Autosomal recessive nonsyndromic hearing impairment in two Finnish families due to the population enriched CABP2 c.637+1G>T variant

Abstract Background The genetic architecture of hearing impairment in Finland is largely unknown. Here, we investigated two Finnish families with autosomal recessive nonsyndromic symmetrical moderate‐to‐severe hearing impairment. Methods Exome and custom capture next‐generation sequencing were used to detect the underlying cause of hearing impairment. Results In both Finnish families, we identified a homozygous pathogenic splice site variant c.637+1G>T in CAPB2 that is known to cause autosomal recessive nonsyndromic hearing impairment. Four CABP2 variants have been reported to underlie autosomal recessive nonsyndromic hearing impairment in eight families from Iran, Turkey, Pakistan, Italy, and Denmark. Of these variants, the pathogenic splice site variant c.637+1G>T is the most prevalent. The c.637+1G>T variant is enriched in the Finnish population, which has undergone multiple bottlenecks that can lead to the higher frequency of certain variants including those involved in disease. Conclusion We report two Finnish families with hearing impairment due to the CABP2 splice site variant c.637+1G>T.


| INTRODUCTION
Over 70 autosomal recessive (AR) nonsyndromic (NS) hearing impairment (HI), genes have been identified, including CABP2 (OMIM: 607314) that encodes Ca 2+binding protein (CaBP) 2. It belongs to the CaBP family that is related to calmodulin and is expressed in the cochlea and hair cells. CABP2 facilitates normal functioning of the peripheral auditory system by inhibiting Ca V 1.3 Ca 2+ -channel inactivation (Cui et al., 2007;Picher et al., 2017;Yang et al., 2018). Four different CABP2 variants have been reported to cause ARNSHI (DFNB93) ( Table 1). For the Finnish population within the Genome Aggregation Database (gnomAD) (Karczewski et al., 2020), GJB2 has the most frequent ARNSHI pathogenic variants, followed by CABP2 (NM_016366.3) c.637+1G>T (allele frequency [AF] = 3.3 × 10 −3 ). CABP2 has not yet been reported to be involved in ARNSHI in Finland. We report on two Finnish families with ARNSHI due to the splice site variant c.637+1G>T. This variant is classified as pathogenic according to the American college of medical genetics (ACMG) guidelines. The Finnish population has a unique genetic architecture in which certain variants are enriched while the variant diversity is reduced compared to other populations Chheda et al., 2017).

| Familial and clinical examination
The study was approved by the ethics committee of the Oulu University Hospital (EETTMK: 55/2018 and 186/2020) and from the institutional review board (IRB) Columbia University (IRB-AAAS2343). Two children in family FINHEAR1 and one child in family FINHEAR2 with HI were born to parents of Finnish origin from the Kainuu and Central Ostrobothnia regions, respectively. No other family members were reported to be hearing impaired. Available medical records as well as familial and clinical history were evaluated. Physical examination, transient otoacoustic emissions (TEOAE), and pure-tone audiometry were performed at 125-8000 Hz for the affected children of FINHEAR1. TEOAE, auditory steadystate response (ASSR), and auditory brainstem response (ABR) were evaluated for the affected child of FINHEAR2. DNA samples were obtained from the unaffected parents and their affected children.

| Exome sequencing and data analysis
Genomic DNAs from the affected members of FINHEAR1 and FINHEAR2 were screened for pathogenic ARNSHI variants in the coding exon of GJB2 via Sanger sequencing. DNA samples from II:1 and II:4 of family FINHEAR1 and II:3 of family FINHEAR2 ( Figure 1a) underwent sequencing using exome and a custom capture array, respectively. For FINHEAR1, library construction and exon capture were completed using BGISEQ/Agilent SureSelect Human All Exon V6 kit (60. For one child prelingual onset could not be confirmed. Table S1 for a list of the genes). An average sequence read depth of 100× was obtained for II:3. Read alignment, variant calling, annotation, and filtering were performed as previously described (Schrauwen et al., 2020). The variants were validated and tested for segregation by Sanger sequencing using DNA samples from the affected members and unaffected parents.

| RESULTS
FINHEAR1 has two affected family members, II:1 and II:4 ( Figure 1a). II:1 initially passed TEOAE newborn hearing screening. He was seen again at 6 years of age due to purulent otitis media and suspected HI. Observations during the operative procedures (tympanostomy and adenotomy) indicated chronic otitis. HI persisted even after otitis media treatment and resolution. HI was confirmed by pure-tone audiometry at 6 years of age ( Figure  1b). For II:4, bilateral HI was detected at 17 days after birth through TEOAE newborn screening. Audiograms taken at the ages of 6.0 and 13.7 years for II:1 and at 2.4, 3.0, and 3.5 years for II:4 ( Figure 1c) feature symmetrical, moderate-to-severe non-progressive HI that is more conspicuous in the mid-frequencies forming a U-shaped audiogram (Figure 1b of age, TEOAE showed no responses, and ASSR ( Figure  1d) and ABR results revealed responses on average at 40-55 dB levels which led to a diagnosis of moderate sensorineural HI. For neither family was there any indications that HI was part of a syndrome. The only rare homozygous variant which was observed in the sequence data was the splice site CABP2 c.637+1G>T variant (Table S2). Additionally, no potential compound heterozygous variants were observed. The c.637+1G>T variant is enriched in the Finnish population (Figure 1e). Although six of the eight previously published families with CABP2-related HI were consanguineous, the parents in these Finnish families reported to be unrelated as far as they know.
The c.637+1G>T variant is suggested to result in skipping of exon 6 which in turn is predicted to cause a shifted reading frame and a truncated protein in the absence of RNA degradation via nonsense-mediated mRNA decay. Functional studies demonstrate that the truncation impairs but does not completely obliterate its ability to modulate Ca v 1.3 (Picher et al., 2017;Schrauwen et al., 2012).
The AF of the CABP2 c.637+1G>T variant within the Finnish population is 3.3 × 10 −3 (N = 12,516) (95% confidence interval [CI] 2.6 × 10 −3 , 4.1 × 10 −3 ) in gnomAD with no homozygous individuals. FinnGen (2021), which includes genotype and imputed genome data obtained on 218,792 individuals, the imputed c.637+1G>T variant has an AF of 4.2 × 10 −3 (95% CI 4.0 × 10 −3 , 4.4 × 10 −3 ). There are four individuals who are likely homozygous for the imputed c.637+1G>T variant. FinnGen reports no association between the c.637+1G>T variant and sensorineural hearing loss (15,952 cases and 196,592 controls) (p value = 4.4 × 10 −1 ). Although this variant is enriched in the Finnish population, CABP2 is likely a rare cause of HI in Finland. The estimated prevalence of HI in Finland due to homozygous c.637+1G>T variants ranges from 0.7 to 1.9 per 100,000 individuals. As the allelic spectrum of pathogenic CABP2 variants in Finland is unknown, the frequency of HI due to CABP2 could be higher if there are other yet to be identified causal variants.
The Finns are an isolated population and their genetic architecture has been molded by several ancient and recent migratory and multiple bottleneck events as well as rapid population growth in the last 10 generations (Chheda et al., 2017). These events have shifted proportion and frequency of variants and contributed to decreased genetic diversity. The history of this population may have caused a higher prevalence of 36 monogenic Mendelian disorders in Finland compared with the rest of the world. These Mendelian diseases include epilepsy, muscular dystrophy, and Usher syndrome and are classified as Finnish heritage diseases (Norio, 2003).
In conclusion, we identified the CABP2 c.637+1G>T variant that segregates with moderate-to-severe ARNSHI in two families of Finnish origin. To the best of our knowledge, these are the first reported cases of CABP2-related ARNSHI in Finland.

ACKNOWLEDGMENTS
We would like to thank the families for participating in the study and the participants and investigators of the FinnGen study. The research was supported by the National Institute of Health and other Communication Disorders (grant nos. R01 DC011651 and R01 DC003594 to SML).

CONFLICT OF INTEREST
All authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon request. The variant has been submitted to ClinVar database at https:// www.ncbi.nlm.nih.gov/clinv ar/ with accession number SCV001622777.1.