Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative conditions. They are characterized by progressive spastic paralysis of the legs as a result of selective, length-dependent degeneration of the axons of the corticospinal tract. Mutations in 3 genes encoding proteins that work together to shape the ER into sheets and tubules - receptor accessory protein 1 (REEP1), atlastin-1 (ATL1), and spastin (SPAST) - have been found to underlie many cases of HSP in Northern Europe and North America. Applying Sanger and exome sequencing, we have now identified 3 mutations in reticulon 2 (RTN2), which encodes a member of the reticulon family of prototypic ER-shaping proteins, in families with spastic paraplegia 12 (SPG12). These autosomal dominant mutations included a complete deletion of RTN2 and a frameshift mutation predicted to produce a highly truncated protein. Wild-type reticulon 2, but not the truncated protein potentially encoded by the frameshift allele, localized to the ER. RTN2 interacted with spastin, and this interaction required a hydrophobic region in spastin that is involved in ER localization and that is predicted to form a curvature-inducing/sensing hairpin loop domain. Our results directly implicate a reticulon protein in axonopathy, show that this protein participates in a network of interactions among HSP proteins involved in ER shaping, and further support the hypothesis that abnormal ER morphogenesis is a pathogenic mechanism in HSP.

OBJECTIVE:Charcot-Marie-Tooth (CMT) disease comprises a large number of genetically distinct forms of inherited peripheral neuropathies. The relative uniform phenotypes in many patients with CMT make it difficult to decide which of the over 35 known CMT genes are affected in a given patient. Genetic testing decision trees are therefore broadly based on a small number of major subtypes (eg, CMT1, CMT2) and the observed mutation frequency for CMT genes. Since conventional genetic testing is expensive many rare genes are not being tested for at all. METHODS:Whole-exome sequencing has recently been introduced as a novel and alternative approach. This method is capable of resequencing a nearly complete set of coding exons in an individual. We performed whole-exome sequencing in an undiagnosed family with CMT. RESULTS:Within over 24,000 variants detected in 2 exomes of a CMT family, we identified a nonsynonymous GJB1 (Cx32) mutation. This variant had been reported previously as pathogenic in X-linked CMT families. Sanger sequencing confirmed complete cosegregation in the family. Affected individuals had a marked early involvement of the upper distal extremities and displayed a mild reduction of nerve conduction velocities. INTERPRETATION:We have shown for the first time in a genetically highly heterogeneous dominant disease that exome sequencing is a valuable method for comprehensive medical diagnosis. Further improvements of exon capture design, next-generation sequencing accuracy, and a constant price decline will soon lead to the adoption of genomic approaches in gene testing of Mendelian disease.

Polg mtDNA mutator mice are important models for investigating the role of acquired mtDNA mutations in aging. Despite extensive study, there remains little consensus on either the etiology of the progeroid phenotype or the mtDNA mutation spectrum induced by disrupted polymerase-γ function. To investigate the latter, we have developed a novel, pragmatic approach we term “Mito-seq,” applying next-generation sequencing to enriched, native mtDNA. Regardless of detection parameters we observed an increase of at least two orders of magnitude in the number of mtDNA single nucleotide variants in Polg mutator mice compared to controls. We found no evidence for the accumulation of canonical mtDNA deletions but multimers of the mtDNA control region were identified in brain and heart. These control region multimers (CRMs) contained heterogeneous breakpoints and formed species that excluded the majority of mtDNA genes. CRMs demonstrate that polymerase-γ 3′-5′ exonuclease activity is required for preserving mtDNA integrity.

Hereditary peripheral neuropathies present a group of clinically and genetically heterogeneous entities. All known forms, including the various forms of Charcot-Marie-Tooth disease (CMT) are characterized as Mendelian traits and over 35 genes have been identified thus far. The mutational mechanism of the most common CMT type, CMT1A, is a 1.5 Mb chromosomal duplication at 17p12 that contains the gene PMP22. Only recently it has been realized that such copy number variants (CNV) are a widespread phenomenon and important for disease. However, it is not known whether CNVs play a wider role in hereditary peripheral neuropathies outside of CMT1A. In a phenotypically heterogeneous sample of 97 patients, we performed the first high-density CNV study of 34 genomic regions harboring known genes for hereditary peripheral neuropathies including the 17p12 duplication region, with comparative genomic hybridization (CGH) microarrays. We identified three CNVs that affected coding exons. A novel shorter form of a PMP22 duplication was detected in a CMT1A family previously tested negative in a commercial test. Two other CNVs in MTMR2 and ARHGEF10 are likely not disease associated. Our results indicate that CNVs are a rare cause for non-CMT1A CMT. Their potential relevance as disease modifiers remains to be evaluated. The present study design cannot rule out that specific CMT forms exist where CNVs play a larger role.

Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.