Novel Usher syndrome pathogenic variants identified in cases with hearing and vision loss

The study involved three families from the Newfoundland population, including two multiplex families. Clinical evaluations included air conduction thresholds using pure-tone audiometry, noting the audiogram configuration, severity, onset and progression. Vision was assessed with ocular examination, visual acuity and visual field testing, electroretinography (ERG) and fluorescein angiography of the retina.

For Family R2100, hearing loss (HL) is present in three sibships with varying audioprofiles, including two sisters who are the product of a consanguineous union (Fig. 1a). The proband (PID V-2) diagnosed with hearing loss at 3 years, presents by age 7 with a mild to moderate bilateral sensorineural HL, and her younger sister (PID V-3) was diagnosed at age 3 with a similar audioprofile (Fig. 1b).

We also recruited a case from our local medical genetics’ clinic (Family R4110) of a child diagnosed at 3 months (following newborn hearing screening), who presents by age 3 with mild to moderate bilateral sensorineural HL (Fig. 1 c, d). In another multiplex family (R0723), the proband and his brothers (PIDs II-5, II-3 and II-6, respectively) were first diagnosed with RP in mid 5th decade when their central vision decreased to the point that they met criteria for legally recognized blindness (Fig. 1e). They reported reduced night vision since the mid-second decade, and hearing loss since young childhood. The proband had been fitted for hearing aids for moderate to severe hearing loss. In R0273, the proband and his brothers (PIDs II-5, II-3 and II-6 respectively) had reported experiencing reduced night vision since their mid-third decade and were all diagnosed with RP in the mid-fifth decade when their central vision decreased. Throughout the course of ongoing clinical assessment, it was noted that the proband was fitted for hearing aids due to a moderate to severe HL and although the age of onset was unknown, he had HL at a young age. Two nieces with early hearing loss were also diagnosed with RP on follow-up, which prompted targeted genetic testing for known USH genes (Fig. 1d).

In the case of family R0723, a targeted gene panel for 13 USH genes (CEI Molecular Diagnostic Laboratory, Portland, OR, USA) was offered through a research study on hereditary vision loss. In the clinical case of the child who failed newborn hearing screening (Family R4110), the family was offered targeted screening (158 syndromic and non-syndromic hearing loss genes, Blueprint Genetics, Comprehensive Hearing Loss and Deafness Panel, version 1, San Francisco, CA, USA). To validate variants of interest and check for co-segregation with RP and/or HL trait in the families, genomic DNA was amplified using custom primers and sequenced in both directions using standard touchdown PCR protocols (ABI PRISM 3500XL DNA Analyzer; Applied Biosystems, Foster City, CA, USA). Sequence traces were analyzed using Mutation Surveyor Software (version 5.00, SoftGenetics LLC State College, PA 16803).

We initially screened the proband of Family 2100 for deafness alleles previously identified in the NL population and submitted representative audiograms to Audiogene, a program comparing these to average audiograms of 34 deafness loci [12]. As this targeted approach failed to solve Family 2100, a traditional linkage study was done. For the linkage analysis, we selected three affected and two unaffected relatives (PID V-2, V-3, III-9, and IV-3, IV-4 respectively) (Fig. 1a) and genotyped 17,407 polymorphic markers with the Illumina Human610-Quad chip. Multipoint linkage analysis (Merlin v1.1.2) [13] was performed under a recessive model with a disease allele frequency of 0.07 and a penetrance of 99%. In order to screen candidate genes within linked regions, whole exome sequencing was carried out on 5 family members (two affected offspring and their parents: PID V-2, V-3, IV-3, IV-4 respectively) using the Ion Torrent AmpliSeq RDY Exome Kit (Life Technologies, Cat. #A27193). Purified libraries were loaded onto an Ion Proton PI v3 chip and sequenced with the Ion Torrent Proton. Only rare variants (MAF < 1%) that mapped to linked regions, had a depth of coverage >20X and were of medium to high impact were validated by Sanger sequencing and selected for cascade screening. Population frequencies were determined using 124 ethnically-matched controls.

For variants of interest that reside within canonical +/− 1 or 2 splice sites, we conducted in silico analyses using Alamut Visual (Interactive Biosoftware Inc., Rouen, France), a program that provides a splicing alteration report by linking to the MaxEnt, NNSPPLICE, SplicSiteFinder, and GeneSplicer algorithms.

In order to experimentally validate splicing predictions, we extracted total RNA from B-cell lymphocytes using standard TRIzol-based methods (Thermo-fisher, Cat. #15596026) and prepared cDNA libraries with the Superscript III First Strand Synthesis System (Thermo-fisher, Cat. #18080093). RT-PCR was carried out with primers that spanned candidate splicing regions, followed by TOPO TA-Cloning Kit for Sequencing with One Shot TOP10 Chemically Competent E. coli (Invitrogen, #K457540) according to the manufacturer’s protocol. RT-PCR products were Sanger sequenced and then analyzed using Mutation Surveyor Software (version 5.00, SoftGenetics LLC State College, PA 16803).

In the step-wise analysis of hearing loss Family 2100, the proband (PID V-2; Fig. 1a) screened negative for all hearing loss variants previously identified in the NL population. Genome-wide linkage analysis (assuming autosomal recessive inheritance) yielded positive LOD scores suggestive of linkage for 8 genomic regions and the theoretical maximum LOD (1.68) for regions on chromosomes 3, 4, 5, 6 and 15 (Table 1). Subsequently, exome sequencing identified 278 variants that were shared between the proband (PID V-2) and her affected sister (PID V-3). Of these, only eight variants remained after filtering for rare variants (MAF < 1%) of medium to high impact that mapped to linked regions and had a depth of coverage >20X (Table 2). Seven of these variants were shown to be false positive INDELs (did not validate with Sanger sequencing) or did not reside within genes associated with syndromic or non-syndromic HL [14]. The remaining candidate (ADGRV1 c.17062C > T; p.Arg5688*) is a nonsense variant associated with USH2C (Fig. 2a). Co-segregation analysis confirmed that the affected sisters were homozygous for ADGRV1 c.17062C > T and their parents were unaffected carriers. The only other available affected relative for cascade sequencing was a maternal uncle (PID III-9; Fig. 1a) who was wild-type (two normal copies) and subsequently confirmed to have acquired his hearing loss after a serious diving accident. The nonsense ADGRV1 variant is predicted to create a premature stop codon nearing the N-terminus of ADGRV1, preventing the translation of all 7 transmembrane domains. Furthermore, the ADGRV1 c.17062C > T variant is absent in the population controls and has a single heterozygous entry in ExAC browser from the European (Non-Finnish) population. According to ACMG guidelines, the ADGRV1 nonsense variant should be classified as pathogenic as it meets the following criteria: PVS1, PM2, PM3, PP1, PP3.

At the start of this study, we were aware of hearing loss (HL) in three sibships with varying audioprofiles, including two sisters who are the product of a consanguineous union. On the basis of the pending molecular diagnosis of Usher syndrome and the serious prognosis, the clinic contacted the sisters in order to request a visual examination (PID V-2 and V-3; Fig. 1a). The sisters are now in their late third and early fourth decade. Both women report impaired vision for some years. Ophthalmology reports on both sisters indicated definite features of RP. Further testing of PID V-3 identified bone spicule pigmentation of the retina (Fig. 2b) and a significant reduction in peripheral visual acuity (Fig. 2c), which are consistent with “typical RP”. These findings prompted the clinic to counsel the women regarding their new diagnosis of USH2C.

The comprehensive gene panel that was offered to the clinical case of the 3-year-old child diagnosed with isolated hearing loss at 3 months (Family 4110; Fig. 1c) identified two novel USH2A splicing variants: c.5777-1G > A (Fig. 2d) and c.10388-2A > G (Fig. 2e). Cascade sequencing confirmed the maternal contribution as c.5777-1G > A and the paternal contribution as c.10388-2A > G, and verified these novel variants reside in trans. However, given this child’s young age and the novelty (variants of unknown significance) of the USH2A variants, the genetic testing results are of limited value.

Fortuitously, the targeted USH gene panel offered to Family R0723 identified these same USH2A splicing variants. The proband (PID II-5) and his brother (PID II-3) are homozygous for UHS2A c.5777-1G > A, their nieces (PIDs III-1 and III-2) are compound heterozygotes (c.5777-1G > A; c.10388-2A > G; Fig. 1e). Even though the deceased brother (PID II-6) was not available for genetic testing, he is likely a USH2A c.5777-1G > A homozygote, given the strong family history of RP. Retrospective audiological data on the proband’s niece, PID III-1, from mid-third decade to mid-fifth decade show a stable hearing loss according to GenDeaf guidelines (Fig. 1d) [15]. The proband (PID II-5) has moderate to severe hearing loss in his seventh decade, not significantly worse than his younger niece (PID III-1) in her late fifth decade (Fig. 1d). His other niece, PID III-2, reveals a similar clinical phenotype (data not shown). With respect to RP, the proband (PID II-5) and his two brothers (PID II-3 and II-6) reported decreased night vision by their late 20s (Fig. 1e); however, RP as seen in retinal photographs of the proband was not diagnosed in the brothers until their late 40’s (Fig. 2f). Following the diagnosis of RP in the uncles, their nieces who had documented hearing loss were closely monitored, and reduced visual fields noted at age 14 in PID III-2, indicating the first symptoms of RP. Abnormal dark adaptation and ERG responses were recorded in both nieces in the third decade and retinal photographs of PID III-2 illustrate arterial attenuation, a characteristic sign of early RP (Fig. 2f).

Cascade sequencing revealed that both USH2A c.5777-1G > A and USH2A c.10388-2A > G co-segregate with disease in families R0723 and R4110. In silico analyses using Alamut Visual suite of algorithms predicted that both variants cause exon skipping (MaxEnt: -100.0%, NNSPLICE: -100.0% and SSF: − 100.0%). Using patient-derived cells, Sanger sequencing of cDNA confirmed that USH2A c.5777-1G > A causes the skipping of exon 29 leading to an in-frame deletion (p. Glu1926_Ala1952del) in an affected individual (PID III-2) compared with a control sample (Fig. 2d; Fig. 3a). The sequencing of patient cDNA also determined that USH2A c.10388-2A > G activates a cryptic acceptor site 14 bps downstream of the canonical splice site (Fig. 2e; Fig. 3b), resulting in a premature stop codon (p.Asp3463Alafs*6). Based on cascade sequencing within these families and subsequent RNA analysis, USH2A c.5777-1G > A and c.10388-2A > G can both be classified as pathogenic variants according to the ACMG guidelines (PVS1, PS3, PM2, PM3, PP1, PP3) [16].