How a paternal uniparental isodisomy of chromosome 17 leads to autosomal recessive limb-girdle muscular dystrophy R3.

Uniparental isodisomy is a condition where both chromosomes of a pair are inherited from one parental homologue. If a deleterious variant is present on the duplicated chromosome, its homozygosity can reveal an autosomal recessive disorder in the offspring of a heterozygous carrier. Limb-girdle muscular dystrophy (LGMD) R3 is an autosomal recessive inherited disease that is associated with alpha-sarcoglycan gene (SGCA) variants. We report the first published case of LGMDR3 due to a homozygous variant in SGCA unmasked by uniparental isodisomy. The patient is an 8-year-old who experienced delayed motor milestones but normal cognitive development. He presented with muscle pain and elevated plasma creatine kinase. Sequencing of the SGCA gene showed a homozygous pathogenic variant. Parents were not related and only the father was heterozygous for the pathogenic variant. A chromosomal microarray revealed a complete chromosome 17 copy number neutral loss of heterozygosity encompassing SGCA, indicating paternal uniparental isodisomy.

Non-invasive prenatal diagnosis of single gene disorders by paternal mutation exclusion: 3 years of clinical experience.

OBJECTIVES: Cell-free fetal DNA (cffDNA) analysis is performed routinely for aneuploidy screening, RhD genotyping or sex determination. Although applications to single gene disorders (SGD) are being rapidly developed worldwide, only a few laboratories offer cffDNA testing routinely as a diagnosis service for this indication. In a previous report, we described a standardised protocol for non-invasive exclusion of paternal variant in SGD. Three years later, we now report our clinical experience with the protocol. DESIGN: Descriptive study. SETTING: Multi-centre French. POPULATION: Indications for referral included pregnancies at risk of 25% or 50% of paternally inherited SGD, and pregnancies associated with an increased risk of SGD due to a de novo variant, either from strongly suggestive ultrasound findings or from a possible parental germinal mosaicism in the context of a previously affected child. METHODS: Non-invasive prenatal diagnosis was performed using custom assays for droplet digital PCR. Feasibility, diagnostic performance and turn-around time were evaluated. RESULTS: Mean time for a new assay design and validation was evaluated at 14 days, and mean result reporting time was 6 days. All referred pathogenic variants could be targeted except one located in a complex genomic region. A result was obtained for every 198 referrals except two. CONCLUSION: This service was successfully implemented as a routine laboratory practice. It has been widely adopted by French clinicians and patients for paternal variant exclusion in various disorders. TWEETABLE ABSTRACT: A robust approach to non-invasive prenatal exclusion of paternal pathogenic variant in a diagnosis setting.

X-linked muscular dystrophy in a Labrador Retriever strain: phenotypic and molecular characterisation.

BACKGROUND: Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the Golden Retriever muscular dystrophy (GRMD) model remains the most used in preclinical studies. Here, we report a new spontaneous dystrophinopathy in a Labrador Retriever strain, named Labrador Retriever muscular dystrophy (LRMD). METHODS: A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing. RESULTS: The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2-Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability. CONCLUSIONS: This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.

Non Random Distribution of DMD Deletion Breakpoints and Implication of Double Strand Breaks Repair and Replication Error Repair Mechanisms.

BACKGROUND: Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR). OBJECTIVE: Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair. METHODS: Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences. RESULTS: Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions. CONCLUSIONS: Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.

Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

BACKGROUND: Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. METHODS: To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. RESULTS: We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. CONCLUSIONS: This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address. METHODS: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts. RESULTS: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model. CONCLUSIONS: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.

When a mid-intronic variation of DMD gene creates an ESE site.

Duchenne and Becker muscular dystrophy are X-linked allelic disorders caused by mutations in the DMD gene. The majority (65%) of these mutations are intragenic deletions/duplications that often lead to frameshift errors. Among the remaining ones, we find the mid-intronic mutations that usually create cryptic exons by activating potential splice sites. In this report, we identified, in a Becker muscular dystrophy patient, a mid-intronic variation that creates two ESE sites in intron 26 of DMD gene resulting in the insertion of a new cryptic exon in mRNA. Despite the out of frame character of this mutation, we observed the production of a reduced amount of full-size dystrophin which could be explained by the alternation between normal and altered splicing of dystrophin mRNA in this patient. To our knowledge, this is the first case report describing this novel pathogenic mechanism of mid-intronic variations of DMD gene.

Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes.

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs.

Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression.

The presence of variable degrees of cognitive impairment, extending from severe mental retardation to specific deficits, in patients with dystrophinopathies is a well-recognized problem. However, molecular basis underlying mental retardation and its severity remain poorly understood and still a matter of debate. Here, we report one of the largest study based on the comparison of clinical, cognitive, molecular and expression data in a large cohort of 81 patients affected with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) bearing mutations predicted to affect either all dystrophin products, including Dp71 or all dystrophin products, except Dp71. In addition to the consistent data defining molecular basis underlying mental retardation in DMD, we show that BMD patients with MR have mutations that significantly affect Dp71 expression or with mutations located in exons 75 and 76. We also show that mutations upstream to exon 62, with DMD phenotype, predicted to lead to a loss-of-function of all dystrophin products, except Dp71 isoform, are associated, predominantly, with normal or borderline cognitive performances. Altogether, these reliable phenotype-genotype correlations in combination with Dp71 mRNA and protein expression studies, strongly indicate that loss-of-function of all dystrophin products is systematically associated with severe form of MR, and Dp71 deficit is a factor that contributes in the severity of MR and may account for a shift of 2 SD downward of the intelligence quotient.

Detection of exonic copy-number changes using a highly efficient oligonucleotide-based comparative genomic hybridization-array method.

Genomic copy-number variations (CNVs) involving large DNA segments are known to cause many genetic disorders. Depending on the changes, they are predicted to lead either to decreased or an increased gene expression. However, the ability to detect smaller exonic copy-number changes has not been explored. Here we describe a new oligonucleotide-based comparative genomic hybridization (CGH)-array approach for high-throughput detection of exonic deletions or duplications and its application to deletion/duplication analyses of the genes encoding CFTR, six sarcoglycans (SGCA, SGCB, SGCG, SGCD, SGCE, and SGCZ), and DMD. In this work we show the successful development of an array format containing 158 exons that collectively span eight genes and its clinical application for the rapid screening of deletions and duplications in a diagnostic setting. We have analyzed a series of 35 DNA samples from patients affected with cystic fibrosis (CF), Duchenne and Becker muscular dystrophies (DMD/BMD), or sarcoglycanopathies, and have characterized exonic copy-number changes that have been validated with other methods. Interestingly, even heterozygous deletions and duplications of only one exon, as well as mosaic deletions, were detected by this CGH approach. Our results showed that the resolution is very high, as abnormalities of about 1.5-2 kb could be detected. Since this approach is completely scalable, this new molecular tool will allow the screening of combinations of genes involved in a particular group of clinically and genetically heterogeneous disorders such as mental retardation, muscular dystrophies and brain malformations.

Revised spectrum of mutations in sarcoglycanopathies.

To define the spectrum of mutations in alpha-, beta-, gamma-, and delta-sarcoglycan (SG) genes, we analyzed these genes in 69 probands with clinical and biological criteria compatible with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. For 48 patients, muscle biopsies were available and multiplex western blot analysis of muscle proteins showed significant abnormalities of alpha- and gamma-SG. Our diagnostic strategy includes multiplex western blot, sequencing of SG genes, multiplex quantitative-fluorescent PCR and RT-PCR analyses. Mutations were detected in 57 patients and homozygous or compound heterozygous mutations were identified in 75% (36/48) of the patients with abnormal western blot, and in 52% (11/21) of the patients without muscle biopsy. Involvement of alpha-SG was demonstrated in 55.3% of cases (26/47), whereas gamma- and beta-SG were implicated in 25.5% (12/47) and in 17% (8/47) of cases, respectively. Interestingly, we identified 25 novel mutations, and a significant proportion of these mutations correspond to deletions (identified in 14 patients) of complete exon(s) of alpha- or gamma-SG genes, and partial duplications (identified in 5 patients) of exon 1 of beta-SG gene. This study highlights the high frequency of exonic deletions of alpha- and gamma-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the beta-SG gene. In addition, protein analysis by multiplex western blot in combination with mutation screening and genotyping results allowed to propose a comprehensive and efficient diagnostic strategy and strongly suggested the implication of additional genes, yet to be identified, in sarcoglycanopathy-like disorders.

Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene.

Straightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations.

Dysferlin mutation in a Chinese pedigree with Miyoshi myopathy.

OBJECTIVES: Mutations in the dysferlin gene cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. The purpose of this study was to diagnose a Chinese pedigree with the autosomal recessive form of muscular dystrophy and conduct mutational screening. METHODS: The pedigree was diagnosed accurately by using two-point linkage analysis and multi-Western blot analysis. Mutations were determined by reverse transcriptase polymerase chain reaction (RT-PCR) followed by DNA sequencing. RESULTS: Two-point linkage analysis showed significant LOD scores with makers from chromosome 2p13. Multi-Western blot analysis confirmed dysferlin deficiency of muscle specimen from the propositus. Mutation analysis of the dysferlin gene revealed a novel mutation, 6429delG, on exon 53. CONCLUSIONS: We identified an inbred Chinese pedigree with Miyoshi myopathy caused by the 6429delG mutation in the dysferlin gene. This mutation is predicted to result in premature termination of translation contributing to Miyoshi myopathy.

Prenatal diagnosis of recessive congenital methaemoglobinaemia type II: novel mutation in the NADH-cytochrome b5 reductase gene leading to stop codon read-through.

A case of type II recessive congenital methaemoglobinaemia (RCM) observed in a Lebanese subject with a novel mutation in NADH-cytochrome b5 reductase gene is described. A homozygous mutation CAC to AA identified at Thr 295 with an out-of-frame 1-bp deletion leads to a frameshift with translational read-through of the natural stop codon. The molecular mechanism is demonstrated by an in vitro translation study. The model of mutated cytochrome b5 reductase protein possessing 46 additional amino acids was obtained by homology modelling. The mutation causes an alteration of hydrophobicity in the carboxyl-terminal portion, resulting in the conformation being drastically disturbed by the presence of 46 supplementary amino acids. The identical mutation was found in the heterozygous state in the patient's parents and sister. Identification of this new mutation enabled us to perform the molecular prenatal diagnosis of type II RCM at the DNA level.

[Dysferlin deficiency: the cause of limb-girdle muscular dystrophy 2B and Miyoshi myopathy in a Chinese pedigree].

OBJECTIVE: To identify an inbred Chinese pedigree with autosomal recessive muscular dystrophy and analyze the molecular defects. METHODS: Linkage analysis was conducted using short tandem repeat(STR) markers from the regions associated with limb-girdle muscular dystrophy type 2A(LGMD2A) through 2H. Multi-Western blot was performed with anti-calpain-3, anti-dysferlin, anti-gamma-sarcoglycan, anti-alpha-sarcoglycan, and anti-dystrophin monoclonal antibodies. Mutation was determined by reverse transcriptase-polymerase chain reaction and sequencing. RESULTS: Two-point linkage analysis showed significant Lod scores with markers from chromosome 2p13, the highest two-point Lod scores were obtained with D2S337 (Z(max)=1.86 at theta=0). Multi-Western blot confirmed dysferlin deficiency of muscle specimen from the proband. Mutation analysis revealed a novel 6429delG mutation on exon 53 of the DYSF gene for the proband. CONCLUSION: The authors identified an inbred Chinese pedigree with Miyoshi myopathy caused by a 6429delG on the DYSF gene. This mutation is predicted to result in premature termination of translation.

Dystrophinopathy caused by mid-intronic substitutions activating cryptic exons in the DMD gene.

In the course of a mutation search performed by muscle dystrophin transcript analysis in 72 Duchenne and Becker Muscular Dystrophies (DMD/BMD) patients without gross gene defect, we encountered four unrelated cases with additional out-of-frame sequences precisely intercalated between two intact exons of the mature muscle dystrophin mRNA. An in silico search of the whole dystrophin genomic sequence revealed that these inserts correspond to cryptic exons flanked by one strong and one weak consensus splice site and located in the mid-part of large introns (introns 60, 9, 1M, and 62, respectively). In each case we identified an intronic point mutation activating the cryptic donor or acceptor splice site. The patients exhibited a BMD/intermediate phenotype consistent with the presence of reduced amounts of normally spliced transcript and normal dystrophin. The frequency of this new type of mutation is not negligible (6% of our series of 65 patients with 'small' mutations). It would be missed if the exploration of the DMD gene is exclusively performed on exons and flanking sequences of genomic DNA.