Suzanne de Bruijn

243 Exploring the missing heritability in subjects with hearing loss and EVA Table 2. WGS revealed two heterozygous splice variants in SLC26A4 Case Class Genome cDNA Protein In-house AF (%) gnomAD AF (%) CADD_ PHRED SIFT PPH2 Mutation Taster SpliceAI ACMG SLC048 M1 Chr7:107335064A>C c.1342-2A>C p.Ser448Leufs*3 0.00 - 21.7 NA NA NA 0.99 (AS loss) UV5 SLC085 M1 Chr7:107314664C>T c.471C>T p.Gly139Alafs*6,= - 0.00 0.725 NA NA NA 0.59 (AS gain) UV5 Whole genome sequencing (WGS) revealed two potentially splice altering variants in SLC26A4 . Variants are selected based on an allele frequency of ≤0.5% in gnomAD and the in-house database. Scores thatmeet the thresholds for pathogenicity as described in themethods section are indicated in red. The predicted effect on splicingwas confirmed in an in vitro splice assay that was performed in HEK293T cells ( Figure S1 ). Genome; Genomic position according to GRCh37/hg19; In-house AF, allele frequency (%) in an in-house database (~7,500 exomes); GnomAD AF, allele frequency (%) in gnomAD database V.2.1.1; CADD_PHRED, Combined Annotation Dependent Depletion PHRED score; SIFT, Scale-Invariant Feature Transform; PPH2, PolyPhen-2 score; MutationTaster (prob), MutationTaster score with probability (0-1); spliceAI, splicing prediction score; AS, acceptor site; ACMG, variant classification according to the American College of Medical Genetics and Genomics (ACMG) classification guidelines 51 ; UV5, pathogenic; NA, not applicable. Table 3. Rare variants identified in EPHA2 , FOXI1 and KCNJ10 Case Class Gene Transcript cDNA Protein In-house AF (%) gnomAD AF (%) CADD_ PHRED SIFT PPH2 Mutation Taster SpliceAI ACMG SLC017 M0 EPHA2 NM_004431.4 c.2627G>A p.(Arg876His) 2.36 1.70 32 0 0.769 NA 0.03 UV2 SLC039 M0/CEVA FOXI1 NM_012188.4 c.677C>T p.(Thr226Ile) 0.56 0.37 11 0.14 0.109 P 0.03 UV2 SLC052 M0 EPHA2 NM_004431.4 c.1941G>T p.(Thr647=) 1.09 0.55 7.309 NA NA NA 0.05 UV2 SLC052 M0 EPHA2 NM_004431.4 c.1896G>A p.(Leu632=) 0.76 0.05 3.197 NA NA NA 0.05 UV2 SLC052 M0 FOXI1 NM_012188.4 c.677C>T p.(Thr226Ile) 0.56 0.37 11 0.14 0.109 P 0.03 UV2 SLC069 M0 FOXI1 NM_012188.4 c.677C>T p.(Thr226Ile) 0.56 0.37 11 0.14 0.109 P 0.03 UV2 Available sequencing datasets of monoallelic (M1, M0/CEVA) and zeroallelic (M0) individuals were screened for variants in EPHA2, FOXI1 and KCNJ10 with an allele frequency of ≤5% in gnomAD (V.2.1.1). Scores that meet the thresholds for pathogenicity as described in themethods section are indicated in red. In-house AF, allele frequency (%) in in-house database (~7,500 exomes); GnomAD AF, allele frequency (%) in gnomAD database V.2.1.1; CADD_PHRED, Combined Annotation Dependent Depletion PHRED score; SIFT, Scale-Invariant Feature Transform; PPH2, PolyPhen-2 score; MutationTaster (prob), MutationTaster score with probability (0-1); spliceAI, splicing prediction score; ACMG, variant classification according to the American College of Medical Genetics and Genomics (ACMG) classification guidelines 51 ; UV2, likely benign; NA, not available; P, polymorphism.