Single Exon Deletions Research Articles

VarGenius-HZD Allows Accurate Detection of Rare Homozygous or Hemizygous Deletions in Targeted Sequencing Leveraging Breadth of Coverage.

Homozygous deletions (HDs) may be the cause of rare diseases and cancer, and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking. We present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness, we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms that each missed at least one event. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis. We provide VarGenius-HZD either stand-alone or integrated within our recently developed software, enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes.

MYO5B-associated diseases: Novel liver-related variants and genotype-phenotype correlation.

Biallelic pathogenic variants in MYO5B cause microvillus inclusion disease (MVID), or familial intrahepatic cholestasis (FIC). The reported FIC patients are scarce and so the genotype-phenotype correlation has not been fully characterised. This study aimed to report more MYO5B-associated FIC patients and correlate genotypes to phenotypes in more detail. The phenotype and genetic data of 12 newly diagnosed MYO5B-associated (including 11 FIC) patients, as well as 118 previously reported patients with available genotypes, were summarised. Only patients with biallelic MYO5B variants were enrolled. Nonsense, frameshift, canonical splice sites, initiation codon loss, and single exon or multiexon deletion were defined as null MYO5B variants. Phenotypically, 50 were isolated MVID, 47 involved both liver and intestine (combined), and 33 were isolated FIC (9 persistent, 15 recurrent, 3 transient, and 6 un-sub-classified) patients. The severity of intestinal manifestation was positively correlated to an increased number of null variants (ρ=0.299, P=.001). All FIC patients carried at least one non-null variant, and the severity of cholestasis was correlated to the presence of a null variant (ρ=0.420, P=.029). The proportion of FIC patients (16/29, 55%) harbouring missense/in-frame variants affecting the non-motor regions of MYO5B was significantly higher than that of MVID (3/25, 12%, P=.001) and combined patients (3/31, 10%, P=.000). 10 of the 29 FIC patients harboured missense/in-frame variants at the IQ motifs comparing to none in the 56 MVID and combined patients (P=.000). The phenotype of MYO5B deficiency was associated with MYO5B genotypes, the nullity or the domain affected.

Long-term effect of conventional phosphate and calcitriol treatment on metabolic recovery and catch-up growth in children with PHEX mutation.

Hereditary hypophosphatemic rickets (HR) is conventionally treated with phosphate and calcitriol. Exploring genotype and phenotypic spectrum of X-linked hypophosphatemic rickets (XLHR), focusing on short-term, long-term, and pubertal impact of conventional treatment was aimed. Sixteen patients from 12 unrelated families with HR were analyzed for phosphate regulating endopeptidase homolog X-linked (PHEX) mutation. Initially Sanger sequencing analysis was performed. If PHEX mutation was not detected, multiplex ligation-dependent probe amplification (MLPA) was performed. If molecular defect was detected, first-degree relatives were analyzed. Thirteen patients (81%) and five first-degree relatives with XLHR were evaluated for genotype-phenotype or gender-phenotype correlation. Clinical characteristics and response to conventional treatment were determined retrospectively. Nine different PHEX mutations were identified; four splice-site, three point mutations, and two single exon deletions. Four were novel mutations. Despite conventional treatment, median adult height was lower than median height on admission (-3.8 and-2.3 SDS, respectively), metabolic and radiographic recovery were not achieved, adherence was low (30%). Although mean adult height was better in compliant patients than noncompliants (-2.6 vs.-3.7 SDS, respectively), they were still short. Correlation between phenotype and genotype or gender could not be shown. Median phosphate decreased significantly throughout puberty (p=0.014). Median pubertal height was lower than prepubertal height (-4.4 vs.-3.6 SDS; respectively), pubertal growth spurt was not observed. Among five patients with a follow-up longer than five years, three had nephrocalcinosis (60%), two had hyperparathyroidism (40%), 4/6 (33%) required correction osteotomy. Conventional treatment appears to have limited effect on metabolic, clinical and radiographic recovery in XLHR. Metabolic control and growth worsened during puberty. Although, long-term adverse effects are yet to be seen, introduction of burosumab as first-line treatment may be an alternative after infancy.

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.

Two new RHD alleles with deletions spanning multiple exons.

The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5' Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5' Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Morpholino Oligomer-Induced Dystrophin Isoforms to Map the Functional Domains in the Dystrophin Protein

Dystrophin plays a crucial role in maintaining sarcolemma stability during muscle contractions, and mutations that prevent the expression of a functional protein cause Duchenne muscular dystrophy (DMD). Antisense oligonucleotide-mediated manipulation of pre-messenger RNA splicing to bypass Duchenne-causing mutations and restore functional dystrophin expression has entered the clinic for the most common DMD mutations. The rationale of “exon skipping” is based upon genotype-phenotype correlations observed in Becker muscular dystrophy, a milder allelic disorder generally characterized by in-frame deletions and internally truncated but semi-functional dystrophin isoforms. However, there is a lack of genotype-phenotype correlations downstream of DMD exon 55, as deletions in this region are rare and most single exon deletions would disrupt the reading frame. Consequently, the amenability of mutations in this region of the DMD gene to exon skipping strategies remains unknown. Here, we induced “Becker muscular dystrophy-like” in-frame dystrophin isoforms in vivo by intraperitoneal injection of peptide-conjugated phosphorodiamidate morpholino oligomers targeting selected exons. The dystrophin isoform encoded by the transcript lacking exons 56+57 appears to be more functional than that encoded by the 58+59-deleted transcript, as determined by higher dystrophin expression, stabilized β-dystroglycan, and less severe dystrophic pathology, indicating some potential for the strategy to address Duchenne-causing mutations affecting these exons.

Alternative mRNA splicing can attenuate the pathogenicity of presumed loss-of-function variants in BRCA2

PurposeCurrent interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants in the canonical splice site sequences ofBRCA2, to be associated with high cancer risk. However, some variant alleles produce alternative transcripts that encode (partially) functional protein isoforms leading to possible incorrect risk estimations. For accurate classification of variants it is therefore essential that alternative transcripts are identified and functionally characterized. MethodsWe systematically evaluated a large panel of human BRCA2 variants for the production of alternative transcripts and assessed their capacity to exert BRCA2 protein functionality. Evaluated variants included all single-exon deletions, various multiple-exon deletions, intronic variants at the canonical splice donor and acceptor sequences, and variants that previously have been shown to affect messenger RNA (mRNA) splicing in carriers. ResultsMultiple alternative transcripts encoding (partially) functional protein isoforms were identified (e.g., ∆[E4–E7], ∆[E6–E7], ∆E[6q39_E8], ∆[E10], ∆[E12], ∆E[12–14]). Expression of these transcripts did attenuate the impact of predicted LoF variants such as the canonical splice site variants c.631+2T>G, c.517-2A>G, c.6842-2A>G, c.6937+1G>A, and nonsense variants c.491T>A, c.581G>A, and c.6901G>T. ConclusionThese results allow refinement of variant interpretation guidelines for BRCA2 by providing insight into the functional consequences of naturally occurring and variant-related alternative splicing events.

Molecular Analysis of Algerian Patients with Duchenne and Becker Muscular Dystrophy

Duchenne and Becker muscular dystrophy (DMD/BMD) are the most com-mon neuromuscular diseases caused by mutations in the dystrophin gene also called (DMD gene), located at Xp21. We report the clinical and genetic analysis of 74Algerian DMD/BMD patients from 62unrelated families who attended the neuromuscular unit of the University Hospital Center of Con-stantine between 2014 and 2017. After informed consent, multiplex poly-merase chain reaction (mPCR)was established to identify deletions. All pa-tients presented a classical phenotype dominated by a bilateral and symmetrical motor deficit predominantly proximal with calf hypertrophy in 91.6% of patients, with very high serum CK levels. Molecular analysis of the DMD gene showed, large deletions at all patients. Most of the deletions were between exons 44 and 53, the most frequent were deletions of exons 45-48, with ex-on 45 as the most common single exon deletion. Our study had generated considerable data on deletions that may promote future experimental thera-pies in Algeria.

P338Dystrophin deficient cardiomyopathy: predictors associated with the cardiac phenotype in a Duchenne registry population - A guide for device therapy

Abstract Background Patients with Duchenne muscular dystrophy (DMD) typically exhibit cardiac dysfunction. With increasing life expectancy due to advances in respiratory support, cardiomyopathy and associated dsyrhythmia are fast becoming the primary cause of morbidity and mortality in this patient group. Despite advances, the correlation between genotype and cardiac phenotype remains poorly understood and individual registries small, with implementation of device therapy often delayed due to poor diagnostic imaging. Methods and Aims: A single-center registry for DMD patients was established and data including genotyping, medical therapy and investigations such as cardiac MRI, nt-Pro BNP levels, echocardiogram were analysed. The aim was to potentially identify predictors associated with a more severe cardiac phenotype and thus help to guide provision of device therapy. Results A total of 19 patients (age 17 - 31) with DMD were reviewed (demographics table 1). All patients were evaluated with echocardiography on at least one occasion (mean EF 46.5%). Cardiac MRI was attempted in 6 patients however only completed in 2 due to contractures preventing access to scanning. 11 of 19 patients (58%) demonstrated an impaired left ventricular ejection fraction (EF) <50% (mean EF 42%). Proximal "hot spot" mutations/deletions (exon 2-19) were associated with a more pronounced reduction in EF, all patients with proximal mutations demonstrating an EF < 45% (mean 41%). 5 of 6 (83%) of those with >1 exon deletion mutations demonstrated more severely impaired EF (mean EF 41%) compared to those with single exon deletions (mean EF 52%). Interestingly, one patient with a proximal mutation (exon 3-6 deletion) remains mobilising to distances of 70 meters, however cardiac MRI revealed a moderate degree of fibrosis with an EF of 43%. Correlation between nt-Pro BNP levels with reduced EF was not uniform, however a level < 100 was associated with an EF >55% in 89% of patients studied. of those on steroid regimes, 6 (54%) had impaired LVEF compared to 5 (83%) of those not on steroid therapy. All patients were on at least one class of heart failure modification, with 79% on two and 37% on three agents. Only one patient in the registry has had an ICD implanted. He has had a device for 10 years in and in this time there have been no therapies delivered however runs of non-sustained ventricular tachycardia have been noted. Conclusions Correlation between predictors and cardiac phenotype in a Duchenne population remains unreliable. Location and size of exon alteration appears to be indicative of more markedly impaired LV function, however larger studies are required to characterise this further and challenges remain with regard to accurate assessment of EF. The use of predictors in future may help to guide appropriate provision of device therapy.

Molecular analysis among a group of Egyptian Duchenne muscular dystrophy patients using real-time PCR

<br><b>Background</b><br>Duchenne muscular dystrophy (DMD) is a neuromuscular disease of children caused by dystrophin protein deficiency encoded by dystrophin gene that is localized to the short arm of the X chromosome, position 21.1q. dystrophin gene mutation results in this disorder. Dystrophin is necessary for keeping the integrity of both skeletal and smooth muscles.<br><b>Aim</b><br>Identify mutations in the group of Egyptian DMD patients, namely exons 48, 49, and 51, and to create a genotype–phenotype correlation from analyzing these patients clinically as well as genetically.<br><b>Patients and methods</b><br>The study included 50 children, 25 DMD patients selected from the Neuropediatric Outpatient Clinic, Abo El Reesh Hospital, Cairo University, selected based on clinical, biochemical, and electromyography findings suggestive of a myopathic picture and 25 normal healthy children matched for age and sex constituted the control group, analysis was done using q-real-time PCR.<br><b>Results</b><br>About 75% (18/24) showed single-exon deletion, while 25% (6/24) showed multiple-exon deletions, of which 12.5% were double deletions and 12.5% were triple deletions, the clinical severity of the condition seems to be independent on the number of deleted exons. The most common single deletion was that of exon 48 followed by exon 51.<br>

Deletion and duplication mutations spectrum in Duchenne muscular dystrophy in the southwest of Iran

BackgroundDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by the mutation within the dystrophin gene. Objectivesthe primary objective of this study was to evaluate the deletion and duplication mutations in DMD patients. Materials and methodsOur study has identified 33 Iranian patients collected from Narges Genetic lab, southwest of Iran, Ahvaz. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase (CPK) level, and DNA analysis. DNA samples were analyzed by multiplex-ligation-dependent probe amplification (MLPA). ResultsIn this study, deletion rate was 75% (25/33) and more frequent in the distal end exons whereas duplication rate was 25% (8/33) and it is more frequent at proximal exons. Most of the deletion (15/25, 60%) were located on the distal hot spot region (exon 44–55). Majority of exon duplications 62.5% (5/8) were located at the proximal hot spot region (exon 1–16) and 37.5% (3/8) were located at the distal hot spot region (exon 50–62). Single exon deletions were present in 10/25 patients (40%) which are mostly were in exon 45, 51 and 52 with similar frequencies (2/25, 8% each). One patient had a de novo single exon deletion (exon 51) and whole dystrophin gene was deleted in only one patient. ConclusionThis finding indicates that as with deletions, duplications occur non-randomly but with a dramatically different distribution. Duplication frequency is highest near the 5′ end of the gene.

The Immune Response to Murine Factor VIII in Single Exon Deletion and Total Gene Deletion Murine Models of Hemophilia a

Introduction: Though a vital therapy for patients with hemophilia A, exposure to factor VIII (fVIII) can lead to the development of neutralizing anti-fVIII IgG. Interestingly, not all patients with hemophilia A form inhibitors, with approximately 20-30% of patients with severe hemophilia A and 5% of patients with mild to moderate hemophilia A developing inhibitors. Several factors have been hypothesized to govern the propensity of patients to develop inhibitors, including the type of F8 gene mutation a patient possesses. Approximately, 60-70% of hemophilia A patients with large deletions (greater than 50 base pairs) of the F8 gene form inhibitors, while only 20-30% of patients with smaller deletions develop inhibitors. However, no significant difference in inhibitor formation has been observed between single exon 16 (E16) and complete (TKO) F8 gene deficient hemophilia A mice exposed to human fVIII (hfVIII). As E16 mice are on a mixed S129-B6 background and strain differences can impact the immunological outcome to a given immunogen, it is possible that the mixed background influenced the humoral immune response to hfVIII in E16 mice. However, E16 mice that are backcrossed &gt;10 generations onto the same B6 background as TKO mice (B6-E16) were found to develop a comparable IgG response to hfVIII as B6-TKO and S129/B6-E16 mice. As human and murine B domain deleted (BDD) fVIII are 85% identical, we hypothesize that the diversity between human and murine fVIII may not allow for elucidation of immune differences that may exist between hemophilia A mice with large or small F8 mutations. Accordingly, we herein sought to compare the relative immunogenicity of murine fVIII (mfVIII) in these distinct hemophilia A mice. Methods:S129/B6 E16, B6-E16 and B6-TKO mice were administered four weekly retro-orbital infusions of 1 μg recombinant BDD mfVIII or hfVIII. Two weeks later, mice were challenged with a 2 μg dose of recombinant mfVIII or hfVIII. One week prior to challenge and one-week post challenge, plasma was collected for examination of anti-fVIII IgG titers by an enzyme linked immunosorbent assay. Results:Consistent with previous reports, no statistically significant difference in anti-hfVIII IgG titers were observed between S129/B6-E16, B6-E16 and B6-TKO mice. When exposed to mfVIII the majority of B6-TKO mice developed inhibitors (87.5%), while the single exon B6-E16 or S129/B6-E16 mice had 50% and 56% rates of inhibitor development, respectively. The B6-E16 mice had a lower median anti-mfVIII IgG ELISA titer of 12.3 (range 1 to 190.3) compared to S129/B6-E16 and B6-TKO that had median ELISA titers of 473 and 289, respectively. When assessing the anti-mfVIII IgG titers only in the mice that had detectable titer levels, the IgG titers for S129/B6-E16 mice ranged from 34.2 to 1519.4 and for B6-TKO mice ranged from 31.3 to 1377. Conclusion: Given the difference in rates of immune response to mfVIII in mice carrying a single exon or total F8 gene deletion, mfVIII may be useful to elucidate the correlation between F8 gene mutations and the propensity of an individual to respond to factor replacement therapy, and in particular modulators of the immune response in lower risk F8 gene mutations. Disclosures Batsuli: Genentech: Other: Advisory board participant; Octapharma: Other: Advisory board participant; Bayer: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

P.148Long-term natural history data in Duchenne muscular dystrophy ambulant patients with mutations amenable to skip exons 44, 45, 51 and 53

Recent clinical trials using dystrophin restoration approaches, such as antisense oligonucleotides for exons skipping or approaches targeting non-sense mutations have shown encouraging results. The interpretation of the long-term results of the clinical trials however is complicated by the paucity of long-term prospective natural history data in patients with distinct genotypes. Retrospective data on loss of ambulation and other features of disease progression in patients with deletions amenable to skip exon 44, 45, 51 and 53 have reported prospectively collected longitudinal data suggesting that the changes in the individual subgroups becomes to be more evident on a longer follow up (24 months). As there are a number of ongoing or planned studies targeting deletions amenable to skip exon 44, 45, 51 and 53 it has become important to obtain more detailed information on the patterns of progression related to different genotypes. The aim of this international collaborative effort was to obtain longitudinal changes over 36 months in the six minutes walking test (6MWT) in a large cohort of DMD ambulant patients amenable to skip exons 44, 45, 51 and 53. Of the 92 patients included in the study, 24 had deletions amenable to skip exon 44, 27 exon 45, 18 exon 51, and 28 exon 53. Five patients with a single deletion of exon 52 were counted in both subgroups skipping exon 51 and 53. The difference between subgroups amenable to skip different exons was not significant at 12 months but became significant at both 24 (p<0.05) and 36 months (p<0.01). Mutations amenable to skip exon 53 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had better results both at baseline and at follow up. Deletions amenable to skip exon 45 were associated with a more variable pattern of progression. Single exon deletions were more often associated with less drastic changes but this was not always true in individual cases. Our results confirm that the progression of disease can differ between patients with different deletions, although the changes only become significant from 24 months onwards. This information is relevant because there are current clinical trials specifically targeting patients with these subgroups of mutations.

Genetic analysis of 1051 Chinese families with Duchenne/Becker Muscular Dystrophy

BackgroundDuchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations.MethodsIn this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations.ResultsDMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis.ConclusionOur results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.

Differing disease phenotypes of Duchenne muscular dystrophy and Moyamoya disease in female siblings of a Korean family.

BackgroundVariable disease phenotypes can be influenced by several factors such as allelic variation, environmental factors, genetic modifiers, and genotype–environment interaction. Herein to the best of our knowledge, this is the first report of the coexistence of DMD and RNF213 gene mutations in a Korean family with differing disease phenotypes of Duchenne muscular dystrophy (DMD) and Moyamoya disease (MMD) in each female sibling.MethodsDeletion or duplication of the exon in DMD was screened using multiplex ligation‐dependent probe amplification (MLPA). Subsequently, single exon deletion or duplication identified by MLPA was confirmed by Sanger sequencing. On the other hand, a common missense mutation [NM_001256071.2:c.14429G>A (p.Arg4810Lys)] related to MMD in exon 60 of RNF213 was also identified by Sanger sequencing.ResultsThree female family members carried the same disease‐causing mutations, c.9953_9954delAG of DMD and c.14429G>A of RNF213. Two (II‐2 and II‐3) of these siblings suffer from the disease but exhibited different DMD or MMD symptoms, while the mother (I‐2) seemed almost unaffected.ConclusionThis report illustrates the difficulty that might be encountered in the interpretation of complex clinical manifestations when different genetic defects affecting neuromuscular and vascular diseases coexist.

Long-term natural history data in Duchenne muscular dystrophy ambulant patients with mutations amenable to skip exons 44, 45, 51 and 53.

IntroductionThe aim of this international collaborative effort was to report 36-month longitudinal changes using the 6MWT in ambulant patients affected by Duchenne muscular dystrophy amenable to skip exons 44, 45, 51 or 53.Materials and methodsOf the 92 patients included in the study, 24 had deletions amenable to skip exon 44, 27 exon 45, 18 exon 51, and 28 exon 53. Five patients with a single deletion of exon 52 were counted in both subgroups skipping exon 51 and 53.ResultsThe difference between subgroups amenable to skip different exons was not significant at 12 months but became significant at both 24 (p≤0.05) and 36 months (p≤0.01).DiscussionMutations amenable to skip exon 53 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had better results both at baseline and at follow up. Deletions amenable to skip exon 45 were associated with a more variable pattern of progression. Single exon deletions were more often associated with less drastic changes but this was not always true in individual cases.ConclusionOur results confirm that the progression of disease can differ between patients with different deletions, although the changes only become significant from 24 months onwards. This information is relevant because there are current clinical trials specifically targeting patients with these subgroups of mutations.

Deletion of exons 16–17b of CFTR is frequently identified in Korean patients with cystic fibrosis

Cystic fibrosis (MIM #219700) is one of the most common autosomal recessively inherited diseases in Caucasians and is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. However, this disease is much less frequent in Asian populations. Here, we performed a clinical characterization of, and genetic analysis of CFTR in, Korean patients with cystic fibrosis. Six Korean patients from five families (two females and four males; median age, 12.5 years) were enrolled. Clinical data were assessed by retrospective review of medical records. The genetic variants of CFTR were analysed by sequencing analysis and multiple ligation-dependent probe amplification (MLPA). Among the six patients, five had at least one allele with a deletion of exons 16–17 b: four had a heterozygous deletion and one had a homozygous deletion. Six of 12 alleles (50%) showed 16–17 b multi-exon deletion. All six patients had a classical cystic fibrosis phenotype and presented with chronic steatorrhea and malabsorption from infancy, resulting in growth failure and chronic recurrent respiratory symptoms, including chronic sinusitis, mucus plugging, and bronchiectasis. All patients survived with supportive care. Early diagnosis and management are important for improving the clinical outcomes of patients with cystic fibrosis. Because of the high frequency of multi- or single-exon deletions in CFTR, we suggest that molecular investigation for identifying exon deletions should be performed to establish an early confirmative diagnosis in Asian populations, including populations in Korea and Japan.

Genetic analysis and prenatal diagnosis of Duchenne or Becker muscular dystrophy

Objective: To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods: Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children's Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results: Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions: MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.

Resolving the full spectrum of human genome variation using Linked-Reads.

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as “Linked-Reads”. This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2. Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.