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IntroductionRapid advancements in Next Generation Sequencing (NGS) and bioinformatics tools have allowed physicians to obtain genetic testing results in a more rapid, cost-effective, and comprehensive manner than ever before. Around 50% of pediatric sensorineural hearing loss (SNHL) cases are due to a genetic etiology, thus physicians regularly utilize targeted sequencing panels that identify variants in genes related to SNHL. These panels allow for early detection of pathogenic variants which allows physicians to provide anticipatory guidance to families. Molecular testing does not always reveal a clear etiology due to the presence of multigenic variants with varying classifications, including the presence of Variants of Uncertain Significance (VUS). This study aims to perform a preliminary bioinformatics characterization of patients with variants associated with Type II Usher Syndrome in the presence of other multigenic variants. We also provide an interpretation algorithm for physicians reviewing molecular results with medical geneticists.MethodsReview of records for multigenic and/or VUS results identified several potential subjects of interest. For the purposes of this study, two ADGRV1 compound heterozygotes met inclusion criteria. Sequencing, data processing, and variant calling (the process by which variants are identified from sequence data) was performed at Invitae (San Francisco CA). The preliminary analysis followed the recommendations outlined by the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) in 2015 and 2019. The present study utilizes computational analysis, predictive data, and population data as well as clinical information from chart review and publicly available information in the ClinVar database.ResultsTwo subjects were identified as compound heterozygotes for variants in the gene ADGRV1. Subject 1's variants were predicted as deleterious, while Subject 2's variants were predicted as non-deleterious. These results were based on known information of the variants from ClinVar, multiple lines of computational data, population databases, as well as the clinical presentation.DiscussionEarly molecular diagnosis through NGS is ideal, as families are then able to access a wide range of resources that will ultimately support the child as their condition progresses. We recommend that physicians build strong relationships with medical geneticists and carefully review their interpretation before making recommendations to families, particularly when addressing the VUS. Reclassification efforts of VUS are supported by studies like ours that provide evidence of pathogenic or benign effects of variants.

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP. [ABSTRACT FROM AUTHOR]

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP.

• A Novel mouse model of USH developed using KM and CBA/J mice. • KMush/ush mice exhibit an USH phenotype based on various assessment tools. • A new mouse model contains Adgrv1 mutations and can be used for audiological research. Previously, we discovered a strain of Kunming mice, referred to as the KMush/ush strain, that exhibited notably abnormal electroretinogram (ERG) readings and elevated thresholds for auditory brainstem responses (ABRs), which resembled the characteristics of Usher Syndrome (USH). We successfully identified the pathogenic genes, Pde6b and Adgrv1 , after KMush/ush crossbred with CBA/CaJ mice, referred to as CBA-1ush/ush, CBA-2ush/ush or CBA-2ush/ush. In this investigation, we crossbred KMush/ush and CBA/J mice to establish novel recombinant inbred lines and analysed their phenotypic and genotypic characteristics. ERG readings, ABR testing, fundus morphology, histological examination of the retina and inner ear, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, western blotting, DNA sequence analysis and behavioural experiments were performed to assess the phenotypes and genotypes of the progeny lines. No obvious waveforms in the ERG were detected in F1 hybrid mice while normal ABR results were recorded. The F2 hybrids, which were called J1ush/ush or J2ush/ush, exhibited segregated hearing-loss phenotypes. J1ush/ush mice had a retinitis pigmentosa (RP) phenotype with elevated ABR thresholds, whereas J2ush/ush mice exhibited only the RP phenotype. Interestingly, J1ush/ush mice showed significantly higher ABR thresholds than wild-type mice at 28 days post born (P28), and RT-qPCR and DNA-sequencing analysis showed that Adgrv1 gene expression was significantly altered in J1ush/ush mice, but histological analysis showed no significant structural changes in the organ of Corti or spiral ganglia. Further elevation of ABR-related hearing thresholds by P56 manifested only as a reduced density of spiral ganglion cells, which differed significantly from the previous pattern of cochlear alterations in CBA-2ush/ush mice. We successfully introduced the hearing-loss phenotype of inbred mice with USH into CBA/J mice, which provides a good animal model for future studies on the important physiological roles of the Adgrv1 gene in inner-ear structure and for therapeutic studies targeting Adgrv1 -mutated USH. [ABSTRACT FROM AUTHOR]