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

ABSTRACT Duchenne muscular dystrophy (DMD) is a recessive disorder associated with the chromosome X caused by mutations in the dystrophin gene It affects mainly boys 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 With many advantages such as rapid, sensitive, cost effective, reliable so MLPA is used as 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 Using the MLPA method, we detected out of 10 female carriers in 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 the DMD gene Most of these mutations are located in the ‘hot spot’ regions In addition, we also detected one patient with duplication exons 11-20 and 51-60 in the gene MLPA assays are performed according to manufacturer recommendations TÓM TẮT Bệnh loạn dưỡng Duchenne (Duchenne Muscular Dystrophy- DMD) bệnh di truyền lặn liên kết với nhiễm sắc thể X gây đột biến gen dystrophin.Theo số nghiên cứu, khoảng hai phần ba số trường hợp gen khiếm khuyết truyền sang trai từ người mẹ có nhiễm sắc thể X bị lỗi; chẩn đoán người nữ dị hợp tử (mẹ, cô, dì, chị gái) để phát đột biến cho phép nhân viên y tế tư vấn di truyền trước sinh giải pháp hiệu để giảm tỷ lệ mắc bệnh Với nhiều ưu điểm nhanh, nhạy, chi phí hiệu quả, độ tin cậy cao nên MLPA sử dụng lựa chọn phương pháp định lượng hữu ích cho việc phát đột biến phân tích nam giới bị bệnh người nữ dị hợp tử Trong nghiên cứu này, thành công việc áp dụng phương pháp MLPA để xác định người nữ dị hợp tử Sử dụng phương pháp MLPA, phát phụ nữ có mang gen dị hợp tử số 10 phụ nữ gia đình bị ảnh hưởng bệnh DMD Bốn người số họ có đột biến xóa đoạn dị hợp tử exon 45-52, 8-43, 3-47 48-53 Ba người nữ lại có đột biến dị hợp tử lặp đoạn exon 11-20 51-60 gen DMD Hầu hết đột biến phát vùng “hot spot” Ngoài ra, phát bệnh nhân có đột biến lặp đoạn exon 11-20 51-60 gen dystrophy Kỹ thuật MLPA thực theo khuyến nghị nhà sản xuất PREFACE Duchenne muscular dystrophy (DMD) is one of the most common fatal genetic disorders affecting children around the world The causes of DMD are mutations in the dystrophin gene on chromosome X; hence, it is diagnosed mostly in males Although DMD is the most common fatal genetic disorder to affect children, at the moment no cures have been found Researchers are still looking for treatments to alter the course of the disease and improve the quality of life for patients Previous studies have shown that two thirds of patients receive the mutation from their mothers and the other one third has new mutation [14], [19], [47], [54] Thus, detection of the heterozygous status of mother as well as other female members of family with suitable consequent antenatal screening of the fetus at risk is highly appreciated active prevention (reduces new incidence of the disease) In recent years, many studies show that Multiplex Ligation- dependent Probe Amplification (MLPA) is a rapid and accurate technique, which allows high-throughput screening mutations, especial deletions and duplications, in DMD and other genetic diseases This is a molecular biology method based on the basic principle of PCR; nonetheless, it uses one pair of primer, two reactions to detection mutation in all 79 exons Thus, within 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 Probe Amplification” CHAPTER 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 more common in males than females DMD is a progressive disease in which the patient's muscle injury is due to a lack of dystrophin protein The dystrophin gene can be passed on from the carrier woman to her child (complies with the rules of the genetic X- linked inheritance) According to Mendelian inheritance, there is 50% chance a mother who carries the DMD gene can pass the X chromosome carrying DMD mutated gene to the sons and they will develop disease and 50% chance that her daughters will carry the gene A carrier mother may or may not pass on the gene with the mutation Normally, the majority of female carriers usually have no symptom However, 2-20% of carriers have symptoms of muscle weakness or clinical signs of disease 1.1.2 Treatment and management of DMD It is nearly 30 years since the discovery of the genetic defect causing DMD, but the disease has yet to be cured To date only one treatment, 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 codon strategies, stem cell therapy, vival vectors and utrophin [59] However, these methods are only of partial support for patients and they are not the complete cures Therefore, diagnosis of women with a high risk and genetic counseling for female carriers in patients’ families 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 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 alternative 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 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- roddomain, the cystein- rich domain, he carboxy- terminal domain 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], [42] 1.4 Mutations in the dystrophin gene 1.4.1 Deletion mutations Deletion one or several exons is common mutations in patients with DMD, accounting for 60-65% of DMD disease-causing mutations [9] Deletion mutations in DMD genes are the most commonly 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 largest mutation in the dystrophin gene after deletion mutations [43] Most point mutations in the DMD gene created stop codon and caused seriously disease Point mutations are located along the entire gene and this is a major 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 'end and 20% in the center In addition, a small percentage of DMD patients have small mutations scattered along 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 especially important role in the process of mutation detection For example, multiplex PCR is appreciated in the diagnosis of deletion and about 98% of deletions are easily detectable using multiplex PCR in affected males [3] 1.5.2 Southern blot method Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples The principle in this method combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size- separated DNA to a filter membrane for probe hybridization Prior (2005) used this method to detect mutations in DMD patients as well as female carriers [39] 1.5.3 Fluorescence in situ hybridization - FISH FISH (fluorescence in situ hypridization) is a molecularcytogenetic technique that permits DNA sequences to be detected on metaphase chromosomes, in interphase nuclei, in a tissue section, or in blastomeres and gametes [40] This method uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity 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] 1.5.4 Sequencing method Today, with advancement of technology, both of Sanger dideoxy method and Maxam-Gilbert chemical cleavage method are replaced by modern sequencing equipment (automatic sequencing) The new technology is based on the same principles of Sanger's method but four different fluorescent dye-labelled ddNTPs are used Thus each fluorescent label can be detected by its characteristic spectrum The products are separated by automated electrophoresis and the bands detected by fluorescence spectroscopy For DMD, sequence analysis of the dystrophin gene is a rapid way to detect small mutation that nearly entire of the 79 exons but this method is very costly and time consuming 1.5.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method In the MLPA technique, genomic DNA is hybridized in solution to probe sets, each of which consists of two oligonucleotides: one short synthetic oligonucleotide and one long probe oligonucleotide The short synthetic oligonucleotide contains a target-specific sequence (20–30 nucleotides) at the 3’ end and a common sequence (19 nucleotides) that is the primer binding sites, at the 5’ end The long MLPA probe contains the 25-43 nucleotides target-specific sequence at the 5’ end, a 36 nucleotides sequence that contains primer binding sites and is common to all probes, at the 3’ end and a suffer sequence (19–370 nucleotides)- a variable length random fragment in between to generate the length differences The different lengths of the products allow separation on an automated capillary sequencer, and the peak areas are quantified [46] MLPA assay has become a widely used technique in laboratories performing genetic testing for the molecular diagnosis in general and DMD in particular Compared to other techniques, MLPA is rapid, sensitive, reliable and very simple to perform Therefore, MLPA is highly recommended by scientists for detecting DMD and others genetic deseases 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 Several previous 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 Vietnam, MLPA has been used commonly because MLPA is rapid, sensitive and accurate The long term objective of this research is to apply MLPA to detect DMD mutated gene in carriers and from which, we aim to further develop the project into early diagnosis prenatal program To achieve the goal, we propose to pursue the following specifics aim: Apply MLPA to detect the carriers in families affected by DMD CHAPTER MATERIALS AND METHODS 2.1 Patient, female carrier and normal control male patient; 10 female relatives from different families characterized by DMD patients with exons 45-50, 11-20, 51-60, 347, 8-43 and 48-50 dystrophin deletions and duplications; healthy female and healthy male without family history of dystrophinopathies 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 (20 mg/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, 3140, 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 (Eppendorf branch); Nano drop spectrophotometer; Vortex machine; Timer, Freezer (2-8oC, -20oC); 0.5, 0.2 ml PCR tubes 10 2.3 Methods 2.3.1 Sampling process Blood samples were collected in EDTA vacutainer tube (3 ml quantities) from female relative, patients and matched control The process ensures absolute sterility 2.3.2 DNA extraction from blood The DNA was extracted from peripheral blood by phenol/chloroform method Then, the DNA will be tested by measurement of optical density at wavelength of 260nm and 280 nm DNA samples, which has to concentrations > 20ng/μl 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 on Spectrophotometric method A ratio of OD260nm/OD280nm in the range of 1.8 – indicates highest -quality DNA whereas lower values indicate protein contamination Higher values indicate RNA contamination [45] 2.3.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method The MLPA DMD test kit (SALSA P34/P035) was purchased from MRC Holland, Amsterdam, the Netherlands The procedure was performed according to the protocol provided by the manufacturer’s recommendation [6] Before carry out of MLPA, prepare DNA sample: 20 ng/ml (DNA working stock) and 11 thermocycler (Eppendorf branch) The process is described as follows: DNA denaturation Add μl of DNA working stock to each PCR tube Denature sample DNA for minutes at 98°C and cool the samples to 25°C Hybridization reaction - Prepare hybridisation master mix for hybridization reaction - Add μl of the hybridisation master mix to each PCR sample tube above Continue incubate for minute at 95 °C, then for 16 – 20 hours at 60 °C following the thermocycler program Ligation reaction - Prepare ligase mix for ligation reaction - Continue the thermocycler program: pause at 54°C When the samples are at 54°C, add 32 µl of ligase mix and incubate 15 minutes at 54oC for ligation; - Heat to inactivate the ligase for minutes at 98 oC and then pause at 15 oC 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 μl of each ligation product to its corresponding PCR tube Spin - Prepare PCR master mix (in icebox) 12 - Continue the thermocycler program: pause at 60 °C and place the PCR tubes in the thermocycler - While these tubes are in a thermocycler at 60 °C, add 10 μl mastermix to each tube Mix by pipetting gently (up and down) and continue the thermocycler program immediately: 35 cycles: 30 seconds 95 °C; 30 seconds 60 °C; 60 seconds 72 °C End with 20 minutes incubation at 72 °C and then pause at 15 °C Table 2.1 PCR program for the MLPA reaction Stage Temperature Time 98 °C minutes 25 °C pause Hybridisation 95 °C minute reaction 60 °C pause 54 °C pause 54 °C 15 minutes 98 °C minutes 15 °C pause 60 °C pause DNA denaturation Ligation reaction PCR reaction 35 cycles 95 °C 30 seconds 60 °C 30 seconds x 35 cycles 72 °C 60 seconds 72 °C 20 minutes 15 °C pause Capillary electrophoresis PCR products were analyzed on the GenomeLabTM GeXP Genetic Analysis System (Applied Biosystems) using Genemarker software 13 v.1.95 (Softgenetic, State College, PA, USA) The peaks achieved after capillary sequencing could easily be differentiated and assigned to particular exons on the basis of their different lengths representing the variability of their stuffer sequences The company protocol recommends that the relative probe signals of each probe was detemined by dividing each measured peak area (As) by the sum of all 45 peaks area (ΣAs) of that sample To obtain the relative peak ratio, the relative peak area (As/ΣAs) is then divided by the relative peak area of the corresponding probe obtained from a control DNA sample For analysis of the test samples, the relative peak areas of the amplified probes were analyzed using Microsoft Excel and the peak area of each fragment was compared to that of a control sample As the DMD gene is located on the X chromosome, deletion of one or more exons will result in a complete lack of one or more of the corresponding peaks in male patients and a 35-55% reduction of the relative peak ratio of the expected value in female carriers Duplications will give an approximately two-fold greater relative peak area in male patients and a 30-55% increase in female carriers Identified deletions and duplications were regarded as reliable when they involved two or more adjacent exons [25] CHAPTER RESULTS AND DISCUSSION 3.1 DNA extraction 14 DNA was extracted from 14 samples: 10 female relatives and patient from different families characterized by DMD and healthy male and healthy female as positive control Then we determined the quality, concentration and purity of DNA by using the spectrophotometric method All samples were obtained with high purity (concentrations > 100 ng/μl and purity- A260/A280 from 1.8 to 2.0) 3.2 MLPA results 3.2.1 MLPA results of patient A’s family a MLPA result of patient A’s mother (D1) Figure 11: MLPA results of DMD female control (C2) and mother (D1) in using DMD Probe set P034 the dystrophin gene b MLPA result of the patient’s second aunt (D2) 15 Figure 12: MLPA results of DMD female control (C2) and second aunt (D2) in using DMD Probe set P034 the dystrophin gene c MLPA result of the first aunt’s daughter (D3) Figure 13: MLPA results of DMD female control (C2) and first aunt’s daughter (D3) in using DMD Probe set P034 the dystrophin gene In the family of patient A, analysis of the MLPA results of the mother, the second aunt and the first aunt’s daughter (figure: 11, 12, 13) show that the peak height of exons 45-52 (reflecting the concentration in the PCR product) of the sample D1 (mother) decreased by approximately a half compared with that of the female control sample; the peak height of exons 45-52 of the sample D2 (second aunt), D3 (first aunt’s daughter) equals the height of corresponding exons in the female control sample According to Hwa H L (2007), the female samples were considered pathological (female carrier) when the following criteria was fulfilled: the peak height of the mutation exons in the female decreased by approprimately a half (3565%) compared with the peak height of corresponding to exons in the healthy female control [20] In addition, according to Lai K.K (2006), 16 the height of the peak signal from the mother reduced by 35-55% compared with the peak signal of female control sample meaning that the mother is heterozygous female [25] Thus, based on the results of the MLPA analysis (figure 11, 12, 13) it is confirm that the mother of patient A is a female carrier and the aunt, first aunt’s daughter are healthy females It is proved that the mutation of the dystrophin gene was transmitted to the patient from his mother 3.2.2 MLPA results of patient B’s family a MLPA result of patient B Figure 15: MLPA results of DMD male control (C1) and patient B (D8) in using DMD Probe set P035 the dystrophin gene b MLPA result of patient's first aunt (D4) 17 Figure 16: MLPA results of DMD female control (C2) and first aunt (D4) in using DMD Probe set P035 the dystrophin gene c MLPA result of patient's second aunt (D5) Figure 17: MLPA results of DMD female control (C2) and second aunt (D5) in using DMD Probe set P035 the dystrophin gene d MLPA result of first aunt’s daughter (D6) 18 Figure 18: MLPA results of DMD female control (C2) and first aunt’s daughter (D6) in using DMD Probe set P035 the dystrophin gene e MLPA result of daughter of second aunt (D7) Figure 19: MLPA results of DMD female control (C2) and second aunt’s daughter (D7) in using DMD Probe set P035 the dystrophin gene In this family, MLPA results (Figure 16,17,18) of the female members (D4, D5, D6) showed that these members have duplication mutations in exons 11-20 and 51-60 because the peak height of exons 11-20 and 51-60 increased by 30 to 50% compare with the height of these corresponding exons in the female control samples According to K.K Lai (2006), the height of the peak signal from the mother increased by 30-50% compared with the peak signal of female control sample meaning that the mother has deletions mutation exon corresponding to that peak signal [25] This proves that D4, D5 and D6 are female carirrers In addition, as mentioned above, based on 19 pedigree we found that the second aunt is an enforced heterozygous carrier so that it is confirmed that the female carries the heterozygous genotype again In particular, the daughter of the second aunt (D7) has a mother carrier but MLPA results did not show the peak height of this sample exon increasing or decreasing compared with the corresponding peak of female control sample (Figure 19) Thus, it was considered the daughter of the second aunt is normal and not a carrier From the family pedigree of patient B, we found that the patient has a cousin affected (deceased), this proved to be genetic factors in this family So that the mother and patient's second aunt are obligate carriers and their sons receive gene from mothers, this is a perfect fit when genetic analysis of patients with mom and second aunt So, five members in the patient's family are female carriers, particularly grandmother, she transmitted gene for all of her daughters All daughters will be unaffected, but each has a 50% chance of being a carrier like her mother However, in family of patient B all of the daughters of his grandmother (his mother and two aunts) are female carriers (heterozygous gene) This suggests the possibility of genetic of the disease of mother carriers has been spreading quite high and quite fast in the community This family should be genetic counseling thorough and detailed, especially provide the advices about prenatal diagnosis when the females want pregnant to avoid giving birth to children continue to be affected or carriers, in order to reduce the burden on families and society Fortunately, the daughter of the second aunt is healthy female, although her mother is a carrier This is of great significance, psychological help relieve anxiety for her and her family However, in the family with so many people which are female carriers 20 (grandmother, mother and aunts), after marriage she should perform prenatal diagnosis methods when she has pregnancy to help give birth healthy babies 3.2.3 MLPA result of patient E’s mother (D9) Figure 20: MLPA results of DMD female control (C3) and patient E’s mother (D9) in using DMD Probe set P035 the dystrophin gene 3.2.4 MLPA result of patient F’mother (D10) Figure 21: MLPA results of DMD female control (C3) and patient F’s mother (D10) in using DMD Probe set P035 the dystrophin gene 21 3.2.5 MLPA results of patient G’s mother (D11) Figure 22: MLPA results of DMD female control (C3) and patient G’s mother (D11) in DMD Probe set P034 the dystrophin gene 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 1120, 31-40 and 48-50 decreased by approximately 35-50% 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 22 Table 3.2 Results of multiplex ligation-dependent probe amplification (MLPA) Patient A Female relative Mutation Mother (D1) Carrier deletion exons 45-52 Second aunt (D2) None carrier First aunt’s daughter (D3) None carrier Carrier duplication exons First aunt (D4) 11-20 and exons 51-60 B Carrier duplication exons Second aunt (D5) 11-20 and exons 51-60 First aunt’s daughter (D6) Second aunt’s daughter Carrier duplication exons 11-20 and exons 51-60 None carrier (D7) E Mother (D9) Carrier deletion exons 8-43 F Mother (D10) Carrier deletion exons 3-47 G Mother (D11) 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 45-52, 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 4453) CONCLUSION AND SUGGESTION 23 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 24 [...]... (D1) in using DMD Probe set P034 the dystrophin gene b MLPA result of the patient’s second aunt (D2) 15 Figure 12: MLPA results of DMD female control (C2) and second aunt (D2) in using DMD Probe set P034 the dystrophin gene c MLPA result of the first aunt’s daughter (D3) Figure 13: MLPA results of DMD female control (C2) and first aunt’s daughter (D3) in using DMD Probe set P034 the dystrophin gene In. .. aunt (D4) in using DMD Probe set P035 the dystrophin gene c MLPA result of patient's second aunt (D5) Figure 17: MLPA results of DMD female control (C2) and second aunt (D5) in using DMD Probe set P035 the dystrophin gene d MLPA result of first aunt’s daughter (D6) 18 Figure 18: MLPA results of DMD female control (C2) and first aunt’s daughter (D6) in using DMD Probe set P035 the dystrophin gene e... OD260nm/OD280nm in the range of 1.8 – 2 indicates highest -quality DNA whereas lower values indicate protein contamination Higher values indicate RNA contamination [45] 2.3.4 Multiplex Ligation- dependent Probe Amplification (MLPA) method The MLPA DMD test kit (SALSA P34/P035) was purchased from MRC Holland, Amsterdam, the Netherlands The procedure was performed according to the protocol provided by the manufacturer’s... mother (D9) in using DMD Probe set P035 the dystrophin gene 3.2.4 MLPA result of patient F’mother (D10) Figure 21: MLPA results of DMD female control (C3) and patient F’s mother (D10) in using DMD Probe set P035 the dystrophin gene 21 3.2.5 MLPA results of patient G’s mother (D11) Figure 22: MLPA results of DMD female control (C3) and patient G’s mother (D11) in DMD Probe set P034 the dystrophin gene The... aunt’s daughter (D7) in using DMD Probe set P035 the dystrophin gene In this family, MLPA results (Figure 16,17,18) of the female members (D4, D5, D6) showed that these members have duplication mutations in exons 11-20 and 51-60 because the peak height of exons 11-20 and 51-60 increased by 30 to 50% compare with the height of these corresponding exons in the female control samples According to K.K Lai (2006),... and well mix by pipetting - At room temperature, transfer 10 μl of each ligation product to its corresponding PCR tube Spin - Prepare PCR master mix (in icebox) 12 - Continue the thermocycler program: pause at 60 °C and place the PCR tubes in the thermocycler - While these tubes are in a thermocycler at 60 °C, add 10 μl mastermix to each tube Mix by pipetting gently (up and down) and continue the thermocycler... diagnosis is extremely important and meaningful to prevent birth of affected children 22 Table 3.2 Results of multiplex ligation- dependent probe amplification (MLPA) Patient A Female relative Mutation Mother (D1) Carrier deletion exons 45-52 Second aunt (D2) None carrier First aunt’s daughter (D3) None carrier Carrier duplication exons First aunt (D4) 11-20 and exons 51-60 B Carrier duplication exons Second... 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... 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,... tube above Continue incubate for 1 minute at 95 °C, then for 16 – 20 hours 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 the samples 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 98 oC and then pause at ... 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... muscle cells and is divided into four domains [23]: the amino-terminal domain, the central- roddomain, the cystein- rich domain, he carboxy- terminal domain Dystrophin plays an important structural... OD260nm/OD280nm in the range of 1.8 – indicates highest -quality DNA whereas lower values indicate protein contamination Higher values indicate RNA contamination [45] 2.3.4 Multiplex Ligation- dependent Probe