A splice-site variant (c.3289-1G>T) in OTOF underlies profound hearing loss in a Pakistani kindred

Abstract

Background: Hearing loss/deafness is a common otological disorder found in the Pakistani population due to the high prevalence of consanguineous unions, but the full range of genetic causes is still unknown.

Methods: A large consanguineous Pakistani kindred with hearing loss was studied. Whole-exome sequencing and Sanger sequencing were performed to search for the candidate gene underlying the disease phenotype. A minigene assay and reverse transcription polymerase chain reaction was used to assess the effect of splicing variants.

Results: The splicing variants of OTOF (NM_194248, c.3289-1G>T) cosegregated with the disease phenotype in this Pakistani family. The substitution of a single base pair causes the deletion of 10 bp (splicing variant 1) or 13 bp (splicing variant 2) from exon 27, which results in truncated proteins of 1141 and 1140 amino acids, respectively.

Conclusion: Our findings reveal an OTOF splice-site variant as pathogenic for profound hearing loss in this family.

Keywords: Hearing loss; Minigene; OTOF; Splice acceptor site.

Fig. 1

Pedigree and Sanger sequence analysis. a A pedigree showing five generations of a consanguineous family; rectangles indicate males, circles indicate females, Roman numerals represent generations, and Arabic numerals indicate the position of each individual in the family. Filled rectangles and circles, respectively, represent affected males and females; a horizontal line above a rectangle or circle shows that the subject was clinically examined (PTA and impedance testing or tympanometry); an asterisk above a symbol indicates that WES was performed; a slash (/) indicates a deceased person; the same line between two individuals represents consanguinity; +/+ represents homozygosity for the wild-type allele, represents ± heterozygosity, and −/− represents homozygosity for the mutant allele; an arrow indicates the proband. b Sanger sequencing of three subjects: an unaffected normal individual (IV-10), a heterozygous individual (IV-16) and an affected individual (IV-2). The arrow in the chromatogram shows the position of the splice-site variant (NM_194248:c.3289-1G>T)

Fig. 2

Pure-tone audiometry and tympanometry. a Pure-tone audiograms of a normal subject (IV-16) and an affected subject (IV-2). The vertical axis indicates the sound level in decibels (dB), and the horizontal axis shows frequency in hertz (Hz). Audiograms of both ears are presented. ‘x’ represents air conduction, and <> indicates bone conduction. b Tympanometry or tympanogram. (i) Normal subject (IV-16); his right-ear peak was 0.4 lower than the left-ear peak of 0.7 cm³ but remained within the normal range (type ‘A’). (ii) Affected subject (IV-9); his right ear peak was 0.1 cm³ (type ‘As’), but his left ear peak was 0.4 cm³ and belonged to type ‘A’. The vertical axis shows compliance or flexibility in cm³ (1.5 cm³). The horizontal axis shows air pressure in decaPascals (daPa), ECV = ear canal volume, GR = gradient. R = right, L = left,

Fig. 3

Minigene assay for the targeted variant. a Minigene construction; the vertical dashed red line shows the position of OTOF on chromosome 2p23.3. The next dashed underline represents OTOF, including its introns and exons. The minigene target sequence is from exon 25 to exon 29; this sequence was cloned in a CAGGS plasmid with internal ribosome entry sites (IRES), and enhanced green fluorescent protein (EGFP). b Sanger sequencing of the wild type (WT) and the mutant for splicing variants 1 and 2; the dashed line indicates the exon junction between exons 26 and 27. Splicing variant 1 deleted 1–10 bp from exon 27, and splicing variant 2 deleted 1–13 bp from the same exon. c The protein translation of the WT sequence and splicing variants 1 and 2. The splicing acceptor site mutation produced truncated proteins of 1141 amino acids (p.Ile1097Glnfs45) in splicing variant 1 and 1140 amino acids (p.Ile1097Glyfs44) in splicing variant 2, instead of the normal mature protein of 1998 amino acids. Ile = isoleucine, Gln = glutamine, Gly = glycine