Proteasome subunit variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress

The ubiquitin–proteasome system degrades ubiquitin‐modified proteins to maintain protein homeostasis and to control signalling. Whole‐genome sequencing of patients with severe deafness and early‐onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient\u27s fibroblasts was however unaffected. Nevertheless, patient\u27s cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient\u27s fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient\u27s cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development

Is glycosylation of viral structural proteins involved in CABYV aphid transmission?

National audienceCucurbit aphid borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) is a plant virus, localized in phloem cells and obligatory transmitted by aphids. Previous studies have shown that post-translational modifications such as N-linked glycosylation can affect polerovirus transmission by aphids. We analyzed the glycosylation status of the 2 structural proteins of CABYV (Coat Protein-CP: major capsid protein, ReadThrough-RT: minor capsid protein) by using different approaches to determine more precisely how this modification may affect aphid transmission. We first constructed four CABYV mutants modified in potential N-linked glycosylation sites (named Ngly-1, -2, -3, -4). Two of them (Ngly-1 and -2) contained a mutation in the major CP, whereas the two others were affected in the RT. When electroporated to plant protoplasts, the four mutants replicated as efficiently as the wild-type virus suggesting that the mutations introduced did not affect virus replication. When introduced in plants, the mutant Ngly-2 was almost totally impeded in long-distance movement whereas Ngly-1, -3, -4 showed a viral accumulation similar to the wild-type virus in systemic leaves. Analysis of the viral progeny in infected plants showed that mutations are maintained and no reverse nor compensatory mutation appeared. Aphid transmission of Ngly-1, -3, -4 mutants was assessed using infected plants as virus source. We observed a reduction in aphid transmission of the 3 mutants using either Aphis gossypii or Myzus persicae, two species known to be efficient vectors of CABYV. In order to conclude if the reduction in transmission efficiency of these viruses was due to a reduction in viral accumulation in plants or to a direct effect in vector interactions, aphid transmission experiments are being performed using purified virus particles as virus source. However, at this stage, we cannot correlate the effects on systemic movement or aphid transmission to modifications of the glycosylation status of the virion. We then analyzed by Mass-Spectrometry if post-translational modifications are present on the two structural proteins of CABYV. So far, our results showed that no glycosylated peptide is present on the viral structural proteins. Glycosylation of virions was also assessed by immunodetection with antibodies specific to complex plants glycanes, and by using the lectin Concanavalin A. No N-glycane could be identified on the viral structural proteins. However, a 90kDa protein (p90), an α-glucosidase, that co-purify with polerovirus was identified and shown to be modified by oligomannosidic and complex N-glycanes. The role in aphid transmission of this plant protein is currently being investigated as well as the potential interaction between p90 and viral particles. We also analysed the behaviour of the virus in A. thaliana mutant, whose homologous p90 protein is not expressed

Is glycosylation of viral structural proteins involved in CABYV aphid transmission?

National audienceCucurbit aphid borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) is a plant virus, localized in phloem cells and obligatory transmitted by aphids. Previous studies have shown that post-translational modifications such as N-linked glycosylation can affect polerovirus transmission by aphids. We analyzed the glycosylation status of the 2 structural proteins of CABYV (Coat Protein-CP: major capsid protein, ReadThrough-RT: minor capsid protein) by using different approaches to determine more precisely how this modification may affect aphid transmission. We first constructed four CABYV mutants modified in potential N-linked glycosylation sites (named Ngly-1, -2, -3, -4). Two of them (Ngly-1 and -2) contained a mutation in the major CP, whereas the two others were affected in the RT. When electroporated to plant protoplasts, the four mutants replicated as efficiently as the wild-type virus suggesting that the mutations introduced did not affect virus replication. When introduced in plants, the mutant Ngly-2 was almost totally impeded in long-distance movement whereas Ngly-1, -3, -4 showed a viral accumulation similar to the wild-type virus in systemic leaves. Analysis of the viral progeny in infected plants showed that mutations are maintained and no reverse nor compensatory mutation appeared. Aphid transmission of Ngly-1, -3, -4 mutants was assessed using infected plants as virus source. We observed a reduction in aphid transmission of the 3 mutants using either Aphis gossypii or Myzus persicae, two species known to be efficient vectors of CABYV. In order to conclude if the reduction in transmission efficiency of these viruses was due to a reduction in viral accumulation in plants or to a direct effect in vector interactions, aphid transmission experiments are being performed using purified virus particles as virus source. However, at this stage, we cannot correlate the effects on systemic movement or aphid transmission to modifications of the glycosylation status of the virion. We then analyzed by Mass-Spectrometry if post-translational modifications are present on the two structural proteins of CABYV. So far, our results showed that no glycosylated peptide is present on the viral structural proteins. Glycosylation of virions was also assessed by immunodetection with antibodies specific to complex plants glycanes, and by using the lectin Concanavalin A. No N-glycane could be identified on the viral structural proteins. However, a 90kDa protein (p90), an α-glucosidase, that co-purify with polerovirus was identified and shown to be modified by oligomannosidic and complex N-glycanes. The role in aphid transmission of this plant protein is currently being investigated as well as the potential interaction between p90 and viral particles. We also analysed the behaviour of the virus in A. thaliana mutant, whose homologous p90 protein is not expressed

WGS Revealed Novel BBS5 Pathogenic Variants, Missed by WES, Causing Ciliary Structure and Function Defects

Bardet–Biedl syndrome (BBS) is an autosomal recessive ciliopathy that affects multiple organs, leading to retinitis pigmentosa, polydactyly, obesity, renal anomalies, cognitive impairment, and hypogonadism. Until now, biallelic pathogenic variants have been identified in at least 24 genes delineating the genetic heterogeneity of BBS. Among those, BBS5 is a minor contributor to the mutation load and is one of the eight subunits forming the BBSome, a protein complex implied in protein trafficking within the cilia. This study reports on a European BBS5 patient with a severe BBS phenotype. Genetic analysis was performed using multiple next-generation sequencing (NGS) tests (targeted exome, TES and whole exome, WES), and biallelic pathogenic variants could only be identified using whole-genome sequencing (WGS), including a previously missed large deletion of the first exons. Despite the absence of family samples, the biallelic status of the variants was confirmed. The BBS5 protein’s impact was confirmed on the patient’s cells (presence/absence and size of the cilium) and ciliary function (Sonic Hedgehog pathway). This study highlights the importance of WGS and the challenge of reliable structural variant detection in patients’ genetic explorations as well as functional tests to assess a variant’s pathogenicity

Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140

International audienceCiliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7 kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders

Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress

International audienceThe ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cuta-neous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The protea-some content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 tran-scriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts , suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid-specific SINE-R/VNTR/Alu type F (SVA-F) insertion in exon 13 of BBS1 in eight families. In six families, the repeat insertion was found in trans with c.1169 T > G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10 800 control individuals from gnomAD-SV) and due to a founder effect. Its 2435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient-derived cell lines confirmed that the BBS1 SVA-F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation

10q26 deletion syndrome: a French cohort study

International audience10q26 deletion syndrome (OMIM #609625) is a rare autosomal dominant genetic disorder with about 100 patients reported. Most cases are sporadic. Global development delay, short stature, microcephaly and typical facial appearance with triangular face, large forehead, low-set malformed ears, hypertelorism, prominent nose and a thin vermilion of the upper lip constitute the main clinical features. The clinical spectrum is very heterogeneous and neurobehavioral manifestations, deafness, limb malformations, cardiac and urogenital abnormalities can be associated. Thus, patients with 10q26 chromosomal deletion need multidisciplinary management strategies from birth. One of the main reasons for this heterogeneity is the variety of 10qter region chromosomal deletions summarized into the “10q26 deletion syndrome”. Various studies proposed critical regions to explain the main phenotype (Yatzenko et al., 2009; Choucair et al., 2015; Lin S et al., 2016) or more specific features (Vera-Carbonell et al., 2015; Choucair et al., 2015). In addition, these studies proposed about 20 genes of interest such as DOCK1 and FGFR2 to explain the different clinical features observed. We report a French ACLF cohort of 35 patients from 9 centers presenting 10q26 complete or partial deletions (size: 64kb to 12.5Mb), complex chromosomal rearrangement and derivative chromosomes diagnosed using DNA-array, to bring a further insight of the genotype/phenotype correlation

Am J Med Genet A

A small but growing body of scientific literature is emerging about clinical findings in patients with 19p13.3 microdeletion or duplication. Recently, a proximal 19p13.3 microduplication syndrome was described, associated with growth delay, microcephaly, psychomotor delay and dysmorphic features. The aim of our study was to better characterize the syndrome associated with duplications in the proximal 19p13.3 region (prox 19p13.3 dup), and to propose a comprehensive analysis of the underlying genomic mechanism. We report the largest cohort of patients with prox 19p13.3 dup through a collaborative study. We collected 24 new patients with terminal or interstitial 19p13.3 duplication characterized by array-based Comparative Genomic Hybridization (aCGH). We performed mapping, phenotype-genotype correlations analysis, critical region delineation and explored three-dimensional chromatin interactions by analyzing Topologically Associating Domains (TADs). We define a new 377 kb critical region (CR 1) in chr19: 3,116,922-3,494,377, GRCh37, different from the previously described critical region (CR 2). The new 377 kb CR 1 includes a TAD boundary and two enhancers whose common target is PIAS4. We hypothesize that duplications of CR 1 are responsible for tridimensional structural abnormalities by TAD disruption and misregulation of genes essentials for the control of head circumference during development, by breaking down the interactions between enhancers and the corresponding targeted gene