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 ACKNOWLEDGEMENTS 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 i ABBREVIATIONS 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 Amplification mRNA RNA messenger OD Optical Density PCR Polymerase Chain Reaction SDS Sodium Dodecyl Sulfate ii LIST OF FIGURES Figure 1: Typical progression of clinical symptoms with age of DMD patients .8 Figure 2: Genetic of DMD Figure 3: A: location of DMD gene B: protein products of the DMD gene [35] 11 Figure 4: The dystrophin- associated glycoprotein complex (DGC) in skeletal muscular [4] 12 Figure 5: The steps in PCR 15 Figure 6: Test hybridization on a metaphase from the 18 Duchenne muscular dystrophy carrier [29] 18 Figure 7: Sequencing of the dystrophin gene .19 Figure 8: Outline of the MLPA reaction [46] .20 Figure 9: MLPA results of control and DMD male patient 21 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 .21 1, 2, 3: female at risk of being healthy carrier of DMD (D1, D2, D3) 37 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 MLPA electropherograms of D1 showing 40-60% reduce of peak areas in DMD Probe set P034 (MRC- Holland) of the Dystrophin gene representing heterozygous deletion of the exons 45-50 as compared to the peak areas of MLPA electropherograms of C2 (control sample) The heterozygous deletion of exons 51, 52 in DMD was detected after probe hybridization with DMD Probe set P035 (data does not shown) Each peak represents one exon of 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 4, 5, 6, 7: female at risk of being healthy carrier of DMD (D4, D5, D6, D7) 42 iii 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 MLPA electropherograms of DMD male patient (D8) showing approximately two-fold increase of peak areas in DMD Probe set P035 (MRC- Holland) representing duplications of exons 11-20 and 51-60 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C1 (control sample) Each peak represents one exon of 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 MLPA electropherograms of D4 showing 30-50% increase of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous duplications of the exons 11-20 and 51-60 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C2 (control sample) Each peak represents one exon of 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 MLPA electropherograms of D5 showing 30-50% increase of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous duplications of the exons 11-20 and 51-60 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C2 (control sample) Each peak represents one exon of 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 MLPA electropherograms of D6 showing 30-50% increase of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous duplications of the exons 11-20 and 51-60 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C2 (control sample) Each peak represents one exon of 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 the dystrophin gene .47 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 MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P035 (MRC- Holland) of the Dystrophin gene representing heterozygous deletion of the exons 11-20 and 31-40 as compared to the peak areas of MLPA iv electropherograms of C3 (control sample) The heterozygous deletion of exons 8-10 and 41-43 in DMD were detected after probe hybridization with DMD Probe set P034 (data does not shown) Each peak represents one exon of the dystrophin gene .50 50 51 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 MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous deletion of the exons 11-20 and 31-40 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C3 (control sample) The heterozygous deletions of exons 3-10 and 41-47 in DMD were detected after probe hybridization with DMD Probe set P034 (data does not shown) Each peak represents one exon of the dystrophin gene .51 Figure 22: MLPA results of DMD female control (C3) and patient G’s mother (D11) in DMD Probe set P034 the dystrophin gene 52 MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P034 (MRC- Holland) of the Dystrophin gene representing heterozygous deletion of the exons 48-50 as compared to the peak areas of MLPA electropherograms of C3 (control sample) Each peak represents one exon of the dystrophin gene 52 LIST OF TABLE vi Table 1.1 Genotype of parent and rate of affected in offspring .7 Table 1.2 Comparision between Multiplex Ligation dependent Probe Amlification (MLPA) and other methods .22 Table 2.1 PCR program for the MLPA reaction 33 Table 3.1 Results of DNA extraction 36 49 50 v 52 52 In all three families, the mothers have a son with DMD so when they intend to have more children, the prenatal diagnosis is extremely important and meaningful to prevent birth of affected children .53 Table 3.2 Results of multiplex ligation-dependent probe amplification (MLPA) 54 vi CONTENTS Hanoi, 2013 i ACKNOWLEDGEMENTS i ABBREVIATIONS ii LIST OF FIGURES iii LIST OF TABLE .v ABSTRACT TÓM TẮT .3 PREFACE .5 CHAPTER INTRODUCTION 1.1 Duchenne muscular dystrophy 1.1.1 Characteristics of DMD .7 1.1.2 Treatment and management of DMD 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 MATERIALS AND METHODS 24 2.1 Samples 24 2.2 Reagents and equipment 24 2.2.1 Reagents 24 2.2.2 Equipment 26 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 RESULTS AND DISCUSSION 35 3.1 DNA extraction 35 3.2 MLPA results 36 3.2.1 MLPA results of patient A’s family 36 3.2.2 MLPA results of patient B’s family 41 3.2.3 MLPA result of patient E’s mother (D9) 49 3.2.4 MLPA result of patient F’mother (D10) .50 3.2.5 MLPA result of patient G’s mother (D11) 51 CONCLUSION AND SUGGESTION 55 BIBLIOGRAPHY 56 ABSTRACT Duchenne muscular dystrophy (DMD) is a recessive disorder associated with the chromosome X caused by mutations in the dystrophin gene It mostly affects boys, and is characterized by a rapidly progressive muscle weakness that almost always results in death, usually by 20 years of age When a family member has DMD, all member of the family are affected by caregiving demands and emotional reactions In Vietnam the socioeconomic burden of disease is very high, as the parents have to cover the cost 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 the defective gene is passed on to a son through the mother’s faulty X chromosome; the diagnosis of female carriers (mother, aunt, sister) to detect mutations which would enable medical staff to provide prenatal genetic counseling for them is the most effective solution to reduce incidence Many methods have been used to help the detection of heterozygous females, such as PCR, Southern blot, FISH, etc, in which, MLPA is increasingly become widely used in laboratories worldwide With many advantages such as rapid, sensitive, cost effective and reliable so MLPA is the first option and is a useful quantitative method for detecting mutation for the analysis of both affected males and female carriers In this study, we have succeeded in the application of the MLPA method to identify female carriers We detected out of 10 female carriers in affected DMD families Four of them show heterozygous deletion exons 4552, 8-43, 3-47 and 48-53, in the DMD gene, respectively The remaining three Figure 20: MLPA results of DMD female control (C3) and patient E’s mother (D9) in using DMD Probe set P035 the dystrophin gene MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P035 (MRC- Holland) of the Dystrophin gene representing heterozygous deletion of the exons 11-20 and 31-40 as compared to the peak areas of MLPA electropherograms of C3 (control sample) The heterozygous deletion of exons 8-10 and 41-43 in DMD were detected after probe hybridization with DMD Probe set P034 (data does not shown) Each peak represents one exon of the dystrophin gene 3.2.4 MLPA result of patient F’mother (D10) 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 MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous deletion of the exons 11-20 and 31-40 of the Dystrophin gene as compared to the peak areas of MLPA electropherograms of C3 (control sample) The heterozygous deletions of exons 3-10 and 41-47 in DMD were detected after probe hybridization with DMD Probe set P034 (data does not shown) Each peak represents one exon of the dystrophin gene 3.2.5 MLPA result of patient G’s mother (D11) 51 Figure 22: MLPA results of DMD female control (C3) and patient G’s mother (D11) in DMD Probe set P034 the dystrophin gene MLPA electropherograms of D9 showing 40-60% reduce of peak areas in DMD Probe set P034 (MRC- Holland) of the Dystrophin gene representing heterozygous deletion of the exons 48-50 as compared to the peak areas of MLPA electropherograms of C3 (control sample) Each peak represents one exon of the dystrophin gene 52 The patients in these families (E, F and G) have been found with deletion mutations in the other study MLPA results (Figure 20, 21, 22) of the mothers (D9, D10, D11) of patients E, F, G, respectively, showed that these mothers have deletion mutations in exons 11-20, 31-40 and 48-50 because the peak height of exons 11-20, 31-40 and 48-50 decreased by approximately 3550% comparison with the peak height of these corresponding exons in the female control samples According to K.K Lai (2006), in female heterozygotes, a 35-55% reduced relative peak area of the amplification product of that probe is expected [25] It is proves that D9, D10 and D11 are female carriers and they transmit gene to their sons In all three families, the mothers have a son with DMD so when they intend to have more children, the prenatal diagnosis is extremely important and meaningful to prevent birth of affected children 53 Table 3.2 Results of multiplex ligation-dependent probe amplification (MLPA) Patient A Female relative Mutation Carrier deletion exons 45-52 None carrier None carrier Carrier duplication exons 11-20 Mother (D1) Second aunt (D2) First aunt’s daughter (D3) First aunt (D4) and exons 51-60 Carrier duplication exons 11-20 B Second aunt (D5) and exons 51-60 Carrier duplication exons 11-20 First aunt’s daughter (D6) E F G Second aunt’s daughter (D7) Mother (D9) Mother (D10) Mother (D11) and exons 51-60 None carrier Carrier deletion exons 8-43 Carrier deletion exons 3-47 Carrier deletion exon 48-50 Table 3.2 shows that out of 10 female members have heterozygous mutation of the DMD gene: four cases have heterozygous deletion exons 4552, 8-43, 3-47 and 48-50, respectively; three cases have heterozygous duplication exons 11-20, 51-60 Deletions and duplications can happen almost anywhere in the dystrophin gene However, deletion mutations are the most commonly found in two “hot spot” regions In this study, most of deletions were confined to these regions (exons 2-20 and exons 44-53) The detection of deletions or duplications of the DMD gene in female members in general and in heterozygous mothers in particular is meaningful because it will help the genetic counseling and prenatal diagnosis to prevent the birth of children affected by this disease 54 CONCLUSION AND SUGGESTION We have successfully applied MLPA technique to detect the carriers in ten female members of DMD patient families Seven out of them were detected to have heterozygous deletion or duplication (4 heterozygous deletions, heterozygous duplications) Carrier detection is important for genetic counseling in order to reduce the incidence of this disease The study was only carried out on a small sample size of Vietnamese DMD female carriers and their family We emphasize the need for applying the MLPA technique further for studying and detecting female carriers in order to provide better diagnostic, prognostic and prenatal services to the suffering patients and their families 55 BIBLIOGRAPHY Aartsma-Rus A., 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pp.236-247 Websites 60 http://duchennemusculardystrophy-2.wikispaces.com/ 61 http://nature.ca/genome/ 62 http://prosensa.eu/hc-professionals/duchenne-muscular-dystroph 63 http://www.dmd.nl/ 64 http://www.mlpa.com/ 65 http://www.patient.co.uk/ 8,9,11,12,15,18,19,20,21,38-40,43-47,50-52 1-7,10,13-14,16-17,22-37,41-42,48-49,53-63 [...]... dystrophin gene will be amplified in 2 reactions Thus, within 1 week, MLPA can screen all mutations in the dystrophin gene This is a particular advantage of the MLPA compared with other methods In order to carry out the diagnosis, 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. .. cystein- rich domain • The carboxy- terminal domain 11 The first domain is the amino-terminal domain It has homology with αactin and contains between 232 and 240 amino acid residues depending on the isoform The larget domain of dystrophin is the central-rod- domain and it is a succession of 25 triple-helical repeats similar to spectrin and contains about 3000 residues The third domain is a cysteine-... and time consuming 18 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 the Multiplex Ligation- dependent Probe Amplification. .. dependent Probe Amplification 6 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 was discovered a long time ago, as recently as the early 1980s, people still did not understand the cause of any form of muscular dystrophy In 1986,... signal loss can also contribute to causing the disease [17], [41] When dystrophin is absent, the DGC is destabilized leads to muscle wasting which characterizes Duchenne muscular dystrophy 1.4 Mutations in the dystrophin gene Mutations in the large dystrophin gene, which consists of 79 exons generally causes a disruption of the open reading frame in the dystrophin protein production process which leads... dystrophinopathies 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 427 kDa, and contains about 3685 amino acids [17], [23] This protein is located in the plasma membrane of muscle cells and is divided into four domains [23]: • The amino-terminal domain • The central- rod- domain... 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 muscle cytoskeleton and extracellular matrix to maintain muscle integrity [7],... factors involved Particularly in the case of DMD, PCR plays an especially important role in the process of mutation detection Several techniques such as multiplex PCR, Nested PCR, RT-PCR have been used to detect mutations are based on the principle of PCR Among these methods, multiplex PCR is appreciated in the diagnosis of deletion and about 98% of deletions are easily detectable using multiplex PCR in affected. .. [7], [42] Many membrane proteins associated with dystrophin protein complex are made up of DGC (dystrophin-glycoprotein complex) One of the other main roles of the DGC is to stabilise the sarcolemma and to protect muscle fibres 12 from long-term contraction-induced damage and necrosis [35] Moreover, the DGC may also play a role in cell signaling by interacting with proteins that send and receive chemical... Accordingly, before using, the potential risks and benefits of this medical intervention need to be adequately investigated In addition, researchers have made great advances in their knowledge of DMD and continue to search for a cure At present, some areas in which research is being focused include: gene therapy, read-through stop codon strategies, stem cell therapy, vival vectors and utrophin [59] ... 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 INTRODUCTION... rod- domain • The cystein- rich domain • The carboxy- terminal domain 11 The first domain is the amino-terminal domain It has homology with αactin and contains between 232 and 240 amino acid... aunt (D5) in using DMD Probe set P035 the dystrophin gene 45 MLPA electropherograms of D5 showing 30-50% increase of peak areas in DMD Probe set P035 (MRC- Holland) representing heterozygous