ABSTRACT
The vast majority of deeply intronic genomic variants are benign, but some extremely rare or private deep intronic variants lead to exonification of intronic sequence with abnormal transcriptional consequences. Damaging variants of this class are likely underreported as causes of disease for several reasons: Most clinical DNA and RNA testing does not include full intronic sequences; many of these variants lie in complex repetitive regions that cannot be aligned from short-read whole-genome sequence; and, until recently, consequences of deep intronic variants were not accurately predicted by in silico tools. We evaluated the frequency and consequences of rare deep intronic variants for families severely affected with breast, ovarian, pancreatic, and/or metastatic prostate cancer, but with no causal variant identified by any previous genomic or cDNA-based approach. For 10 tumor-suppressor genes, we used multiplexed adaptive sampling long-read DNA sequencing and cDNA sequencing, based on patient-derived DNA and RNA, to systematically evaluate deep intronic variation. We identified all variants across the full genomic loci of targeted genes, applied the in silico tools SpliceAI and Pangolin to predict variants of functional consequence, and then carried out long-read cDNA sequencing to identify aberrant transcripts. For eight of the 120 (6%) previously unsolved families, rare deep intronic variants in BRCA1, PALB2, and ATM create intronic pseudoexons that are spliced into transcripts, leading to premature truncations. These results suggest that long-read DNA and cDNA sequencing can be integrated into variant discovery, with strategies for accurately characterizing pathogenic variants.
ABSTRACT
Importance: In the US, most childhood-onset bilateral sensorineural hearing loss is genetic, with more than 120 genes and thousands of different alleles known. Primary treatments are hearing aids and cochlear implants. Genetic diagnosis can inform progression of hearing loss, indicate potential syndromic features, and suggest best timing for individualized treatment. Objective: To identify the genetic causes of childhood-onset hearing loss and characterize severity, progression, and cochlear implant success associated with genotype in a single large clinical cohort. Design, Setting, and Participants: This cross-sectional analysis (genomics) and retrospective cohort analysis (audiological measures) were conducted from 2019 to 2022 at the otolaryngology and audiology clinics of Seattle Children's Hospital and the University of Washington and included 449 children from 406 families with bilateral sensorineural hearing loss with an onset younger than 18 years. Data were analyzed between January and June 2022. Main Outcomes and Measures: Genetic diagnoses based on genomic sequencing and structural variant analysis of the DNA of participants; severity and progression of hearing loss as measured by audiologic testing; and cochlear implant success as measured by pediatric and adult speech perception tests. Hearing thresholds and speech perception scores were evaluated with respect to age at implant, months since implant, and genotype using a multivariate analysis of variance and covariance. Results: Of 406 participants, 208 (51%) were female, 17 (4%) were African/African American, 32 (8%) were East Asian, 219 (54%) were European, 53 (13%) were Latino/Admixed American, and 16 (4%) were South Asian. Genomic analysis yielded genetic diagnoses for 210 of 406 families (52%), including 55 of 82 multiplex families (67%) and 155 of 324 singleton families (48%). Rates of genetic diagnosis were similar for children of all ancestries. Causal variants occurred in 43 different genes, with each child (with 1 exception) having causative variant(s) in only 1 gene. Hearing loss severity, affected frequencies, and progression varied by gene and, for some genes, by genotype within gene. For children with causative mutations in MYO6, OTOA, SLC26A4, TMPRSS3, or severe loss-of-function variants in GJB2, hearing loss was progressive, with losses of more than 10 dB per decade. For all children with cochlear implants, outcomes of adult speech perception tests were greater than preimplanted levels. Yet the degree of success varied substantially by genotype. Adjusting for age at implant and interval since implant, speech perception was highest for children with hearing loss due to MITF or TMPRSS3. Conclusions and Relevance: The results of this cross-sectional study suggest that genetic diagnosis is now sufficiently advanced to enable its integration into precision medical care for childhood-onset hearing loss.