Deletions In Duchenne Muscular Dystrophy Gene Research Articles

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

BackgroundThe phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene.MethodologyGenetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data.ResultsIn the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45–55 and 6–15. Exon 45–60 identified as a novel in-frame variation hotspot. Exon 45–59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations.ConclusionDMD gene deletions and duplications are concentrated in exons 45–55 and 2–20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

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Disrupted structural connectome and neurocognitive functions in Duchenne muscular dystrophy: classifying and subtyping based on Dp140 dystrophin isoform.

Neurocognitive disabilities in Duchenne muscular dystrophy (DMD) children beginning in early childhood and distal DMD gene deletions involving disruption of Dp140 isoform are more likely to manifest significant neurocognitive impairments. MRI data analysis techniques like brain-network metrics can provide information on microstructural integrity and underlying pathophysiology. A prospective study on 95 participants [DMD = 57, and healthy controls (HC) = 38]. The muscular dystrophy functional rating scale (MDFRS) scores, neuropsychology batteries, and multiplex ligand-dependent probe amplification (MLPA) testing were used for clinical assessment, IQ estimation, and genotypic classification. Diffusion MRI and network-based statistics were used to analyze structural connectomes at various levels and correlate with clinical markers. Motor and executive sub-networks were extracted and analyzed. Out of 57 DMD children, 23 belong to Dp140 + and 34 to Dp140- subgroup. Motor disabilities are pronounced in Dp140- subgroup as reflected by lower MDFRS scores. IQ parameters are significantly low in all-DMD cases; however, the Dp140- has specifically lowest scores. Significant differences were observed in global efficiency, transitivity, and characteristic path length between HC and DMD. Subgroup analysis demonstrates that the significance is mainly driven by participants with Dp140- than Dp140 + isoform. Finally, a random forest classifier model illustrated an accuracy of 79% between HC and DMD and 90% between DMD- subgroups. Current findings demonstrate structural network-based characterization of abnormalities in DMD, especially prominent in Dp140-. Our observations suggest that participants with Dp140 + have relatively intact connectivity while Dp140- show widespread connectivity alterations at global, nodal, and edge levels. This study provides valuable insights supporting the genotype-phenotype correlation of brain-behavior involvement in DMD children.

Duchenne Muscular Dystrophy: Genetic and Clinical Profile in the Population of Rajasthan, India.

Background:Duchenne Muscular Dystrophy (DMD) is an X-linked recessive muscular dystrophy that affects young boys and is caused by mutation of the dystrophin gene located over X chromosome.Materials and Methods:In this prospective study, 120 clinically diagnosed DMD patients were tested for exon deletions, duplication or point mutation.Results:Of the 120 clinically suspected DMD patients, the diagnosis of DMD was confirmed by the genetic study or muscle biopsy in 116 patients. The mean age of onset was 3.2 years and the mean age at presentation was 7.2 years. 110/120 cases were confirmed by genetic testing and six were by absence of staining for dystrophin on muscle biopsy. DMD gene deletion was present in 78.5%, duplication in 5.3% and point mutation in 11.2% cases. 70.3% of patients had deletion located at a distal hot spot region. Single exon deletion was found in 16.5%. Distal hotspot exons 47, 48 and 50 were the commonly deleted exons.Conclusions:In our study, 94.8% cases showed genetic change in the DMD gene. Muscle biopsy was the choice of investigation in earlier days. Detection of DMD by DNA based method eliminates the need to do an invasive procedure for diagnosis. Hence the genetic testing should be the investigation of choice in suspected cases of DMD. The pattern of deletion, obtained in the population of Rajasthan was similar when compared with other ethnic groups of the Indian population. It would be helpful for researchers to develop drugs specific to exons or for ongoing mutation-specific therapies.

Mutation spectrum of Duchenne muscular dystrophy patients in Indian population

Background: Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disorder in children. Since the novel, DMD therapies are mutation-specific, so detection of mutation is of paramount importance in planning the treatment of DMD patients. Objective: The objective of this study was to find different mutations present in DMD patients in Indian population. Materials and Methods: This study was a hospital-based retrospective observational study conducted from December 2018 to December 2019 in the pediatric department of a tertiary hospital of western India. A total of 52 children age 2–16 years, presenting with progressive muscle weakness, were included in the study. DMD multiplex ligation-dependent probe amplification (MLPA) for 79 exons was done for detection of deletion/duplication for all patients. Whole DMD gene sequencing was done for those who were found MLPA negative for DMD gene mutation (deletion/duplication). Results: Our study states that most of the DMD patients presented with deletions (84%) or duplications (11%) in the dystrophin gene, and remaining due to point mutation. The study shows that most of the mutations occur due to deletions (67.30%) in DMD gene at distal hotspot 45–52 exons and deletions (15.38%) in DMD gene at proximal hotspot 10–19 exons. In addition, to expanding the mutational spectrum of DMD, these results establish improved mutations data in the Indian population. Conclusion: The novel developed therapies for DMD are mutation-specific, so molecular diagnostic tests are very important in diagnosis and categorization for the prevalent mutations in the Indian population.

DMD-related muscular dystrophy in Cameroon: Clinical and genetic profiles.

BackgroundMost of the previous studies on Duchenne Muscular Dystrophy (DMD) were conducted in Caucasian, Asian, and Arab populations. Therefore, little is known about the features of this disease in Africans. In this study, we aimed to determine the clinical characteristics of DMD, and the common mutations associated with this condition in a group of Cameroonian patients.MethodsWe recruited DMD patients and performed a general physical examination on each of them. Multiplex ligand‐dependant probe amplification was carried out to investigate exon deletions and duplications in the DMD gene (OMIM: 300377) of patients and their mothers.ResultsA total of 17 male patients from 14 families were recruited, aged 14 ± 5.1 (8–23) years. The mean age at onset of symptoms was 4.6 ± 1.5 years, and the mean age at diagnosis was 12.1 ± 5.2 years. Proximal muscle weakness was noted in all patients and calf hypertrophy in the large majority of them (88.2%; 15/17). Flexion contractures were particularly frequent on the ankle (85.7%; 12/14). Wasting of shoulder girdle and thigh muscles was present in 50% (6/12) and 46.2% (6/13) of patients, respectively. No patient presented with hearing impairment. Deletions in DMD gene (OMIM: 300377) occurred in 45.5% of patients (5/11), while duplications were observed in 27.3% (3/11). Both mutation types were clustered between exons 45 and 50, and the proportion of de novo mutation was estimated at 18.2% (2/11).ConclusionDespite the first symptoms of DMD occurring in infancy, the diagnosis is frequently made later in adolescence, indicating an underestimation of the number of cases of DMD in Cameroon. Future screening of deletions and duplications in patients from Cameroon should focus on the distal part of the gene.

561. CRISPR-Induced Deletion (CinDel) Method Allows Permanent and Efficient Restoration of the DMD Gene Reading Frame in Duchenne Patient Myoblasts and Preserves Truncated Dystrophin Structure

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne Muscular Dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. Here we present CRISPR-induced deletion (CinDel), a new gene therapy approach to correct the DMD gene in Duchenne patients with one or more exons deletions. By using adequate pair of gRNAs targeting specifically the exons precede and follow the patient deletion in DMD gene, CinDel induces precise DSB in targeted sequences and allows an additional deletion. The remaining parts of the exons were fusioned by NHEJ to form a hybrid exon and restored the DMD reading frame in 62 % of hybrid exons in vitro in patient myoblasts and in vivo in electroporated muscle hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted dystrophin protein was detected following the formation of myotubes by the unselected treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

Detection of Mutation-Hotspots and Exon-Deletions using Digital Signal Processing in Duchenne Muscular Dystrophy (DMD) Gene

Duchenne muscular dystrophy (DMD) is a life threatening disease which mostly occurs due to deletions in the dystrophin gene. The deletions are reported to be clustered in two main regions in the gene involving nearly one fourth of exons. These regions also represent major meiotic recombination hotspots. The detection of deletions/mutation-hotspots in the coding sequences (exons) is not much possible till date due to the gene specific-potential variations or the complex organization of introns and exons in the gene. A Digital signal processing (DSP) method based on antinotch filter that exploits the period-3 property of a DNA sequence is applied for the identification of exons in DNA sequences by providing concerned peaks in magnitude-plot. A two digit numerical representation has been proposed for generating indicator sequence. In our approach to detect deletion in DMD gene, all-exon-sequence was taken and analyzed through digital filter to obtain normal peaks in the plot, which was than compared with the plots obtained after deletion of hotspot exons one by one. It was found that most of the hotspot exons share the peaks and deletions are marked by significant modifications in peaks. Thus, it was known that the peaks in filter-plot of DMD gene are associated with the location of the hotspots and their modifications/absence can be utilized to detect the deletions of exons.

G.P.77 Deletions in the dystrophin gene predict loss of ambulation before 10 years of age in boys with Duchenne muscular dystrophy

There is considerable variation in the rate of disease progression and response to steroid therapy in males with Duchenne muscular dystrophy (DMD). Genetic influences may explain at least part of this heterogeneity. This retrospective case series explored the relationship between dystrophin gene mutations (DMD) and age at loss of ambulation (LOA). Medical histories (n=144) were examined from two Australian neuromuscular clinics. Data were collected on clinical characteristics including DMD mutation type and location, age at LOA, anthropometry, lung function and steroid use. The relationship between age at LOA and genetic and clinical characteristics including age at chart review, duration of steroid treatment, and BMI z-score was explored via multiple regression analysis. Significant independent variables were also entered in a logistic regression to predict LOA <10years of age. The mean (±SD) age of the cohort was 11.9±4.0years, and 51% of boys had lost the ability to walk independently. Mutation type and duration of steroid treatment were significant independent predictors of LOA <10years in a logistic model explaining 31.2% of the variance in age of LOA. Boys with deletions in the dystrophin gene were six times more likely to stop walking before age 10 compared to boys with duplications, point or unknown mutations. This was reflected in the mean age at LOA in boys with deletions versus other mutations (9.30±1.60 vs 10.97±2.03years, respectively, p<0.0005). A longer duration of steroid therapy was associated with a reduction in risk of LOA <10years. BMI had no relationship with age at LOA. Whilst the negative effects of steroid treatment such as weight gain and decreased bone health need to be considered, early introduction of steroids may provide some functional benefits, especially to boys with a documented deletion in DMD gene.

Carrier Woman of Duchenne Muscular Dystrophy Mimicking Inflammatory Myositis

Carrier woman of Duchenne muscular dystrophy (DMD) can mimic the inflammatory myositis in presenting symptoms. Two diseases should be differentiated by the clinical history, muscle biopsy and genetic study. There are few reports in which both histochemical and genetic study showed the possible link of overlapping inflammatory pathophysiology with dystrophinopathy. We report a 40-yr-old woman who presented with subacute proximal muscle weakness and high serum level of creatine kinase. She had a history of Graves' disease and fluctuation of serum liver aminotransferase without definite cause. MRI, EMG and NCV were compatible with proximal muscle myopathy. Muscle biopsy on vastus lateralis showed suspicious perifascicular atrophy and infiltration of mono-macrophage lineage cells complicating the diagnosis. Dystrophin staining showed heterogeneous diverse findings from normal to interrupted mosaic pattern. Multiple ligation probe amplification and X chromosome inactivation test confirmed DMD gene deletion mutation in exon 44 and highly skewed X inactivation.

Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy.

BackgroundA significant component of the variation in cognitive disability that is observed in Duchenne muscular dystrophy (DMD) is known to be under genetic regulation. In this study we report correlations between standardised measures of intelligence and mutational class, mutation size, mutation location and the involvement of dystrophin isoforms.Methods and ResultsSixty two male subjects were recruited as part of a study of the cognitive spectrum in boys with DMD conducted at the Sydney Children's Hospital (SCH). All 62 children received neuropsychological testing from a single clinical psychologist and had a defined dystrophin gene (DMD) mutation; including DMD gene deletions, duplications and DNA point mutations. Full Scale Intelligence Quotients (FSIQ) in unrelated subjects with the same mutation were found to be highly correlated (r = 0.83, p = 0.0008), in contrast to results in previous publications. In 58 cases (94%) it was possible to definitively assign a mutation as affecting one or more dystrophin isoforms. A strong association between the risk of cognitive disability and the involvement of groups of DMD isoforms was found. In particular, improvements in the correlation of FSIQ with mutation location were identified when a new classification system for mutations affecting the Dp140 isoform was implemented.SignificanceThese data represent one of the largest studies of FSIQ and mutational data in DMD patients and is among the first to report on a DMD cohort which has had both comprehensive mutational analysis and FSIQ testing through a single referral centre. The correlation between FSIQ results with the location of the dystrophin gene mutation suggests that the risk of cognitive deficit is a result of the cumulative loss of central nervous system (CNS) expressed dystrophin isoforms, and that correct classification of isoform involvement results in improved estimates of risk.

Evaluation of Multiplex PCR Assay Using Dual Priming Oligonucleotide System for Detection Mutation in the Duchenne Muscular Dystrophy Gene

Exon deletions of Duchenne muscular dystrophy (DMD) gene account for most of the alterations found in DMD and Becker muscular dystrophy (BMD). This study was to evaluate the usefulness of dual priming oligonucleotide multiplex PCR (DPO PCR) in detection of exon deletions of DMD gene. Thirty-seven DMD or BMD patients who had known exon deletions detected by conventional multiplex PCR (conventional PCR) and nine control subjects were enrolled in this study. When a discrepancy was shown between the results of conventional PCR and DPO PCR, the multiplex ligation-dependent probe amplification (MLPA) technique was performed as a confirmation test. The same deletions previously identified by conventional PCR in 32 out of 37 subjects were also detected by DPO PCR. For the five subjects (13.5%) showing discrepant results between the conventional PCR and DPO PCR, MLPA was performed and its results were found to correlate better with those of DPO PCR. The discrepancies were due to false positive or false negative results of the conventional PCR. DPO PCR shows a high agreement of results with the conventional PCR and is considered an adequate method to be used as a primary genetic test for the diagnosis of DMD. Because of an improved accuracy, especially for determining the boundaries of DMD gene deletions, DPO PCR can be very useful as a supplement to the conventional PCR.

Similarity of DMD gene deletion and duplication in the Chinese patients compared to global populations

BackgroundDNA deletion and duplication were determined as the major mutation underlying Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).MethodApplying multiplex ligation-dependent probe amplification (MLPA), we have analyzed 179 unrelated DMD/BMD subjects from northern China.ResultsSeventy-three percent of the subjects were found having a deletion (66.25%) or duplication (6.25%). Exons 51–52 were detected as the most common fragment deleted in single-exon deletion, and the region of exons 45–50 was the most common exons deleted in multi-exon deletions. About 90% of DMD/BMD cases carry a small size deletion that involves 10 exons or less, 26.67% of which carry a single-exon deletion. Most of the smaller deletions resulted in an out-of-frame mutation. The most common exons deleted were determined to be between exon 48 and exon 52, with exon 50 was the model allele. Verifying single-exon deletion, one sample with a deletion of exon 53 that was initially observed from MLPA showed that there was a single base deletion that abolished the ligation site in MLPA. Confirmation of single-exon deletion is recommended to exclude single base deletion or mutation at the MLPA ligation site.ConclusionThe frequency of deletion and duplication in northern China is similar to global ethnic populations.

Multiplex Ligation-dependent Probe Amplification Identification of Deletions and Duplications of the Duchenne Muscular Dystrophy Gene in Taiwanese Subjects

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations in the DMD gene. We intended to determine the distribution of DMD gene deletions and duplications in local Taiwanese male patients and potential female carriers. A total of 102 unrelated subjects, including 89 unrelated DMD/BMD male patients and another 13 unrelated potential female carriers, were recruited for this study. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect DMD gene deletions and duplications in the 102 subjects. MLPA was informative in 60.7% (54/89) of these patients, identifying deletions in 36.0% (32/89) and duplications in 24.7% (22/89) of these patients. This assay revealed deletions in 30.8% (4/13) and duplications in 30.8% (4/13) of the 13 potential carriers. Deletions and duplications were detected in 35.3% (36/102) and 25.5% (26/102) of a total of 102 affected families, respectively in this series. The "hotspot" regions of the duplications were close to those of the deletions. MLPA was proven to be a powerful tool for the detection of DMD gene deletions and duplications in male patients and female carriers. There was a relatively lower frequency of deletion and a higher frequency of duplication of DMD gene in this population compared to previous reports.

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.

DMD-specific FISH probes are diagnostically useful in the detection of female carriers of DMD gene deletions.

The challenge of Duchenne muscular dystrophy (DMD) carrier identification resides in the ability to identify the presence of a mutant gene over the background contributed by the normal allele. Current diagnosis of carrier status when a deletion has been identified in a proband is based on an analysis of a gene dosage. We present a diagnostic strategy that uses fluorescence in situ hybridization (FISH) to detect female carriers with major deletions in the dystrophin gene. We screened a human X-chromosome-derived genomic library with a full-length dystrophin cDNA and isolated 15 dystrophin-specific cosmids that contain DMD gene exons. Six cosmids were further tested as FISH probes in control individuals and subsequently applied on chromosomes from eight males with DMD and known deletions and on samples from three female carriers. As expected, X chromosomes in normal females displayed four signals, two for the DMD-specific probe and two for the X-chromosome centromeric probe. Hybridization on chromosomal spreads from carriers of deletions revealed only one signal from the DMD-specific probe and two from the control centromeric probe. Males carrying deletions showed no DMD-specific signal for the deleted exons tested. Our data indicate that FISH could represent an alternative method for the detection of female carriers with DMD gene deletions.

THE USE OF cDN A PROBES FOR DETECTION DELETIONS IN DNA OF DUCHENNE MUSCULAR DYSTROPHY BOYS

Duchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disease mapped in band Xp21. Since 1983 linkage of the disease to restriction fragment length polymorphism (RLFP) has been applied for genetic counseling. Recently DMD cDNA has been cloned so the possibility of direct detection of DMD gene mutation appears. The purpose of our study was to examine usefulness of cDNA probes for detection of deletions in DNA of polish patients. Eleven DNA samples of DMD boys from 10 families were digested with enzyme Hind III and hybridized to labelled cDNA probes: 1-2a, 5b-7 and 8, supplied by ATCC. In 5 out of total 11 DNA samples partial DMD gene deletions were found: in 2 patients it was located in the middle and in 3 patients on the proximal portion of the gene. Our study suggests the testing all affected boys with cDNA probes for deletions is more cost-effective and faster than RLFP analysis. Besides in 40% of cases it provides an accurate genetic marker for subsequent prenatal diagnosis in the families.