Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi children

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene (DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe. In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion regions (HFDR’s) in the DMD gene mutations in KSA.     

Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies

Comprehensive molecular testing for mutations in the DMD gene causing Duchenne and Becker muscular dystrophy (DMD/BMD) is challenging because of the large size of the gene and the variety of mutation types. There is an increasing demand for comprehensive DMD gene molecular testing, including deletion/duplication testing of 79 exons and direct sequencing of the 14-kb coding region from genomic DNA, to provide confirmation of clinical diagnoses in affected patients and to determine carrier risk for family members. To determine an efficient strategy to prioritize patients for comprehensive molecular testing of the DMD gene, we tested a consecutive cohort of 165 males referred over a 4-year period because of a suspicion of DMD or BMD using: (1) a new quantitative multiplex polymerase chain reaction (PCR) assay designed to detect deletions or duplications in all exons of the gene and the brain promoter and (2) direct sequencing of the coding region and intron/exon boundaries. For the patients being tested because of a suspicion of DMD, deletion/duplication testing followed by direct sequencing detected pathogenic mutations in 98% (106/108 total patients). However, of the patients tested because of a suspicion of BMD, only 60% (34/57 total patients) had causative mutations identified, all of which were deletions or duplications. Our results suggest that direct genomic sequence analysis of the DMD gene is a useful addition to deletion/duplication testing for diagnosis of DMD, but does not provide an improved sensitivity compared to deletion/duplication analysis alone for the diagnosis of BMD. In addition, due to the relatively common finding of single exon deletions and duplications (22%, 27 of 125 total patients with deletions/duplications), methods to examine all exons of the gene for deletions/duplications should be used as the initial molecular quantitative test for DMD and BMD.     

Molecular deletion patterns in families from southern France with Duchenne/Becker muscular dystrophies

Summary We studied 38 unrelated patients from southern France with Duchenne (DMD) or Decker (BMD) muscular dystrophy for intragenic deletions of the DMD/ BMD gene. We used both multiplex amplification of selected exons and cDNA probes. Of the 26 (68%) unrelated individuals found to have deletions, 24 (92%) were detected by multiplex polymerase chain reaction. All these deletions have been delineated with regard to the exon-containing HindIII fragments revealed by cDNA probes, and in two cases, junction fragments of altered size were seen. The correlation between phenotype and type of deletion agreed with the reading frame theory, except for two BMD and two DMD cases.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.     

Detection of gene deletions in Chinese patients with Duchenne/Becker muscular dystrophy using CDNA probes and the polymerase chain reaction method.

One hundred thirty-eight patients with Duchenne/Becker muscular dystrophy (DMD/BMD) were screened with complete cDNA probes and the multiplex polymerase chain reaction (mPCR) amplification of 18 pairs of oligonucleotide primers. Eighty-six deletions and 4 duplications were detected, the deletion frequency being 62.3%. Eighty-two deletions were detected with the two sets of primers described by Chamberlain et al. and Beggs et al, which was 95.4% of deletions detected by complete cDNA probes. Consistent with the deletion locations described previously, the deletions of dystrophin gene in Chinese individuals are clustered mainly in two high-frequency deletion regions of exons 44-52 (68.6%) of 3' side of the gene central regions and exons 1-19 (26.7%) in the 5' side. The distribution of deletions in dystrophin gene is associated with the phenotype of DMD/BMD. In the 25 cases with in-frame deletions, 15 deletions located in the region of exons 2-47 were milder BMD and intermediate patients, as the location of deletions was not the important region of the dystrophin gene.     

Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction

Summary We describe oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR). When used in conjunction with an existing primer set, these two multiplex reactions detect about 98% of deletions in patients with Duchenne or Becker muscular dystrophy (DMD, BMD). Furthermore, these primers amplify most of the exons in the deletion prone hot spot region around exons 44 to 53, allowing determination of deletion endpoints and prediction of mutational effects on the translational reading frame. Thus, use of these PCR-based assays will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus

Summary A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both×chromosomes, whereas only one×chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.     

Patterns of exon deletions in Duchenne and Becker muscular dystrophy

Summary A panel of patients with Duchenne and Becker muscular dystrophy (DMD and BMD) has been screened with the cDNA probes Cf56a and Cf23a, which detect exons in the central part of the DMD gene. One or more exons were deleted in 60% of patients. The deletions were mapped and prove to be heterogeneous in size and extent, particularly in DMD. Deletions specific to DMD and to BMD are described. Half of all BMD patients have a deletion of one particular small group of exons; smaller deletions within this same group produce the more severe DMD.     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy.

We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.     

Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1–2 (n = 1) or exons 5–7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5′ and/or 3′ breakpoints located within Alu repeat elements. In addition, we identified the 5′ breakpoint of a previously reported deletion of exons 1–2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.     

Genotype-phenotype correlation and germline mosaicism in DMD/BMD patients with deletions of the dystrophin gene

Summary The molecular analysis of 127 DMD/BMD patients showed that 73 of them (57%) had deletions in the dystrophin gene. Two different methods were used in this study: (a) hybridization of HindIII-digested genomic DNA with nine cDNA probes corresponding to the entire 14 kb cDNA of the DMD gene; and (b) simultaneous amplification of nine exons of the DMD gene (multiplex DNA amplification) by the polymerase chain reaction (PCR). When the deletion breakpoints of the intragenic deletions were analyzed with regard to their phenotypic consequences, nine patients were found to represent exceptions to the reading-frame hypothesis. Information regarding mental development was also available for 61 of the 73 deleted patients and for 34 of the 54 non-deleted ones. The proportion of mentally retarded patients was found to be similar in the two groups (deleted, 15%; non-deleted, 18%). Finally, in one family, a junction fragment present in the patient was not found in the peripheral blood DNA of the mother but was present in the sister, thus indicating germline mosaicism in the mother.     

Analysis of deletion mutations of the dystrophin gene by the multiplex polymerase chain reaction method in the diagnosis of Duchenne muscular dystrophy]

The 33 patients suffering from the Duchenne muscular dystrophy (DMD), 7 healthy donors and a DMD risk family were studied by means of polymerase multiplex chain reaction (MPCR) with 6 oligoprimer pairs for 6 different exons of dystrophin gene. The deletions varying in sizes from 1 to 6 exons were detected in 12 out of 33 DMD patients studied (36.3%). The prenatal diagnosis of DMD was carried out by chorionic villus biopsy on the 1st trimester of pregnancy. Contrary to earlier findings, in elder brother with sever DMD manifestation, no visible deletion was detected in the DNA sample from the male foetus and thus the diagnosis of DMD in foetus was rejected. The perspectives of MPCR in pre and postnatal diagnosis of DMD are discussed.     

Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method

Mutation detection in the DMD gene defective in Duchenne (DMD) and Becker muscular dystrophies (BMD) is complicated by the presence of 79 exons. The majority of recognized mutations are, however, copy number changes of individual exons, which traditionally have been identified by three common multiplex polymerase chain reaction (PCR) assays and/or Southern blotting. Here we report the use of the newly developed quantitative assay multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of each of the 79 DMD exons in 182 males and 14 carrier females referred to our diagnostic facility on the clinical suspicion of DMD or BMD. The MLPA method confirmed all previously recognized mutations and identified an additional 28, including four point mutations. Also, the assay reliably identified 7 carrier females, which are usually not easily recognized. In our hands the method is highly reproducible, easy to handle, and has increased our mutation pick-up rate by a total of 33%.     

Analysis of deletions in the dystrophin gene in patients with Duchenne muscular dystrophy in the Bashkir Republic]

The deletion spectrum and distribution of deletion breakpoints (DBs) in 36 patients with Duchenne muscular dystrophy (DMD) from 33 families and in three patients with Becker muscular dystrophy (BMD) from one family from Bashkortostan were studied by amplifying 20 exons of the dystrophin gene by multiplex polymerase chain reaction (mPCR). Eight out of 34 unrelated DMD (BMD) patients (23.2%) were shown to carry a deletion varying in size from one to seven exons. Most DBs (15 out of 16, 93.7%) were in the distal region of the gene, commonly between exons 44-45, 45-47, and 50-52. Thus, high-polymorphic intergenic markers located in introns 44 (STR 44), 45 (STR 45), 49 (STR 49), and 50 (STR 50) can be used for indirect or direct carrier detection among women closely related to DMD patients that carry a deletion with DB located between exons 44-45, 45-47, and 50-52. Prenatal diagnosis of DMD is also possible in these families.     

Screening for DMD/BMD deletion carriers by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) serves as an excellent alternative for direct detection of heterozygous deletions. Using a set of exon-specific cosmid DNA probes representing 18 exons, one-color FISH on metaphase and interphase preparations was performed to identify Duchenne/Becker muscular dystrophy (DMD/BMD) deletion carriers. The peripheral blood samples from 9 normal male or female controls and 5 females of independent DMD/BMD families, as well as 2 amniotic fluid specimens and 2 chorionic villus samples (CVS) from normal pregnant females, were analyzed. Expected signals were displayed in 72-100% of peripheral blood lymphocyte metaphases or interphases, 60-70% of amniocyte interphases, and 95-99% of chorionic villus cell interphases. One suspected female was identified as a deletion carrier and two were excluded. The results indicated that metaphase and interphase FISH were both useful for detection of heterozygous deletions. FISH, in combination with other available techniques, allowed efficient screening of DMD/BMD deletion carriers. The study also offered preliminary results in support of an approach to prenatal diagnosis of potential fetal carriers.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.