Autosomal recessive cerebellar ataxias are a group of neurodegenerative disorders that are characterized by complex clinical and genetic heterogeneity. Although more than 20 disease-causing genes have been identified, many patients are still currently without a molecular diagnosis. In a two-generation autosomal recessive cerebellar ataxia family, we mapped a linkage to a minimal candidate region on chromosome 16p13.3 flanked by single-nucleotide polymorphism markers rs11248850 and rs1218762. By combining the defined linkage region with the whole-exome sequencing results, we identified a homozygous mutation (c.493CT) in CHIP (NM_005861) in this family. Using Sanger sequencing, we also identified two compound heterozygous mutations (c.389AT/c.441GT; c.621C>G/c.707GC) in CHIP gene in two additional kindreds. These mutations co-segregated exactly with the disease in these families and were not observed in 500 control subjects with matched ancestry. CHIP colocalized with NR2A, a subunit of the N-methyl-D-aspartate receptor, in the cerebellum, pons, medulla oblongata, hippocampus and cerebral cortex. Wild-type, but not disease-associated mutant CHIPs promoted the degradation of NR2A, which may underlie the pathogenesis of ataxia. In conclusion, using a combination of whole-exome sequencing and linkage analysis, we identified CHIP, encoding a U-box containing ubiquitin E3 ligase, as a novel causative gene for autosomal recessive cerebellar ataxia.

BackgroundMany applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.ResultsIn the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.ConclusionsNHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1518-x) contains supplementary material, which is available to authorized users.

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BackgroundThere is increasing interest in the concept of atrial cardiomyopathy, but the underlying molecular and mechanistic determinants remain poorly defined. We identified a family with heritable atrial cardiomyopathy manifesting as progressive atrial‐selective electromechanical dysfunction, tachyarrhythmias, and bradyarrhythmias requiring pacemaker implantation. Myosin light‐chain 4 (MYL4), encoding the atrial‐selective essential myosin light chain, was identified as a candidate gene. We used genetically modified rat models to investigate the role of MYL4 in atrial cardiomyopathy.Methods and ResultsExome sequencing and systematic bioinformatic analyses identified a rare missense variant of MYL4 (c.31G>A [p.E11K]) in a large multiplex atrial cardiomyopathy family pedigree. The mutation cosegregated with atrial standstill (selected as the principal presenting trait) with a logarithm of the odds score of 5.3. The phenotype of rats with MYL4 mutation knock‐in confirmed the causative role of the mutation. MYL4 knockout rats showed a similar atrial cardiomyopathy phenotype, whereas rats with an adjacent 4‐amino‐acid deletion showed no phenotype. Both MYL4 p.E11K knock‐in rats and MYL4 knockout rats showed progressive atrial electrophysiological, contractile, and fibrotic abnormalities, similar to affected patients. Biochemical analyses of MYL4 p.E11K mutation rats showed activation of proapoptotic and profibrotic signaling, along with increased atrial‐cardiomyocyte terminal deoxynucleotidyl transferase dUTP nick end labeling staining, suggesting enhanced apoptotic cell death, findings that were mimicked by in vitro adenoviral transfer of the mutant gene to neonatal‐rat cardiomyocytes.ConclusionsLoss‐of‐function MYL4 gene variants cause progressive atrial cardiomyopathy in humans and rats. Our findings identify MYL4 as a key gene required for atrial contractile, electrical and structural integrity. These results improve our understanding of the molecular basis of atrial cardiomyopathy and introduce new models for further mechanistic analysis.

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The isobaric vapor–liquid equilibrium measurements for the systems of 2-butyl acetate with three chemicals (methanol, 2-butyl alcohol, methyl acetate) were determined respectively in a modified Rose still at 101.33 kPa. Thermodynamic consistency of the three binary vapor–liquid equilibrium data had been confirmed by Herington methods. The three groups of experimental data were all correlated with NRTL and Wilson models, respectively. The binary interaction parameters of both models had been obtained by the simplex method. The NRTL and Wilson equations showed low deviation with respect to the experimental data.

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Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management.