Validation of Nanopore long-read sequencing to resolve RPGR ORF15 genotypes in individuals with X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is characterized by progressive vision loss leading to legal blindness in males and a broad severity spectrum in carrier females. Pathogenic alterations of the retinitis pigmentosa GTPase regulator gene (RPGR) are responsible for over 70% of XLRP cases. In the retina, the RPGRORF15 transcript includes a terminal exon, called ORF15, that is altered in the large majority of RPGR-XLRP cases. Unfortunately, due to its highly repetitive sequence, ORF15 represents a considerable challenge in terms of sequencing for molecular diagnostic laboratories. However, in a recent preliminary work Yahya et al. reported a long-read sequencing approach seeming promising. Here, the aim of the study was to validate and integrate this new sequencing strategy in a routine screening workflow. For that purpose, we performed a masked test on 52 genomic DNA samples from male and female individuals carrying 32 different pathogenic ORF15 variations including 20 located in the highly repetitive region of the exon. For the latter, we have obtained a detection rate of 80-85% in males and 60-80% in females after bioinformatic analyses. These numbers raised to 100% for both status after adding a complementary visual inspection of ORF15 long-reads. In accordance with these results, and considering the frequency of ORF15 pathogenic variations in XLRP, we suggest that a long-read screening of ORF15 should be systematically considered before any other sequencing approach in subjects with a diagnosis compatible with XLRP.

Functional analysis of germline VANGL2 variants using rescue assays of vangl2 knockout zebrafish.

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.

Identification and in vivo functional investigation of a HOMER2 nonstop variant causing hearing loss.

DFNA68 is a rare subtype of autosomal dominant nonsyndromic hearing impairment caused by heterozygous alterations in the HOMER2 gene. To date, only 5 pathogenic or likely pathogenic coding variants, including two missense substitutions (c.188 C > T and c.587 G > C), a single base pair duplication (c.840dupC) and two short deletions (c.592_597delACCACA and c.832_836delCCTCA) have been described in 5 families. In this study, we report a novel HOMER2 variation, identified by massively parallel sequencing, in a Sicilian family suffering from progressive dominant hearing loss over 3 generations. This novel alteration is a nonstop substitution (c.1064 A > G) that converts the translational termination codon (TAG) of the gene into a tryptophan codon (TGG) and is predicted to extend the HOMER2 protein by 10 amino acids. RNA analyses from the proband suggested that HOMER2 transcripts carrying the nonstop variant escaped the non-stop decay pathway. Finally, in vivo studies using a zebrafish animal model and behavioral tests clearly established the deleterious impact of this novel HOMER2 alteration on hearing function. This study identifies the fourth causal variation responsible for DFNA68 and describes a simple in vivo approach to assess the pathogenicity of candidate HOMER2 variants.

CEP162 deficiency causes human retinal degeneration and reveals a dual role in ciliogenesis and neurogenesis.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Identification of the First Single GSDME Exon 8 Structural Variants Associated with Autosomal Dominant Hearing Loss.

GSDME, also known as DFNA5, is a gene implicated in autosomal dominant nonsyndromic hearing loss (ADNSHL), affecting, at first, the high frequencies with a subsequent progression over all frequencies. To date, all the GSDME pathogenic variants associated with deafness lead to skipping of exon 8. In two families with apparent ADNSHL, massively parallel sequencing (MPS) integrating a coverage-based method for detection of copy number variations (CNVs) was applied, and it identified the first two causal GSDME structural variants affecting exon 8. The deleterious impact of the c.991-60_1095del variant, which includes the acceptor splice site sequence of exon 8, was confirmed by the study of the proband's transcripts. The second mutational event is a complex rearrangement that deletes almost all of the exon 8 sequence. This study increases the mutational spectrum of the GSDME gene and highlights the crucial importance of MPS data for the detection of GSDME exon 8 deletions, even though the identification of a causal single-exon CNV by MPS analysis is still challenging.

The Study of a 231 French Patient Cohort Significantly Extends the Mutational Spectrum of the Two Major Usher Genes MYO7A and USH2A.

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.

When Familial Hearing Loss Means Genetic Heterogeneity: A Model Case Report.

We describe a family with both hearing loss (HL) and thrombocytopenia, caused by pathogenic variants in three genes. The proband was a child with neonatal thrombocytopenia, childhood-onset HL, hyper-laxity and severe myopia. The child's mother (and some of her relatives) presented with moderate thrombocytopenia and adulthood-onset HL. The child's father (and some of his relatives) presented with adult-onset HL. An HL panel analysis, completed by whole exome sequencing, was performed in this complex family. We identified three pathogenic variants in three different genes: MYH9, MYO7A and ACTG1. The thrombocytopenia in the child and her mother is explained by the MYH9 variant. The post-lingual HL in the paternal branch is explained by the MYO7A variant, absent in the proband, while the congenital HL of the child is explained by a de novo ACTG1 variant. This family, in which HL segregates, illustrates that multiple genetic conditions coexist in individuals and make patient care more complex than expected.

SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect.

The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.