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Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain-containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein-coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.

ATP8A2 -related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea, tremor, optic atrophy and cerebellar atrophy (CARMQ4). Through a multi-centric collaboration, we identified six point mutations (one splice site and five missense mutations) involving ATP8A2 in six individuals from five families. Two patients from one family with the homozygous p.Gly585Val mutation had a milder presentation without encephalopathy. Expression and functional studies of the missense mutations demonstrated that protein levels of four of the five missense variants were very low and lacked phosphatidylserine-activated ATPase activity. One variant p.Ile215Leu, however, expressed at normal levels and displayed phospholipid-activated ATPase activity similar to the non-mutated protein. We therefore expand for the first time the phenotype related to ATP8A2 mutations to less severe forms characterized by cerebellar ataxia without encephalopathy and suggest that ATP8A2 should be analyzed for all cases of syndromic or non-syndromic recessive or sporadic ataxia.

Cystic fibrosis (CF) is a rare genetic disease that affects the respiratory and digestive systems. Lung disease is variable among CF patients and associated with the development of comorbidities and chronic infections. The rate of lung function deterioration depends not only on the type of mutations in CFTR, the disease-causing gene, but also on modifier genes. In the present study, we aimed to identify genes and pathways that (i) contribute to the pathogenesis of cystic fibrosis and (ii) modulate the associated comorbidities. We profiled blood samples in CF patients and healthy controls and analyzed RNA-seq data with Weighted Gene Correlation Network Analysis (WGCNA). Interestingly, lung function, body mass index, the presence of diabetes, and chronic P. aeruginosa infections correlated with four modules of co-expressed genes. Detailed inspection of networks and hub genes pointed to cell adhesion, leukocyte trafficking and production of reactive oxygen species as central mechanisms in lung function decline and cystic fibrosis-related diabetes. Of note, we showed that blood is an informative surrogate tissue to study the contribution of inflammation to lung disease and diabetes in CF patients. Finally, we provided evidence that WGCNA is useful to analyze-omic datasets in rare genetic diseases as patient cohorts are inevitably small.

Background Lung disease is variable among patients with cystic fibrosis (CF) and depends on genetic and environmental factors. To better understand the molecular determinants of lung disease variability, we carried out an epigenome-wide association study (EWAS) in sputum samples from patients with CF. Methods We profiled 64 sputum samples using Human Methylation EPIC BeadChips and assessed the correlation between DNA methylation levels and four clinical traits: lung function (FEV1 pp ), lung function variation (FEV1 pp slope), presence and number of pulmonary exacerbations. Sputum samples were collected at four time points over an 18-month follow-up period. Selected CpG sites were reassessed in independent sputum samples from the same cohort by pyrosequencing. Results In the EWAS, we identified two differentially methylated CpG sites (cg11047325/ SOCS3 , p  = 4 × 10 –6 ; cg18608055/ SBNO2 , p  = 6 × 10 –7 ) that correlated with lung function. They were validated in independent sputum samples from the same cohort using pyrosequencing. Additionally, three CpG sites (cg23107754, cg03209812 and cg09600088) split patients with declining lung function from those whose lung function either improved or remained stable (accuracy = 0.80). Of interest for CF-related diabetes, one of these CpG sites (cg09600088) maps to the BRSK2 gene, which plays a role in pancreatic beta cell function. Finally, a DNA methylation signature of 23 CpG sites predicted patients with pulmonary exacerbation (accuracy = 0.84). Conclusions We provide the first longitudinal assessment of genome-wide DNA methylation in a cohort of patient with CF and identify CpG sites that predict clinical traits of key importance for lung disease. The associated genes play a critical role in inflammation or pancreatic endocrine activity. Overall, our results underscore the emerging role of DNA methylation as a key modulator of disease severity in CF.

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing loss combined with retinitis pigmentosa, and in some cases, vestibular areflexia. Three clinical subtypes are distinguished, and MYO7A and USH2A represent the two major causal genes involved in Usher type I, the most severe form, and type II, the most frequent form, respectively. Massively parallel sequencing was performed on a cohort of patients in the context of a molecular diagnosis to confirm clinical suspicion of Usher syndrome. We report here 231 pathogenic MYO7A and USH2A genotypes identified in 73 Usher type I and 158 Usher type II patients. Furthermore, we present the ACMG classification of the variants, which comprise all types. Among them, 68 have not been previously reported in the literature, including 12 missense and 16 splice variants. We also report a new deep intronic variant in USH2A. Despite the important number of molecular studies published on these two genes, we show that during the course of routine genetic diagnosis, undescribed variants continue to be identified at a high rate. This is particularly pertinent in the current era, where therapeutic strategies based on DNA or RNA technologies are being developed.

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin ( FBN1 ) at 15q21.1 (ref. 1 ). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2 , at 3p25–p24.2 in a large French family (family MS1) 2 . Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-β receptor 2 ( TGFBR2 ) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G→A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-β signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2 , a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.

Peroxisomal biogenesis disorders (PBDs) consist of a heterogeneous group of autosomal recessive diseases, in which peroxisome assembly and proliferation are impaired leading to severe multisystem disease and early death. PBDs include Zellweger spectrum disorders (ZSDs) with a relatively mild clinical phenotype caused by PEX1 , (MIM# 602136), PEX2 (MIM# 170993), PEX6 (MIM# 601498), PEX10 (MIM# 602859), PEX12 (MIM# 601758), and PEX16 (MIM# 603360) mutations. Three adult patients are reported belonging to a non-consanguineous French family affected with slowly progressive cerebellar ataxia, axonal neuropathy, and pyramidal signs. Mental retardation and diabetes mellitus were optional. The age at onset was in childhood or in adolescence (3–15 years). Brain MRI showed marked cerebellar atrophy. Biochemical blood analyses suggested a mild peroxisomal defect. With whole exome sequencing, two mutations in PEX10 were found in the three patients: c.827G>T (novel) causing the missense change p.Cys276Phe and c.932G>A causing the missense change p.Arg311Gln. The phenotypic spectrum related to PEX10 mutations includes slowly progressive, syndromic recessive ataxia.

A number of chronic airway diseases are characterized by high inflammation and unbalanced activation of the immune response, which lead to tissue damage and progressive reduction of the pulmonary function. Because they are exposed to various environmental stimuli, lung cells are prone to epigenomic changes. Many genes responsible for the immune response and inflammation are tightly regulated by DNA methylation, which suggests that alteration of the epigenome in lung cells may have a considerable impact on the penetrance and/or the severity of airway diseases. A major hurdle in clinical epigenomic studies is to gather appropriate biospecimens. Herein, we show that nasal epithelial cells are suitable to analyze DNA methylation in human diseases primarily affecting the lower airway tract.

Accessible information about the presence and effect of specific mutations in cancer-causing genes is essential, but keeping track of these mutations is not simple. This article, using p53 as an example, highlights the difficulties involved in maintaining mutation databases. Between 50,000 and 60,000 mutations have been described in various genes that are associated with a wide variety of diseases. Reporting, storing and analysing these data is an important challenge as such data provide invaluable information for both clinical medicine and basic science. Locus-specific databases have been developed to exploit this huge volume of data. The p53 mutation database is a paradigm, as it constitutes the largest collection of somatic mutations (22,000). However, there are several biases in this database that can lead to serious erroneous interpretations. We describe several rules for mutation database management that could benefit the entire scientific community.