Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment

Abstract

Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow "U" or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.

Figure 1

Mutation Analysis of OTOG (A) Partial sequences are shown of OTOG exon 35 from an affected member and a parent of family W00-384 and a normal control. The amino acids are indicated above the sequence. The c.5508delC variant found is predicted to result in a frameshift and premature stop codon, p.Ala1838ProfsX31 (reference sequence XM_291816.8). (B) Pedigree of family W00-384 and segregation of the c.5508delC variant. (C) Partial sequences of OTOG exon 37 from a normal-hearing control and affected subject S1778 II.2, showing the c.6347C>T (p.Pro2116Leu) substitution. (D) Partial sequences of OTOG exon 38 from a normal-hearing control and affected subject S1778 II.2, showing the c.6559C>T (p.Arg2187X) nonsense mutation. (E) Pedigree of Spanish family S1778 and segregation analysis of the c.6347C>T and c.6559C>T mutations. (F) Localization and conservation of the Pro-2116 residue. Above are shown domains of human otogelin. Red box, N-terminal signal peptide; vWD, von Willebrand factor type D domain; C8, domain containing eight conserved cysteine residues; TIL, trypsin inhibitor-like domain; CT, cystine knot-like domain. The localization of the p.Pro2116Leu missense mutation is indicated by an arrow. Below are shown alignment of diverse vertebrate otogelin (OTOG) and otogelin-like (OTOGL) amino acid sequences (accession numbers are shown on the right). ConSeq conservation scores are shown by the color scale. Residue Pro-2116 (arrowhead), located in the fourth von Willebrand factor type D domain, is highly conserved (score 8).

Figure 2

OTOG Expression Profile in Human Tissues Relative OTOG mRNA levels as determined by qPCR in fetal (A) and adult (B) tissues. Because this was performed for adult and fetal tissues in two separate experiments, fetal inner ear was included in both to be able to compare the expression levels. The delta Ct method was used to calculate the expression levels. To calculate the relative expression, we set the tissue with the lowest detectable expression (Ct < 35) to 1. In the adult tissues, the expression in colon was set to 1 and in fetal tissues the expression in kidney. RNA was isolated from inner ear of an embryo at 8 weeks gestation; the other fetal RNA samples were commercially available (all adult tissues from Stratagene; fetal heart, brain, and kidney from Clontech, fetal skeletal muscle, liver, lung, spleen, thymus, colon, and stomach from Stratagene) and derived from embryos at 20–21 weeks of gestation.

Figure 3

Audiometric Characterization of OTOG Families (A) Binaural mean air-conduction pure tone threshold values of affected members of family W00-384. These represent mean values of audiograms at different ages for each affected individual; II.1 (7.2–19.6 yrs), II.2 (5.4–17.2 yrs), II.3 (3.5–15.3 yrs), and II.4 (3.9–13.8 yrs). (B) Mean air-conduction pure tone threshold values of family S1778. These represent mean values of audiograms at different ages for each affected individual; 5–30 years for subject II.1 and 6–24 years for subject II.2. AD (aurio dextra) and AS (aurio sinister) represent the right and left ear, respectively.