Discussion
Mutations in approximately 30 genes have been implicated in ADNSHL (with variable evidence) [
2]. The respective gene products fall into many different categories and comprise ion channels and transporters, motor molecules, components of the extracellular matrix, the cytoskeleton, adhesion complexes etc. [
1].
Of the five heterozygous variants found in genes from the mapped candidate regions on chromosomes 12 and 20 in our family, the
OSBPL2 frameshift mutation represented a reasonable candidate for the ADNSHL-causing mutation (see results section). OSBPL2 does not belong to any of the above protein classes. It is part of a 12-member, evolutionarily highly conserved family of lipid binding/transfer proteins, the oxysterol binding proteins (OSBPs) and related proteins (OSBPLs) that share the characteristic OSBP signature, EQVSHHPP [
38]. OSBPL proteins play an important role in non-vesicular intracellular transport of lipids, particularly oxysterol, a derivative of cholesterol. OSBPLs serve as sterol sensors and transporters that modulate the lipid composition of cell organelle membranes and assembly of protein complexes, thereby impacting signaling, vesicle transport and lipid metabolism [
39].
Osbpl2 was among 132 mRNAs with 3′UTRs predicted to contain potential target sites for
miR-96 [
32], a microRNA whose mutations cause progressive hearing loss in mice and humans [
32,
40], but we found no upregulation of
Osbpl2 in
Mir96 mutant mice (
diminuendo) (Additional file
1). However, besides its previously reported expression in the mouse organ of Corti at the onset of hearing [
41], there are several lines of evidence that
OSBPL2 is the gene underlying ADNSHL in the family described herein: The frameshift mutation c.141_142delTG very likely represents a loss-of-function allele causing
OSBPL2 haploinsufficiency. It either results in a truncated non-functional protein of 151 residues (wild-type: 480 residues) including 102 unrelated amino acids (p.Arg50Alafs*103) or in an unstable mRNA undergoing nonsense-mediated decay. OSBPL2 interacts with diaphanous homologue 1 (DIAPH1) [
42], the gene mutated in human ADNSHL type 1 (
DFNA1) [
43]. DIAPH1 is a Rho effector protein that regulates cytoskeletal dynamics by interacting with actin, microtubules and other proteins associated with cytoskeleton function [
44,
45]. Mutations in
DIAPH1 are thought to impair the structural integrity of hair cells’ stereocilia, which strongly depends on their actin cytoskeleton, and of the kinocilium, which is built around a microtubular backbone. As is assumed for DIAPH1, OSBPL2 could play a role for the maintenance of hair cells’ cytoskeleton, which would be compatible with the prominent presence of OSBPL2 protein at stereocilia (Figure
4). OSBPL2 binds phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)
P
3) [
46], a phospholipid of the plasma membrane that is crucial for defining neuronal polarity [
47,
48], a possible hint that OSBPL2 could be needed to establish and maintain polarity of hair cells. It remains to be determined if the interaction of both proteins is reflected by (at least partial) cellular co-localization.
Of note, an
OSBPL2 frameshift mutation in close proximity to the nucleotide position affected by the mutation reported herein has recently been described to co-segregate with ADNSHL in a large Chinese family [
49]. Similar to the German
DFNA67 family, age of onset was variable (5 to 32 years), and hearing loss was progressive, ranging from mild to profound. This additional
DFNA67 family strongly supports the association of
OSBPL2 mutations with ADNSHL.
A truncating variant in a gene closely neighboring the chromosome 20 candidate locus, a nonsense mutation in
SLC17A9, p.Ser96*, is unlikely to cause hearing loss because it was present in healthy individuals of both our family and our in-house database of 511 epilepsy exomes, and in the general population.
SLC17A9 encodes a vesicular nucleotide transporter [
50], and heterozygous mutations in this gene have recently been reported to cause disseminated superficial actinic porokeratosis, DSAP [
51]. None of the 12 individuals from our
DFNA67 family who carried the p.Ser96*
SLC17A9
mutation had any skin abnormalities. In conclusion,
SLC17A9 is not only unrelated to hearing loss in this family; its haploinsufficiency does not seem to cause DSAP either. The manifestation of DSAP might thus only result from missense mutations with a dominant-negative effect. On the other hand, our data challenge the assumption that
SLC17A9 mutations cause DSAP, and additional research seems necessary to verify the postulated implication of
SLC17A9 in skin disease.
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Competing interests
TE, CB and HJB are employees of Bioscientia, which is part of a publicly traded diagnostic company. The authors declare that they have no competing interests.
Authors’ contributions
MP, MMH, SM, AG and HJB carried out the clinical characterization of the family. MT and IE carried out the molecular genetic studies apart from exome sequencing. Targeted NGS was carried out and analyzed by TE, CB and HJB. GN, PN and HT performed linkage analysis, exome sequencing and bioinformatic/statistical analysis. UZ and MK determined localization of OSBPL2 in hair cells. MAL and KPS investigated Osbpl2 expression in diminuendo mice. HJB designed the study and wrote the manuscript. All authors have read and approved the final manuscript.