Duchenne Muscular Dystrophy Families Research Articles

Duchenne/Becker muscular dystrophy in the family: have potential carriers been tested at a molecular level?

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease. After identification of the mutation in the index patient, family members can be reliably investigated. Carriers should be informed about their risk of having offspring with the disease and about their own risk for cardiomyopathy for which regular cardiac surveillance is recommended. In a small country like the Netherlands with well-organized genetic services, one would expect that most DMD families are adequately informed about the above mentioned risks for carriers. We have investigated whether women at risk had been tested at a molecular level. In the national Duchenne/Becker database 311 DMD and 99 Becker muscular dystrophy (BMD) patients had been registered up to 1 July 2009. These patients were asked to give information about the number of sisters and maternal aunts of the DMD/BMD patient and anything that was known about their genetic status and that of the mother. This information was compared with the information known at the genetic laboratory. Thirty-five of 104 adult sisters/maternal aunts of DMD patients with a 50% risk of being a carrier and 45 of 148 adult women with a 4.3% risk because of germ line mosaicism for DMD had not been tested by DNA analysis. Our study indicates that about one third of the potential carriers have not been tested. Given the possible far-reaching clinical consequences of being a carrier, further studies are needed to investigate the reasons why potential female carriers have not been tested.

P4.10 Duchenne/Becker in the family: are women aware of the potential risks?

Background: Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease. After identification of the mutation in the index patient, family members can be reliably investigated. Carriers should be informed about their risk of having offspring with the disease and about their own risk for cardiomyopathy for which regular cardiac surveillance is recommended. In a small country like the Netherlands with well organized genetic services, one would expect that most DMD families are adequately informed about the above mentioned risks for carriers. We have investigated whether women at risk had been tested at a molecular level. Method: In the national Duchenne/Becker database 311 DMD and 99 BMD patients/families had been registered up to July 1, 2009. These families were asked to list the number of sisters and maternal aunts of the DMD/BMD patient and anything that was known about their genetic status and that of the mother. This information was compared with the information known at the genetic laboratory. Findings: Thirty-five out of 104 adult sisters/maternal aunts of DMD patients with a 50% risk of being a carrier and 45 out of 148 adult women with a 4.3% risk due to germ line mosaicism for DMD had not been tested by DNA analysis. Conclusion: Our study indicates that about one third of the potential carriers have not been tested. Given the possible far-reaching clinical consequences of being a carrier, further studies are needed to investigate the reasons why potential female carriers have not been tested.

Detection of long-term progression of myocardial fibrosis in Duchenne muscular dystrophy in an affected family: A cardiovascular magnetic resonance study

Background Detection of myocardial fibrosis and left ventricular dysfunction in Duchenne muscular dystrophy (DMD) is the corner stone for further therapeutic studies. Little is known about the ability of cardiac magnetic resonance imaging (CMR) to evaluate progression of myocardial fibrosis. Aim of our study was to provide CMR data in a previously genotyped DMD family and to evaluate whether progression of myocardial fibrosis could be visualized. Methods and results DMD genotypes were available in 14 family members. CMR was performed in 4/5 carrier females, in 2/2 affected males and in one healthy family member with normal genotype. Functional images and late gadolinium enhanced (LGE) images in contiguous short-axis orientation were acquired at baseline and follow-up of 1231 days CMR examination could be repeated in three carrier females, in one affected male and in the healthy subject previously scanned. Mean decrease of left ventricular ejection fraction during the follow-up period was 10.5 ± 11.0%, mean progression of LGE volume 11.7 ± 9.5%. Conclusions Myocardial fibrosis seems to occur prior to global left ventricular dysfunction in DMD diseased males and carrier females. CMR could be used to evaluate progression of myocardial fibrosis and left ventricular function and may thus serve as an important diagnostic tool in the evaluation of therapeutical options in DMD.

Rapid and powerful decaplex and dodecaplex PGD protocols for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a common childhood lethal X-linked recessive disorder, resulting from deletions, duplications and point mutations in the dystrophin gene. Single-cell protocols for preimplantation genetic diagnosis (PGD) still remain challenging due to the enormous size of the gene and the high risk of intragenic recombination, limitations that often lead to sex determination and selection of female embryos. This study describes direct and rapid decaplex and dodecaplex polymerase chain reaction protocols enabling the analysis of five or seven exons and four microsatellite markers scattered along the dystrophin gene, chosen to be located in the two deletion hotspots, and the analysis of amelogenin sequences for gender determination. The dodecaplex protocol may be applied to most of the couples requesting PGD for DMD in whom the female partner is a carrier of a deletion. This generic approach will allow prompt response to the PGD referrals by reducing the pre-clinical PGD work-up. It was successfully applied in three DMD families, resulting in the birth of a girl as well as in a healthy ongoing pregnancy.

Prenatal Diagnosis in Macedonian Duchenne Muscular Dystrophy Families

Duchenne muscular dystrophy (DMD) is an Xlinked recessive disorder caused by mutations in the dystrophin gene at Xp21.2. Mutations include gross deletions (60%), duplications (10%), and point mutations (30%). Duchenne muscular dystrophy is a serious and disabling disease. Progressive muscle wasting, which leads to severe disability and early death, make DMD highly distressing disorders to both patient and family. Since no effective treatment is as yet available, prenatal diagnosis is important for prevention of the disease. In this paper, we present our results from prenatal diagnoses in Macedonian DMD families. For prenatal diagnosis of 15 pregnancies at risk of having a DMD child, we used multiplex polymerase chain reaction (mPCR), multiplex ligation-dependent probe amplification analy sis (MLPA) and DNA linkage analysis, using highly polymorphic intragenic short tandem repeat [STR(CA) n] markers. DNA material was extracted from chorionic villus and amniotic fluid samples. Eight of the fetuses were male, three of whom had deletions in the dystrophin gene and five were normal. Two of the female fetuses were carriers of deletions in the dystrophin gene.

Preimplantation genetic diagnosis (PGD) for Duchenne muscular dystrophy (DMD) by triplex-nested PCR

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with an incidence of approximately 1 in 3500 males, caused by mutation in the DMD gene. About 2/3 of DMD cases are caused by gross DMD gene deletion mutations. The purpose of this study was to develop a series of single-cell multiplex-nested PCR protocols for preimplantation genetic diagnosis (PGD) of the most prevalent DMD deletions. The protocols were developed on single blood leukocytes from normal males and females and patients with known DMD gene deletion. In the first reaction, 2 of 11 different primer sets (exons 4, 8, 12, 13, 17, 46, 47, 49, 50, 52 and intron 52) were used to allow the simultaneous amplification of different DMD loci and the SRY gender marker, in a single triplex-nested polymerase chain reaction (PCR). Aliquots of this reaction were then subjected to nested PCR in which each locus was amplified individually. Following the successful establishment of single-cell triplex-nested PCR in single leukocytes, the technique was employed in five clinical PGD cases. For each DMD locus, more than 50 single leukocytes from healthy controls and more than 100 single leukocytes from affected individuals with known deletions were analyzed. Amplification efficiency for each tested locus was 98-100%. The false-negative rates for each analysis taken separately was <1%. Taken together, however, the results of the triplex-nested PCR analysis had a false-negative rate of 0%. No contamination was detected in all wash-drop blanks tested. We subsequently performed 18 PGD cycles in 5 DMD carriers. A total of 156 embryos were biopsied and successfully analyzed. Of these, 39 affected embryos were detected and 50 unaffected embryos were transferred (mean = 2.9 +/- 1.1 embryos per cycle). These resulted in three biochemical pregnancies and three clinical pregnancies, all of which have culminated in the birth of normal offspring. Triplex-nested PCR using 2 of 11 DMD loci and the SRY gender marker allow PGD for >90% of DMD families with known deletions. These protocols are associated with a high amplification efficiency and accuracy.

Detection of germline mosaicism in two duchenne muscular dystrophy families using polymorphic dinucleotide (CA)n repeat loci within the dystrophin gene

Approximately one-third of new cases of Duchenne muscular dystrophy (DMD) can be attributed to sporadically arising new mutations, however in the majority of cases the DMD mutation has been inherited from the mother. These female carriers can have either a constitutive or mosaic mutation. The aim of this study was to determine the segregation of the at-risk haplotype and to find a deletion in the dystrophin gene of patients. We analyzed individuals from two families with a history of DMD in order to predict the carrier status of related females. In one of these cases the mother had two affected sons, while in the other one son and two grandchildren were affected; therefore we predict that the mother would be an obligatory carrier. Haplotype analysis of the DMD loci revealed that in the two families both the healthy and affected brothers had inherited the same X maternal chromosome. However, the affected brother carried a deletion, which was absent in the unaffected sibling. These findings suggested that the mothers in the two families were germline mosaics for the DMD gene. The results of this study demonstrate the usefulness of the methodology that combine the haplotype analysis with the identification of the mutation in order to detect hidden germline mosaicisms and, thus, improve genetic counseling.

Achievement of successful pregnancy in the preimplantation genetic diagnosis for Duchenne Muscular Dystrophy (DMD), Ornithine Transcarbamylase (OTC) deficiency, and Epidermolysis Bullosa (EB) of single gene disorders

Objective: Preimplantation genetic diagnosis technique is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. We applied PGD to carriers at risk of transmitting Duchenne muscular dystrophy (DMD), ornithine transcarbamylase (OTC) deficiency and epidermolysis bullosa (EB). Design: Analysis of laboratory data and clinical outcome of PGD for single gene disorders in human IVF-ET program. Materials and Methods: Ovarian stimulation, oocyte retrieval, and ICSI procedure was underwent by conventional protocols. Diagnosis for single gene disorders was carried out after biopsy of one or two blastomeres from the embryos on day 3. We performed the duplex nested PCR protocol for the simultaneous amplification of the causative mutation loci as well as the SRY gene on Y chromosome in case of DMD and OTC deficiency. In case of EB, two different loci of ITGB4 gene were amplified by the same protocol. The PCR products were analyzed by direct sequencing or RFLP analysis. Results: Tabled 1 No contamination was detected in all negative controls (n=37). In DMD and OTC deficiency cases, singleton pregnancies were established successfully. They are now in ongoing pregnancy at 14 and 13 weeks of gestation, respectively and amniocentesis are planned within a few weeks. Conclusion: This study shows that single cell duplex nested PCR can be an efficient method in identifying the sex and single gene mutation simultaneously in PGD of DMD, OTC deficiency and EB family members. This technique will allow more flexible options for the selection of embryos regardless of sex in sex-linked recessive disorders and safer diagnosis by excluding female embryos in sex-linked co-dominant disorders.

Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available

In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositaˈs mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.

Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family

Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified...

Molecular analysis of the Duchenne muscular dystrophy gene in Spanish individuals: deletion detection and familial diagnosis.

Deletion studies were performed in 26 Duchenne muscular dystrophy (DMD) patients through amplification of nine different exons by multiplex polymerase chain reaction (PCR). DNA from paraffin-embedded muscle biopsies was analyzed in 12 of the 26 patients studied. Optimization of this technique is of great utility because it enables analysis of material stored in pathology archives. PCR deletion detection, useful in DMD-affected boys, is problematic in determining the carrier state in female relatives. For this reason, to perform familial linkage diagnosis, we made use of a dinucleotide repeat polymorphism (STRP, or short tandem repeat polymorphism) located in intron 49 of the gene. We designed a new pair of primers that enabled the detection of 22 different alleles in relatives in the 14 DMD families studied. The use of this marker allowed familial diagnosis in 11 of the 14 DMD families and detection of de novo deletions in 3 of the probands.

Restoration of half the normal dystrophin sequence in a double‐deletion Duchenne muscular dystrophy family

Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions--one in the 5', proximal deletional hotspot region, and the other in the 3', more distal deletional hotspot region. The second propositus showed only the 5' deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, we show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier's dystrophin genes, giving rise to a son with a partially "repaired" gene retaining only the 5' deletion.

Identification of a point mutation and germinal mosaicism in a duchenne muscular dystrophy family

Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X-linked disorders arising from mutations in the (2.4 Mb) dystrophin gene at Xp21. We have applied the reverse transcriptase-polymerase chain reaction (RT-PCR) to identify a larger than normal dystrophin mRNA from a male with Duchenne muscular dystrophy and his younger affected brother. The increased size of the dystrophin mRNA was due to a splice-site mutation at the exon 26:intron 26 junction where a T to G substitution prevented normal RNA processing. A cryptic splice-site, downstream of the mutation, was activated during processing, resulting in the inclusion of 117 bases of intron 26. This insertion introduced an in-frame stop codon into the mature dystrophin mRNA. An allele-specific test was developed to identify the mutation and was applied to this family. Interestingly, the mother of the two affected boys did not carry the mutation, as determined by allele-specific amplification and direct DNA sequence analysis, indicating gonadal mosaicism. Her eldest daughter, designated as a carrier based upon conventional testing and haplotype analysis, also did not carry the family mutation. Initial haplotyping of the family appeared to be straightforward with gonadal mosaicism becoming evident only after allele-specific analysis. The application of linked markers to identify the disease allele for conventional genetic counselling would have been erroneous in this family and highlights the diagnostic power of precise identification of the disease-causing mutation.

Dystrophin gene analysis and prenatal diagnosis of Duchenne muscular dystrophy in Russia

Of 126 families referred for counselling of Duchenne muscular dystrophy (DMD), DNA analysis has been suggested to 119 families with at least one affected child or with an affected close male relative of the woman at risk of being a DMD carrier. A large proportion (about 80 per cent) of the families were represented by sporadic cases (only one affected individual). By means of multiplex polymerase chain reactions with different sets of oligoprimers providing amplification of 10-11 different exons, altogether 49 dystrophin gene deletions were identified (41 per cent). Eighteen deletions clustered in the 5' 'hot spot' region of DMD cDNA and 36 in the distal half of the central rod domain around exons 43-53. An unusually high frequency (18 per cent) of deletions involving exons 17-19 was discovered. Large deletions extending through both 'hot spot' regions and thus occupying over 30-40 exons were recorded in five cases (10 per cent). Seventy-six of 94 families were found to be informative by RFLP analysis for intragenic or extragenetic DNA probes. Carrier status was ascertained in 20 and rejected in 32 female relatives in 40 DMD families. Eight DMD-affected fetuses were diagnosed prenatally by direct deletion testing or by RFLP analysis. Feasible interpopulation variations in the dystrophin gene deletion pattern are discussed. The prospects for more effective prenatal diagnosis and carrier detection in high-risk DMD families in Russia are briefly outlined.

Multiple mutation in an extended Duchenne muscular dystrophy family.

We have investigated an extended pedigree with three cousins affected by Duchenne muscular dystrophy with apparent transmission through the male line. However, molecular studies have shown that one boy has a de novo duplication, another has a deletion, and the molecular mutation has yet to be defined in the third boy. All three X chromosomes in the affected boys appear to have a different origin. We speculate on the mechanisms by which the Duchenne locus may be particularly prone to mutation in this family and the possible involvement of transposons is discussed. Whatever the mechanism involved, the occurrence of three different mutations in one pedigree is a rare event.

CULTURAL DIFFERENCES IN FAMILY COMMUNICATION ABOUT DUCHENNE MUSCULAR DYSTROPHY

This study compared the patterns of communication and use of professional support systems of Irish and American families with sons with Duchenne muscular dystrophy (DMD). Structured interviews were used with the parents of 34 Irish boys and 21 American boys attending the muscular dystrophy services of Central Remedial Clinic, Dublin, and Children's Hospital, Boston. A matched control group was interviewed in each culture to compare use of professional support systems. Difficulties in communicating about DMD with their spouses and with their affected sons were reported by significantly more Irish parents than by their American counterparts. More Irish parents had never spoken about DMD with their affected sons. American DMD families reported significantly more contact with professional support systems than did the Irish families. Factors which may contribute to these cultural differences are discussed.

Carrier estimations in Duchenne muscular dystrophy families in Northern Ireland using RFLP analysis.

Intragenic RFLP analysis was used to provide carrier risk estimations on 100 possible female carriers from 22 Duchenne muscular dystrophy families. This enabled 78% of possible carriers to be assigned high or low risks (greater than 90% or less than 10%) as opposed to 26% assigned low risk on pedigree data alone. When a single polymorphism is not informative the use of haplotype analysis for carrier estimations is illustrated for one family.

Deletion screening and prenatal diagnosis of Duchenne muscular dystrophy using cDNA probes Cf 23a and Cf 56a

We have screened patients of 14 families at risk for Duchenne muscular dystrophy (DMD) from the northern part of the German Democratic Republic using the cDNA clones Cf 23a and CF 56a. Of the 14 unrelated DMD families, 7 (50%) showed different deletions with these cDNA probes. A prenatal diagnosis by chorionic villi sampling was performed in a DMD family with patients showing a deletion of the 5.4 kb Pst I band detected by the cDNA probe Cf 56a. This band corresponds to a 10 kb exon region of the cDNA probe 8 of Koenig et al. The patient's mother was informative only for the flanking marker 99.6. The male fetus showed the same haplotype and the same deletion as the two patients.

Molecular-genetic study of Duchenne and Becker muscular dystrophies: deletion analyses of 45 Japanese patients and segregation analyses in their families with RFLPs based on the data from normal Japanese females.

This study consisted of 1) molecular deletion analyses in patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) using the entire cDNA for the DMD gene as hybridization probes, 2) RFLP analyses in a large number of Japanese normal women using 11 DMD-linked cloned DNAs as probes, and 3) segregation analyses with these RFLP data in 17 DMD families in which prenatal or carrier diagnosis was required. The deletion study showed that 18 (43%) of 42 male DMD patients had a deletion within the DMD gene, while no detectable deletion was found in 3 BMD patients. These deletions were preferentially observed at the 5' end of the DMD gene, while no deletion was found in the 3' portion of the gene. Of a total of 15 RFLPs detected with the 11 probes, one was a new RFLP (probe/enzyme: P20/MspI). In 6 RFLPs, the allele frequencies in the Japanese were statistically different from those in the Caucasian. Based on the RFLP data combined with the result of the deletion study, an estimated diagnostic rate for prenatal diagnosis and/or carrier detection in the Japanese DMD families was 63%. The real diagnostic rate obtained from the prenatal and carrier diagnoses, which were practically performed in 17 families, corresponded to the estimation. A protocol useful for the diagnosis in Japanese DMD families is presented.

Duchenne muscular dystrophy in Wales: a 15 year study, 1971 to 1986.

A register of families with Duchenne muscular dystrophy (DMD) in Wales was set up in 1973 and has been regularly maintained ever since. All women at significant risk in these families were offered estimation of their carrier status by creatine kinase and pedigree analysis. A total of 225 of the 512 women tested was assigned a risk of carrying the DMD gene of less than 5%. One hundred and twenty live births from this group were notified to the register and only one was an affected male. This was the expected number and shows that the risks given were largely accurate. Women given a risk of 5% or greater were offered fetal sexing with termination of any male. Amniocentesis was of limited value and chorionic villus sampling, introduced in 1984, was acceptable to more women at risk. The incidence of DMD in Wales dropped from one in 3435 at the beginning of the study to one in 4046 by 1982, and the proportion of recurrent cases from 40% to 22%. Common reasons for recurrence were birth of a second affected boy before diagnosis of the first, and failure of families with affected dead members in previous generations to be notified to the register. Maintaining an accurate register of DMD families is an essential tool in their management.