Exploring the missing heritability in subjects with hearing loss, enlarged vestibular aqueducts, and a single or no pathogenic SLC26A4 variant

Abstract

Pathogenic variants in SLC26A4 have been associated with autosomal recessive hearing loss (arHL) and a unilateral or bilateral enlarged vestibular aqueduct (EVA). SLC26A4 is the second most frequently mutated gene in arHL. Despite the strong genotype-phenotype correlation, a significant part of cases remains genetically unresolved. In this study, we investigated a cohort of 28 Dutch index cases diagnosed with HL in combination with an EVA but without (M0) or with a single (M1) pathogenic variant in SLC26A4. To explore the missing heritability, we first determined the presence of the previously described EVA-associated haplotype (Caucasian EVA (CEVA)), characterized by 12 single nucleotide variants located upstream of SLC26A4. We found this haplotype and a delimited V1-CEVA haplotype to be significantly enriched in our M1 patient cohort (10/16 cases). The CEVA haplotype was also present in two M0 cases (2/12). Short- and long-read whole genome sequencing and optical genome mapping could not prioritize any of the variants present within the CEVA haplotype as the likely pathogenic defect. Short-read whole-genome sequencing of the six M1 cases without this haplotype and the two M0/CEVA cases only revealed previously overlooked or misinterpreted splice-altering SLC26A4 variants in two cases, who are now genetically explained. No deep-intronic or structural variants were identified in any of the M1 subjects. With this study, we have provided important insights that will pave the way for elucidating the missing heritability in M0 and M1 SLC26A4 cases. For pinpointing the pathogenic effect of the CEVA haplotype, additional analyses are required addressing defect(s) at the RNA, protein, or epigenetic level.

Fig. 1

Overview of genetic analyses performed in zeroallelic and monoallelic SLC26A4 cases. a, b To explain the missing heritability in zeroallelic (M0, n = 12) and monoallelic (M1, n = 16) SLC26A4 cases, different genetic analyses were performed. First, individuals were screened for the presence of the CEVA haplotype (M0/CEVA, n = 2; M1/CEVA, n = 10). Second, whole-genome sequencing (WGS) was performed in all monoallelic cases (M0/CEVA, M1) to identify potential structural, splice (M2, n = 2) or regulatory variants. Finally, sequencing data were screened for potentially pathogenic variants in the EPHA2, FOXI1 and KCNJ10 genes. Digenic inheritance has been previously suggested for variants in these genes and the SLC26A4 gene. In three cases (M0/FOXI1 (M0^F), n = 2, CEVA/FOXI1 (M1^F), n = 1), a potentially pathogenic variant in FOXI1 (NM_012188.4, c.677C > T) was identified

Fig. 2

Determination of the boundaries of the shared CEVA haplotype. a The CEVA haplotype was detected in 10 individuals, in an additional 2 individuals (SLC040 and SLC071, indicated with *), a smaller haplotype was found, termed V1-CEVA. To determine the boundaries of the CEVA haplotype, VNTR marker analysis was performed. The shared haplotype (0.89 Mb, CEVA; 0.57 Mb V1-CEVA is marked in orange. For marker D7S2420 (light-orange) a deviating CA-repeat length was determined in SLC003. Nevertheless, the marker is still considered to be potentially part of the shared haplotype as a change or repeat length cannot be excluded. Genomic positions (Mb) are according to the UCSC Genome Browser (GRCh37/hg19). b A schematic overview of the identified shared CEVA haplotype (D7S501-D7S2459). Positions of the CEVA-associated SNPs and the genes located within the haplotype region (CEVA, D7S501-D7S2459; V1-CEVA, SNP3-D7S2459) have been indicated. All SNPs are located within intronic or intergenic regions. Genomic positions of the CEVA-associated SNPs are provided in Table S7. SLC26A4 (NM_000441.1) is only partially included (exons 10/21) in the shared haplotype

Fig. 3

Results of audiometric evaluation in affected individuals. PTA_0.5–4 kHz for ears with an EVA. Each dot represents the hearing level of an ear with an enlarged vestibular aqueduct, allocated to genotype class (M2, M1/CEVA, M1, M0/CEVA and M0). The M1/CEVA group also includes subjects with an M1/V1-CEVA genotype. For an objective comparison, the same methods as used by Chao et al. (2019) were applied