiMedical University for their clinical observation and molecular diagnosis In-formed consent was obtained from all patient families for this study

2.2 Reagents and equipment

2.2.1 Reagents

a Reagents for DNA extraction from blood

- Lysis buffer solution: lysis buffer is a buffer solution which is used foraid in the breaking of the cell membrane It contains protease enzymesand essential salts to bring about this process

- K solution: buffer solution

- Proteinase K solution (20 mg/ml): Proteinase K is commonly used inmolecular biology to digest protein and remove contamination frompreparations of nucleic acid

- SDS 10% solution: SDS (Sodium Dodecyl Sulfate) a detergent that isknown to denature proteins It is used in nucleic acid extractionprocedures for the disruption of cell walls and dissociation of nucleic acid.

- Phenol: chloroform: isoamyl (25:24:1): is frequently used to removeproteins from preparations of nucleic acids

- Chloroform: isoamyl (24:1): removes most of the protein

- 100% ethanol (cold):Ethanol is used to precipitate the DNA

- 70% ethanol (cold): Wash precipitate and remove salts from the DNA

- Sodium acetate 3M (pH 5.2)

- TE (Tris- EDTA) buffer or sterile distilled water

Lysis buffer solution

Reagents Concentration of stock

solution Buffer Stocks (200ml)

pH to 8.0 with NaOH, add water up to roughly 200 ml

Note: Lysis buffer solution and K solution must be absolute sterile (wetsteaming at 121 ° C for 15 minutes)

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, TheNetherlands

 SALSA MLPA buffer 200.1 (yellow)

 Ligase-65: 120.1 (green)

 Ligase-65 buffer A: 400.1(transparent)

 Ligase-65 buffer B: 4001 (white)

 SALSA PCR buffer: 600.1 (red)

 SALSA PCR Primer mix 250.1 (brown), mix PCR primers + dNTPs

 SALSA Polymerase: 75.1 (orange)

 SALSA Enzyme dilution buffer: 250.1 (blue)

 SALSA Probe mix 160.1 (black) (P034&P035)

 Mineral oil

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 (Eppendorf branch)

- Nano drop spectrophotometer

2.3.2 DNA extraction from blood

DNA extraction is the first step of the process detection carrier females

in affected families with DMD This is an important step to ensure the success

of the next steps The DNA was extracted from peripheral blood byphenol/chloroform method Blood samples typically were obtained as 2 ml of

whole blood stored in EDTA vacutainer tubes at 2-80C After drawing blood,DNA was extracted as soon as possible, usually within 24 hours to gain thebest DNA.

Step 1: Add 0.5 ml of blood into a 1.5 ml micro centrifuge tube taining 0.5 ml of lysis buffer Vortex immediately to prevent clot formation.Store on ice for 10 minutes Centrifuge at 8000 rpm (revolutions per minute)for 10 minutes at 4oC Remove all of the supernatant

con-Step 2: Add 0.5 ml lysis buffer solution and re-suspend the pellet tex for 30 seconds at the medium speed) Store on ice for 10 minutes Cen-trifuge at 8000 rpm for 10 minutes at 4oC Remove all of the supernatant The pellet should be white to cream in colour If the obtained pellet is red, re-peat the washing step again

(vor-Step 3: Add 0.5 ml K solution, centrifuge at 8000 rpm for 10 minutes at

4oC Remove all of the supernatant

Step 4: Add 0.5 ml lysis buffer, 12.5 µl SDS 10%, 10 µl protease K,vortex briefly and incubate at 56oC during 2-3 hours (or longer)

Step 5: Add 0.5 ml phenol/chloroform/isoamyl alcohol and mix by vertion (do not vortex) Centrifuge the sample at 12.000 rpm for 10 minutes at

in-4oC in a micro centrifuge tube The sample separates into 3 layers: 1st layer isDNA, 2nd layer is protein, 3rd layer is phenol chloroform

Step 6: Carefully transfer the top layer (containing DNA) by pipetting anew clean 1.5 ml microcentrifuge tube Do not include bottom (phenol) or in-terface (protein) layers It is better to leave a bit of the top layer behind than tocontaminate with the bottom or interface layers

- Repeat step 5-6