This study reports variant data from a geographical region which has been understudied to date. State-wide newborn screening (99% coverage), and follow-up GJB2/6 testing in screen positive individuals revealed biallelic and heterozygous carriage in association with predominantly mild hearing loss. The most frequent variant, c.109G > A, has been described with mild phenotypes, and may partially explain the increase in mild hearing loss detected on newborn screening. Consistent with the literature, the c.35delG variant was associated with more severe HL, while the c.101T > C variant was associated with milder HL and U-shaped audiograms. This information provides a more complete picture of the phenotypic spectrum of GJB2/6 associated HL which provides short-term prognostic data and can inform pre- and post-natal counselling for individuals and families found to carry these variants.
Ascertainment in this study differs from most previously reported literature which genetically evaluated individuals being considered for cochlear implants i.e., typically severe levels of hearing loss [24, 25]. In those studies, the most common variants were c.35delG and c.235delC which were identified in populations from European and Asian backgrounds, respectively. The c.109G > A variant has been reported previously, especially from Asian ancestry cohorts [26], where the minor allele frequency is 0.08 [27]. Thus, it is unsurprising that it is prevalent in our agnostically ascertained cohort where the sensitive equipment used for universal newborn hearing screening and aABR (automated auditory evoked brainstem response) as a screening method can lead to capturing patients that may have mild, transient and/or fluctuating hearing profiles.
An artefact of newborn screening is the detection of mild hearing loss. As mild hearing-loss is being increasingly diagnosed at an earlier age [28], this presents prognostic and management uncertainty for both families [29] and clinicians [30]. This study provides evidence for a genetic basis for many mild hearing loss cases adding to the emerging body of literature describing genotypes in mild and moderate hearing loss cohorts [31, 32].
Prior publications have noted milder hearing loss in association with either c.101T > C or c.109G > A alleles [33]. The pathogenic classifications of both variants were initially controversial, but an international consensus paper classified both as pathogenic with variable expressivity and incomplete penetrance [34]. Consistently, in this Australian cohort this variant is associated with a milder phenotype. This phenotypic information is valuable for clinicians and families presented with these results in infancy, prenatally or as part of reproductive carrier screening [35].
Previous research has shown that a heterozygous GJB2 variant is detected in 10–50% of individuals with hearing loss [36], which can complicate and limit clinical interpretation and management. Additionally, some studies have indicated that carriers of certain variants have been reported to be more likely than ‘non-carriers’ to develop hearing impairment when exposed to other environmental factors or genetic defects [37]. In the current study, heterozygosity was identified in 37% of the cohort and the associated hearing loss phenotype was highly variable ranging from normal (in the better ear) to profound. These findings align with previous publications [33] and may be due to (i) the GJB2 variant being coincidental, with hearing loss secondary to variants in another NSHL gene, (ii) failure to detect a second, possibly intronic functionally significant variant in GJB2, (iii) the GJB2 variant modifies the expression of other variants in related hearing loss genes or (iv) the GJB2 variant being coincidental and the hearing loss stemming from a non-genetic aetiology. Comprehensive panel testing and/or whole genome sequencing may help identify the first two possibilities [38], and further research could possibly elucidate the third.
It is important to appreciate that classification of hearing loss in this study is relative to the better hearing ear. Thus, our study detected individuals with normal hearing and asymmetric hearing loss where the hearing of the contralateral ear could range from mild to profound. The fact that genetic testing was offered in these cases implies that the hearing loss was, at the time of testing, considered to be clinically indicated and/or socially significant to the individual or their families. While hearing loss in some cases may have been complicated by transient, conductive overlay, the findings from the present study support that GJB2 variants can be associated with asymmetric hearing loss [8]. The identification of these asymmetric cases (where one ear is classified as ‘normal’ hearing) may be reduced from this point forward given recommendations to only offer genetic testing in cases of bilateral hearing loss [39]. However, a uniform and consistent approach to genetic testing for patients with NSHL is important to mitigate the risk of uncertain findings. Furthermore, this could potentially reduce the financial and psychological costs associated with inappropriate genetic testing.
The natural history in this cohort was predominantly stable but shows both improvements and progression over time. These findings are consistent with those previously reported in the literature [8, 40]. However, it should be noted that audiogram results become more accurate with increasing age in children, thus fluctuation/progression may reflect the young age of this cohort. Importantly, U-shaped hearing loss was identified more frequently at subsequent time points than initial assessments and was associated with c.101T > C. There is a paucity of literature on U-shaped (mid-frequency) hearing loss, an uncommon audiometric finding, more commonly diagnosed in older individuals [41]. Although U-Shaped hearing loss has not been formally associated with GJB2 generally and the c.101T > C variant specifically, in reviewing previously published audiograms in c.101T > C positive individuals [42], we identified cases of U-Shaped hearing loss. This is clinically significant because this mid-frequency loss is associated with greater difficulty understanding speech in a noisy environment such as a classroom setting. Thus, children may function differentially in quiet and noisy environments, which could mask detection, thus increasing the risk of social problems and fatigue, especially if it is a deterioration [43].
Cumulatively, these results demonstrate a broad phenotypic association with GJB2 variants and some genotype-phenotype associations which can provide prognostic value. This data from a population wide cohort, provides prognostic information for preconception, prenatal and paediatric counselling of couples and families carrying these variants. For example, Freeman et al.’s [44] discussion of views regarding genetic testing for deafness in reproductive settings, highlighted that the recent American College of Medical Genetics and Genomics practice guidelines [35] recommended the inclusion of GJB2 variants in prenatal genetic screening on the basis of prevalence and NSHL being categorised as ‘moderately severe’ [45]. If such guidelines were adopted in Australia, the information in studies like this would be invaluable in counselling.
Strengths of this study include the agnostic mode of ascertainment which allowed for identification of a broad phenotypic spectrum. The centralisation of newborn screening, pathology and clinical data allowed for comprehensive phenotypic characterisation. Limitations include a finite sample size and follow up period, lack of detailed data about other potentially contributing factors for HL and the fact that testing was limited to GJB2 coding variants and a single GJB6 deletion.
Future directions to further assist clinicians in providing genetic counselling in this area could include longer follow-up to clarify stability over time, broadening the phenotype to include developmental outcomes including speech and language development and response to intervention e.g., documenting outcomes of children who have required cochlear implants, and comprehensive panel testing for hearing loss. Cumulatively, this information would provide clinicians and families with greater prognostic and management certainty at the time of diagnosis.