To define the spectrum of mutations in a-, b-, c-, and d-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 a-and c-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 a-SG was demonstrated in 55.3% of cases (26/47), whereas c-and b-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 a-or c-SG genes, and partial duplications (identified in 5 patients) of exon 1 of b-SG gene. This study highlights the high frequency of exonic deletions of a-and c-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the b-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.
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.
For the Focus Section on Array-CGHGenomic 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 highthroughput 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. Hum Mutat 29(9), [1083][1084][1085][1086][1087][1088][1089][1090] 2008.
The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient's kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for β-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-β-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis.
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