Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous ...families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126−/− mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu c.2306T>A) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.
Arthrogryposis multiplex congenita (AMC) is characterized by the presence of multiple joint contractures resulting from reduced or absent fetal movement. Here, we report two unrelated families ...affected by lethal AMC. By genetic mapping and whole-exome sequencing in a multiplex family, a heterozygous truncating MAGEL2 mutation leading to frameshift and a premature stop codon (c.1996delC, p.Gln666Serfs∗36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfs∗7) and occurring de novo on the paternal allele of MAGEL2 was identified in the affected individual. In both families, RNA analysis identified the mutated paternal MAGEL2 transcripts only in affected individuals. MAGEL2 is one of the paternally expressed genes within the Prader-Willi syndrome (PWS) locus. PWS is associated with, to varying extents, reduced fetal mobility, severe infantile hypotonia, childhood-onset obesity, hypogonadism, and intellectual disability. MAGEL2 mutations have been recently reported in affected individuals with features resembling PWS and called Schaaf-Yang syndrome. Here, we show that paternal MAGEL2 mutations are also responsible for lethal AMC, recapitulating the clinical spectrum of PWS and suggesting that MAGEL2 is a PWS-determining gene.
Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been ...described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.
Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) ...were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (<10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases.
To identify the underlying etiology of 3 patients in a multiplex family with strokes, chronic immune-mediated peripheral neuropathy, and hemolysis. All had onset in infancy.
We performed genome-wide ...linkage analysis followed by whole exome sequencing (WES) in the proband, Sanger sequencing, and segregation analysis of putative mutations. In addition, we conducted flow cytometry studies to assess CD59 expression.
In a 2-generation-3-affected family with early-onset immune-mediated axonal neuropathy, cerebrovascular event both in the anterior and posterior circulation, and chronic Coombs-negative hemolysis, we detected CD59 deleterious mutation as the underlying cause. Linkage analysis and homozygosity mapping using single nucleotide polymorphism (SNP) microarrays in the family followed by WES in one index case allowed identification of a homozygous missense mutation in the CD59 gene (c.A146T:p.Asp49Val). Sanger sequencing validated the mutation, showing cosegregation with the disease phenotype. Flow cytometry using blood cells in the 3 patients showed a lack of CD59 expression at the cell membrane compared to control and CD55 labeling.
We added to the knowledge base about inherited CD59 deficiency.
Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, ...homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na+ channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies.
Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify ...sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.
Differentiation studies indicated that mRNA‐derived induced pluripotent stem cells (iPSCs) differentiated efficiently into hepatoblasts and that these cells did not load additional copy number ...variations during differentiation. The integration‐free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell‐derived hepatocyte transplantation.
The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA‐based or virus‐based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA‐derived iPSCs with that of retrovirus‐derived iPSCs generated in strictly comparable conditions, by single‐nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA‐derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus‐derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone‐dependent. Furthermore, differentiation studies indicated that mRNA‐derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration‐free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell‐derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.
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