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.     

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.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

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.     

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.     

DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations

Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.     

Regional genomic instability predisposes to complex dystrophin gene rearrangements

Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.     

Screening of Duchenne Muscular Dystrophy (DMD) Mutations and Investigating Its Mutational Mechanism in Chinese Patients

Duchenne muscular dystrophy (DMD) is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2%) and 81 patients with DMD deletions/duplications (del/dup) (68.1%), of which 64 (79.0%) were deletions, 16 (19.8%) were duplications, and one (1.2%) was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot), we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43–55 and exon 10–23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.     

Sarcoglycanopathies: Clinical,Molecular and Genetic Characteristics,Epidemiology, Diagnostics and Treatment Options

Sarcoglycanopathies are a group of autosomal recessive limb-girdle muscular dystrophies (LGMD) caused by mutations in sarcoglycan genes: SGCA (LGMD 2D, MIM 600119), SGCB (LGMD 2E, MIM 604286), SGCG (LGMD 2C, MIM 353700), and SGCD (LGMD 2F, MIM 601287). These genes encode four transmembrane sarcoglycan subunits participating in formation of the large sarcolemmal dystrophin- glycoprotein complex. Clinical symptoms of sarcoglycanopathies resemble the ones in Duchenne/Becker muscular dystrophy and several autosomal recessive LGMD, which causes difficulties in the differential diagnostics between these diseases. This review covers the main aspects of sarcoglycanopathies, such as etiology, spectrum of mutations, clinical features and diagnostics. In addition, we review the fundamental pathogenesis mechanisms leading to sarcoglycanopathies, which can also help to understand the potential options for treatment for patients with muscular dystrophies.     

Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.     

Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0–4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294–304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.     

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.     

Muskeldystrophien Duchenne und Becker

Duchenne muscular dystrophy (DMD) is the most frequent muscular disorder in infancy. The inheritance is X-linked recessive with mutations in the dystrophin gene (about 65% deletions, about 7% duplications, about 26% point mutations, and about 2% unknown mutations). The genetic model is complex. The sex ratio of the mutations is unequal. Point mutations and duplications arise in spermatogenesis, whereas deletions arise in oogenesis. About 33% of all patients are new mutations; however, most new mutations are germline mosaic. Becker muscular dystrophy is allelic to DMD.     

Molecular pathology of Duchenne and Becker muscular dystrophy]

Duchenne and Becker muscular dystrophies (DMD and BMD) are two allelic recessive X-linked disorders. Molecular deletions of various regions of the dystrophin gene are the main mutations detected in DMD and BMD patients. Molecular study of DMD and BMD DNA are instrumental to understand the pathological molecular mechanisms and the function of the protein. We describe here dystrophin and its interaction with a glycoprotein complex and we then focus on two particular patients with partial deletions of the dystrophin gene: 1) a typical Becker patient, who shows an intragenic deletion disrupting the reading frame. We describe in this case alternative splicings restoring the reading frame, which might explain the mild clinical phenotype of this patient, 2) a deletion of the distal part of the DMD gene coding for the carboxyterminal domain of the dystrophin in a young patient. The normal localization of dystrophin at the inner face of the plasma membrane in the muscle of this patient suggests that the last domain of this protein is not sufficient to anchor dystrophin at the membrane.     

Intragenic deletion patterns of dystrophin gene in Duchenne and Becker muscular dystrophy patients from Algeria

Duchenne and Becker muscular dystrophies (DMD and BMD) represent the most frequent neuromuscular diseases in humans (1/3,500–6,000 live male births), characterized by an X-linked recessive pattern of inheritance and therefore affecting mainly male individuals. DMD and BMD are allelic disorders resulting from genetic defects, mostly intragenic deletions, in the dystrophin gene. Using multiplex polymerase chain reaction (PCR), we have analyzed 170 male patients from unrelated families originating from Algeria, showing that 68 % of them harbored deletion events affecting the known 5′ or 3′ hot spot regions. The distal portion was predominantly involved (85 %), whereas 37 distinctive patterns of deletion were identified in our panel. The extent of deletion varied from 1 to 32 exons, although the average number was about four exons. The lack of seven exons (45, 46, 47, 48, 50, 51 and 52), each alone or in combination, represented about 78 % of the alterations encountered, while exon 48 was most frequently involved (50 %). The effect of the deletions showed that the reading frame rule proved mostly true, correlating with the clinical diagnosis suggested. Moreover, the c.525delT mutation in the γ-sarcoglycan gene was present in non-deleted patients (7 %), suggesting that clinical features can still be misleading. Finally, multiplex PCR proved to be a simple, fast and low-cost approach for the molecular diagnosis of dystrophinopathies in Algeria, whereas our data could contribute to the creation of a national registry of DMD/BMD patients in our country, which would give them hope to an access to already available genotype-based therapies.     

Is the Mutation Rate Lower in Genomic Regions of Stronger Selective Constraints?

A study of the plant Arabidopsis thaliana detected lower mutation rates in genomic regions where mutations are more likely to be deleterious, challenging the principle that mutagenesis is blind to its consequence. To examine the generality of this finding, we analyze large mutational data from baker''s yeast and humans. The yeast data do not exhibit this trend, whereas the human data show an opposite trend that disappears upon the control of potential confounders. We find that the Arabidopsis study identified substantially more mutations than reported in the original data-generating studies and expected from Arabidopsis'' mutation rate. These extra mutations are enriched in polynucleotide tracts and have relatively low sequencing qualities so are likely sequencing errors. Furthermore, the polynucleotide “mutations” can produce the purported mutational trend in Arabidopsis. Together, our results do not support lower mutagenesis of genomic regions of stronger selective constraints in the plant, fungal, and animal models examined.     

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.     

Large BRCA1 and BRCA2 genomic rearrangements in Polish high-risk breast and ovarian cancer families

BRCA1 and BRCA2 are two major genes associated with familial breast and ovarian cancer susceptibility. In Poland standard BRCA gene test is usually limited to Polish founder BRCA1 mutations: 5382insC, C61G and 4153delA. To date, just a few single large genomic rearrangements (LGRs) of BRCA1 gene have been reported in Poland. Here we report the first comprehensive analysis of large mutations in BRCA1 and BRCA2 genes in this country. We screened LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification in 200 unrelated patients with strong family history of breast/ovarian cancers and negative for BRCA1 Polish founder mutations. We identified three different LGRs in BRCA1 gene: exons 13-19 deletion, exon 17 deletion and exon 22 deletion. No LGR was detected in BRCA2 genes. Overall, large rearrangements accounted for 3.7 % of all BRCA1 mutation positive families in our population and 1.5 % in high-risk families negative for Polish founder mutation.     

KIT Proto-oncogene Exon 8 Deletions at Codon 419 are Highly Frequent in Acute Myeloid Leukaemia with Inv(16) in Indian Population

The KIT gene is a receptor tyrosine kinase class III expressed by early hematopoietic progenitor cells and plays a significant role in hematopoietic stem cell proliferation, differentiation and survival which is considered to be a remarkable feature in the course of growth of acute myeloid leukaemia (AML). Owing to insufficient study of mutations in the KIT gene, the diagnosis and rate of recurrence of these mutations with divergent subtypes in AML cases in India is of concern. In order to find out the frequency of mutations of KIT gene exon 8 in 109 AML cases, we have performed polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) followed by DNA sequencing and have identified 24 mutations in exon 8 in 13 cases, including deletions at codon 418 (n = 3), 419 (n = 11) and 420 (n = 5) as well as point mutations at codon 417 (n = 1) and 421 (n = 4). In eleven AML cases, exon 8 deletion and point mutations involved the loss at codon Asp419 immoderately conserved cross species placed in the receptor extracellular domain. Frequency elevation of the KIT proto-oncogene exon 8 deletion and point mutations in AML cases allude a crucial function for this region of the receptor extracellular domain. Thus, we report the incidence of acquired mutations in exon 8, with consistent loss at codon Asp419, in 10.09 % of AML cases in a selected Indian population.     

The emerging family of dystrophin-related proteins

Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.