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, lengthdependent 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. IntroductionThe ER is a continuous membrane system comprising the nuclear envelope and a dynamic network of proximal sheets and peripheral tubules. Proteins of 2 classes -the reticulons and the REEP/DP1/yop1p family (referred to herein as the REEPs) -are fundamental to the generation of both sheets and tubules. These proteins share a characteristic sequence feature; in the case of the reticulons this feature is termed the reticulon homology domain (RHD) and consists of 2 long hydrophobic stretches separated by a hydrophilic sequence. Each hydrophobic stretch is thought to sit in the ER membrane as a hairpin loop. Such loop domains have been suggested to generate membrane curvature by occupying more space in the outer leaflet of the membrane than the inner in a process termed "hydrophobic wedging" (1-3). These proteins are thus critical for producing

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.

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. Over 40 chromosomal loci have been identified for autosomal dominant, recessive, and X-linked HSP. Mutations in the genes atlastin, spastin and REEP1 are estimated to account for up to 50% of autosomal dominant HSP and currently guide the molecular diagnosis of HSP. Here we report the mutation screening results of 120 HSP patients from North America for spastin, atlastin, and REEP1, with the latter one partially reported previously. We identified mutations in 36.7% of all tested HSP patients and describe 20 novel changes in spastin and atlastin. Our results add to a growing number of HSP disease associated variants and confirm the high prevalence of atlastin, spastin, and REEP1 mutations in the HSP patient population.

Charcot-Marie-Tooth (CMT) disease is among the most common inherited neurological disorders. Mutations in the gene mitofusin 2 (MFN2) cause the axonal subtype CMT2A, which has also been shown to be associated with optic atrophy, clinical signs of first motor neuron involvement, and early onset stroke. Mutations in MFN2 account for up to 20-30% of all axonal CMT type 2 cases. To further investigate the prevalence of MFN2 mutations and to add to the genotypic spectrum, we sequenced all exons of MFN2 in a cohort of 39 CMT2 patients. We identified seven variants, four of which are novel. One previously described change was co-inherited with a PMP22 duplication, which itself causes the demyelinating form CMT1A. Another mutation was a novel in frame deletion, which is a rare occurrence in the genotypic spectrum of MFN2 characterized mainly by missense mutations. Our results confirm a MFN2 mutation rate of ~ 15-20% in CMT2.

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.

Introduction: Mycoplasma genitalium (Mg) is a sexually transmitted pathogen associated with non-gonococcal urethritis, cervicitis, pelvic inflammatory disease and infertility. Since Mg is very difficult to culture from clinical samples, typing strains relies on the variability of a 281pb fragment of the mgpB gene, encoding the adhesin MgPa. Here we present the analysis of the sequences of 14 Mg strains detected from clinical samples between 2013 and 2016.MethodsThis was a retrospective study in which we analysed all the Mg positive samples diagnosed in our laboratory in the period 2013–2016. Detection of Mg was performed by in-house PCR assay using primers previously described; the resulting 281pb fragments from Mg positive specimens were sequenced by Sanger method. Sequences were analysed and compared with all currently available clinical sequences.ResultsA total of 452 genital samples were tested, from which 17 resulted positive for Mg. Of these, only 14 could be successfully sequenced. The analysis of sequenced samples revealed eight different types of sequences. When compared with published data, four sequence types (representing a total of 10 different strains) resulted identical to previously reported genotypes. The relative frequencies of these genotypes were: 29% genotype 1 (4/14), 29% genotype 2 (4/14), 7% genotype 4 (1/14), and 7% genotype 21 (1/14, 7%). The remaining sequences showed between one and four nucleotide differences compared to already existing variants; in three of them this resulted in amino acid changes. ConclusionThis is the first study to characterise the molecular types of Mg among clinical strains in our country. Through comparative sequence analysis, eight different mgpB region variants were identified, four of which have not been report in the past. This reveals the presence of new sequence variants in Argentina. Further studies are needed to evaluate the association between these sequence variants and clinical/epidemiological data that could help us to understand the dynamics of Mg infection in the region.

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