Introduction

Hearing loss is the most common inherited sensory impairment in humans. More than 50% of prelingual sensorineural hearing loss (SNHL) is attributed to genetic causes, and approximately 80% of these cases are classified as non-syndromic SNHL (NSHL)1. Non-syndromic prelingual SNHL is genetically heterogeneous, but autosomal recessive inheritance accounts for the majority of cases, representing approximately 80%2,3. To date, the most frequent genetic cause of AR-NSHL is a mutation in the GJB2 gene located at the DFNB1 locus4. The second most commonly affected locus is DFNB16, caused by pathogenic variants in the STRC gene. STRC variants are increasingly recognized as a major cause of non-syndromic hearing loss and have been reported at relatively high prevalence across various ethnic populations5,6. In mixed-ethnicity cohorts with hearing loss, STRC deletions or mutations have been reported at frequencies ranging from 5.4% to 16.1%. In the United States and Italy, prevalence rates have been reported at 6–11.2% and 6%, respectively. In Asian populations, the estimated prevalence is 1.7–2.4% in Japan and 10.8% in Korea. Notably, in Korean patients with mild-to-moderate SNHL, STRC mutations were identified as the most common genetic cause, while in a comparable Japanese cohort, they were found to be the second most frequent cause following GJB2 single nucleotide variants7,8,9,10,11,12,13,14.

The STRC gene encodes stereocilin, a protein localized to the stereocilia bundles of outer hair cells. STRC is located on chromosome 15q15.3, within a complex segmental duplication region that also includes three other genes: PPIP5K1 (MIM: 610979), CATSPER2 (MIM: 607249), and CKMT1A (MIM: 613415)15. Genetic testing of STRC is particularly challenging due to the presence of a highly homologous pseudogene, pSTRC, in close proximity. STRC and pSTRC share 99.6% sequence identity in coding regions and 98.9% when intronic sequences are included, making them nearly indistinguishable by conventional sequencing methods. Moreover, pSTRC is a nonfunctional pseudogene due to a nonsense mutation in exon 206,16. These structural complexities necessitate specialized approaches for accurate STRC analysis. Although most STRC-related pathogenic variants are copy number variations (CNVs) involving the entire gene or large portions of it, point mutations, small insertions/deletions, and gene conversions have also been reported. Therefore, variants detected by next-generation sequencing (NGS) must be validated using gene-specific long-range PCR followed by Sanger sequencing to avoid misinterpretation caused by pseudogene contamination. Additionally, for deletion screening, a quantitative competitive fluorescence PCR (QCF-PCR) assay—exploiting length differences between STRC and pSTRC—has been implemented in diagnostic workflows17,18.

Among the various NGS approaches, massively parallel sequencing of targeted deafness genes has become the most frequently used and effective method for patients without biallelic GJB2 mutations. Currently, large-scale NGS is widely applied in clinical practice, enabling accurate diagnosis through dedicated bioinformatics pipelines. In this study, we aimed to identify patients with STRC mutations using NGS-based methods and to characterize their clinical and genetic features, including auditory genotype–phenotype correlations.

Results

We identified and clinically evaluated 14 children and 9 adults with pathogenic variants in the STRC gene (Table 1). Genetic diagnoses were confirmed by a board of otolaryngologists and clinical geneticists according to the hearing loss-specified American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines in the Deafness Variation Database19. STRC gene deletion ,defined as a contiguous loss of exons 19–25 detected by P461-A1 multiplex ligation-dependent probe amplification (MLPA) kit, or one exon deletion in STRC was confirmed pathogenic according to the ACMG/AMP guideline. In all cases with STRC gene deletion, a CATSPER2 gene deletion, defined as a contiguous loss of exons 1,2,4,7, and 12 detected by P461-A1 MLPA kit, was also identified (Table 1). Pure-tone audiometry (PTA) could not be performed in patients younger than 4 years; instead, auditory brainstem response (ABR) and/or auditory steady-state response (ASSR) tests were used. For patients aged 4 to 5 years, behavioral audiometry with headphones was conducted. Due to differences in testing modalities, patients who underwent ASSR instead of PTA—primarily because of their young age—were excluded from the audiometric comparison analysis. To ensure consistency in measurement, only patients who underwent behavioral audiometry were included.

Table 1 Clinical and genetic characteristics of patients with STRC-related hearing loss.
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Table 2 Clinical and Genetic Characteristics of Patients with STRC-Related Hearing Loss.
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Among the 19 patients who underwent PTA, most exhibited flat or gently sloping audiometric configurations, with elevated thresholds particularly in the mid-to-high frequencies. When comparing mean thresholds across frequencies, the threshold at 500 Hz was significantly different from those at higher frequencies. The mean hearing thresholds at each frequency were as follows: right ear – 32.9, 43.4, 44.7, 41.8, and 44.2 dB HL; left ear – 32.9, 43.4, 44.5, 45.0, and 45.3 dB HL (at 500, 1,000, 2,000, 4,000, and 8,000 Hz, respectively) (Fig. 1).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Mean hearing thresholds by frequency in patients with STRC-related hearing loss (n = 19). Only patients who were able to undergo behavioral audiometry were included.

We compared the distribution of average pure-tone hearing thresholds between two genetically defined groups: patients with homozygous STRC/CATSPER2 gene deletion and those with other pathogenic variants (e.g., heterozygous exon deletions, nonsense mutations, or compound heterozygous mutations). Audiometric comparisons were based on the mean initial PTA thresholds at 0.5, 1, 2, and 4 kHz for the better ear (Fig. 2). For the group with homozygous STRC/CATSPER2 gene deletions, individual audiograms were presented alongside family pedigrees (Supplemental Fig. 1).

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Comparison of mean hearing thresholds among STRC genotypic groups. Comparison of mean hearing thresholds (better ear) between genotypic groups: patients with homozygous STRC/CATSPER2 deletions (homo del, n = 10) and those with other pathogenic variants (other, n = 9), including exon deletions, nonsense or missense mutations, and compound heterozygous combination of a STRC gene deletion with another variant (p = 0.549 by Mann–Whitney U test).

Linear mixed model analysis was used to compare serial hearing levels over time. Nineteen ears were assessed at the initial test, 10 ears at the 2-year follow-up, and 6 ears at the 4-year follow-up, analyzing thresholds from the better ear. No statistically significant differences in hearing thresholds were observed over time at any frequency (Fig. 3). In 75% of ears (9 out of 12), the difference in average hearing thresholds between the first and last tests was within 5 dB or not statistically significant. The relationship between age and mean PTA thresholds was analyzed using linear regression, and no significant age-related progression was observed in the better ear across all genotypic subgroups (Fig. 4).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Longitudinal changes in hearing thresholds in patients with STRC mutations. Better ear thresholds at 500–8000 Hz measured at baseline (●, n = 19), 2 years (■, n = 10), and 4 years (▲, n = 6); no significant time effect (linear mixed model, F(2, 4.79) = 1.75, p = 0.268).

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Age-related change in mean pure tone audiometry (PTA) thresholds by genotype. (A-C) Mean PTA thresholds (average of 0.5, 1, 2, and 4 kHz) of the better ear are plotted as a function of age. Solid lines represent best-fit linear regression models, and dashed lines indicate 95% confidence intervals. No statistically significant age-related progression was observed in any group. (A) Better ear thresholds in all patients (n = 19). Regression: Y = − 0.03·X + 39.80 (95% CI: − 0.37 to 0.30, r² = 0.002, p = 0.84). (B) Better ear thresholds in patients with homozygous STRC/CATSPER2 gene deletions (n = 10). Regression: Y = 0.21·X + 35.83 (95% CI: − 0.38 to 0.80, r² = 0.076, p = 0.44). (C) Better ear thresholds in patients with other STRC variants (n = 9). Regression: Y = − 0.29·X + 45.32 (95% CI: − 0.77 to 0.18, r² = 0.237, p = 0.18).

Discussion

In this study, we demonstrated that patients with STRC-related hearing loss predominantly exhibited mild-to-moderate SNHL, characterized by flat or gently sloping audiometric configurations affecting the mid-to-high frequencies. Notably, no significant differences in hearing thresholds were observed between patients with homozygous STRC gene deletions and those with other pathogenic variants, such as heterozygous exon deletions, nonsense mutations, or compound heterozygous mutations. Furthermore, longitudinal audiometric evaluations revealed no statistically significant progression of hearing loss over time.

One of the major challenges in analyzing STRC variants lies in its high sequence similarity with the adjacent pseudogene, STRCP1, which complicates read mapping and variant interpretation when using short-read sequencing technologies. In this study, gene-specific long-range PCR was not employed to confirm the origin of detected variants. For cases involving novel single nucleotide variants, we applied multiple layers of bioinformatic and manual validation. This included visual inspection of mapped reads using Integrative Genomics Viewer (IGV) to assess mapping quality and rule out ambiguous alignments, as well as sequence similarity analysis using UCSC BLAT. In these analyses, the reads demonstrated alignment patterns more consistent with STRC than STRCP1. While these approaches provide reasonable confidence in variant localization, we acknowledge that long-range PCR remains the gold standard for definitive confirmation, especially in cases involving novel or uncertain variants. We recommend that future studies incorporate orthogonal validation methods to further reduce the risk of pseudogene-related misinterpretation, particularly in clinical diagnostic settings.

The STRC gene encodes stereocilin, an extracellular structural protein localized in the stereocilia of outer hair cells (OHCs). Stereocilin plays a crucial role in anchoring the OHC bundle to the tectorial membrane20,21,22. The absence of upper horizontal connectors or abnormalities in tip-link structures between stereocilia may impair cochlear amplification, particularly affecting high-frequency hearing22,23. Consistent with previous findings, our study confirmed a statistically significant threshold difference between 500 Hz and higher frequencies.

Previous reports investigating STRC-related SNHL have identified patients across a wide age range, consistently revealing a high-frequency hearing loss pattern. Most individuals demonstrated mild-to-moderate hearing loss without progression to severe impairment8,9,13,14,24,25. In agreement with these studies, our findings show that thresholds at frequencies ≥ 1,000 Hz were indicative of moderate hearing loss. Prior studies have also demonstrated that this hearing profile typically persists into the fifth or sixth decade of life, with no evidence of substantial progression9,14,25,26. In our cohort, 75% of ears showed stable thresholds (change < 5 dB), aligning with previous reports of similar stability rates25. Although some studies have reported progressive hearing loss, the changes were generally statistically insignificant or occurred at a slow rate (e.g., 0.6 dB/year)27,28.

We further categorized patients based on genotypic patterns and compared their audiologic outcomes. Our data support previous findings that no significant differences in mean hearing thresholds exist between genotypes. Simi et al. reported no statistical difference in baseline pure-tone averages between patients with homozygous STRC deletions and those with compound heterozygous variants28. Similarly, Markova et al. compared average hearing thresholds among three genotype-based subgroups—homozygous deletions, compound heterozygotes with two distinct deletions, and compound heterozygotes with a deletion and a point mutation in trans—and found comparable thresholds across all groups, although a few individuals in the homozygous deletion and deletion/point mutation groups exhibited relatively better hearing25.

This study has several limitations. Audiologic assessments were not uniformly applied across all participants, and hearing loss progression may not have been detected in some patients with shorter follow-up periods. To enhance statistical power, we performed linear mixed model analysis.

Given that STRC-related SNHL typically presents with mild-to-moderate severity and may remain undetected without active screening, the importance of accurate molecular diagnosis is heightened. Compared to severe-to-profound hearing loss, mild-to-moderate SNHL is more likely to be overlooked or underestimated. While severe hearing loss is often identified early, enabling timely intervention that can mitigate significant medical, educational, and socioeconomic burdens, mild-to-moderate SNHL may go undetected and, if unmanaged, result in greater academic challenges than in patients with properly treated hearing loss29,30. The STRC gene is known to be technically challenging to analyze due to the presence of a highly homologous pseudogene and frequent genomic duplications, which limit the sensitivity of standard sequencing methods13,18. Therefore, supplementary genetic testing methods, such as MLPA or CNV analysis, are recommended to ensure accurate detection of pathogenic variants.

In conclusion, this study characterized the clinical and audiologic features of STRC-related SNHL, demonstrating that most patients exhibited mild-to-moderate hearing loss with a stable, non-progressive course. Notably, hearing thresholds were comparable between patients with homozygous STRC gene deletions and those with other genotypes. Given the subtle nature of STRC-related SNHL, such cases may be easily overlooked without active screening. Incorporating STRC analysis into broader genetic testing strategies may facilitate earlier diagnosis and intervention, potentially improving long-term developmental and educational outcomes.

Methods

Patient enrollment

This study enrolled patients from the Yonsei University Hearing Loss (YUHL) cohort, a registry for individuals with genetic hearing loss. Patients included in the cohort either had a family history of hearing loss or voluntarily underwent genetic testing. From this larger cohort, 23 probands were retrospectively selected between 2017 and 2024 based on the identification of biallelic pathogenic variants in the STRC gene. The cohort comprised 6 males and 17 females, with a mean age of 13.4 ± 9.7 years. The study was approved by the Severance Hospital Institutional Review Board (approval no. 4–2015-0659), and written informed consent was obtained from all participants. All procedures performed in this study were in accordance with the ethical standards of the institutional review board and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Eight patients did not have any relatives with hearing loss within two generations (Table 1).

Evaluation of hearing function

Audiologic evaluations were conducted using age-appropriate methods. For children under 4 years of age, ABR and/or ASSR tests were performed. Children aged 4–5 years underwent behavioral audiometry with headphones (play audiometry), while conventional (PTA was used for older patients. Medical and family histories, clinical information, and age of onset of hearing loss were documented. Patients under serial follow-up received repeated audiologic assessments at each visit. Specifically, three children underwent ABR and ASSR, and four children were evaluated using behavioral pure-tone audiometry. Audiologic evaluations were conducted during the same study period, from 2017 to 2024. One patient did not undergo audiologic testing at the time of enrollment; therefore, hearing assessments were available for 22 of the 23 patients.

Genetic analysis

Genetic testing was performed on affected individuals and their family members using NGS, as previously described31,32,33,34,35,36. A two-track approach was employed, utilizing either whole-exome/genome sequencing (WES/WGS) or a targeted deafness gene panel, depending on insurance coverage and patient preference. For panel-based testing, a custom-designed panel targeting 207 deafness-associated genes was used, as previously reported37,38. WES was performed using the Agilent SureSelect V5 enrichment capture kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocol. Sequencing was carried out on the Illumina MiSeq platform (San Diego, CA, USA) using the MiSeq Reagent Kit v2 (300 cycles). Segregation analysis was conducted using Sanger sequencing. Variant detection was performed using the “Basic Variant Caller” function in CLC software, applying thresholds of a minimum of five reads, 20× coverage, and a variant allele frequency ≥ 20%. Variants with minor allele frequencies > 0.5% (for recessive genes) or > 0.05% (for dominant genes) in the dbSNP and gnomAD databases were excluded.

CNVs were detected using MLPA for panel-tested patients and ExomeDepth (v1.1.16) for WES data. CNV calls from WES were confirmed by MLPA using MLPA Probemix P461 and reagent kit (MRC-Holland, Amsterdam, Netherlands). CNV of “STRC gene deletion” was defined as a contiguous loss of exons 19–28 (covered exons by P461 kit) on at least one allele, which we interpret as complete loss of the gene because exons 1–18 cannot be reliably assessed due to pseudogene homology. Similarly, CNV of “CATSPER2 gene deletion” was defined as a contiguous loss of exons 1, 2, 3, and 7 (covered exons by P461 kit). Final genetic diagnoses were made by a multidisciplinary team of otolaryngologists and clinical geneticists in accordance with hearing loss–specific interpretation guidelines from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP), as implemented in the Deafness Variation Database. Patients with one pathogenic/likely pathogenic variant in trans with a VUS were also included when segregation and phenotype supported a molecular diagnosis of STRC-related hearing loss.

Statistical analysis

A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS for Windows, version 20.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation. The Mann–Whitney U test or linear mixed model analysis were used to compare average thresholds between groups and to evaluate longitudinal changes in hearing levels, respectively. In addition, linear regression analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) to assess the relationship between age and hearing thresholds.