Identification of two novel mutations in FAM136A and DTNA genes in autosomal-dominant familial Meniere's disease

Abstract

Meniere's disease (MD) is a chronic disorder of the inner ear defined by sensorineural hearing loss, tinnitus and episodic vertigo, and familial MD is observed in 5-15% of sporadic cases. Although its pathophysiology is largely unknown, studies in human temporal bones have found an accumulation of endolymph in the scala media of the cochlea. By whole-exome sequencing, we have identified two novel heterozygous single-nucleotide variants in FAM136A and DTNA genes, both in a Spanish family with three affected cases in consecutive generations, highly suggestive of autosomal-dominant inheritance. The nonsense mutation in the FAM136A gene leads to a stop codon that disrupts the FAM136A protein product. Sequencing revealed two mRNA transcripts of FAM136A in lymphoblasts from patients, which were confirmed by immunoblotting. Carriers of the FAM136A mutation showed a significant decrease in the expression level of both transcripts in lymphoblastoid cell lines. The missense mutation in the DTNA gene produces a novel splice site which skips exon 21 and leads to a shorter alternative transcript. We also demonstrated that FAM136A and DTNA proteins are expressed in the neurosensorial epithelium of the crista ampullaris of the rat by immunohistochemistry. While FAM136A encodes a mitochondrial protein with unknown function, DTNA encodes a cytoskeleton-interacting membrane protein involved in the formation and stability of synapses with a crucial role in the permeability of the blood-brain barrier. Neither of these genes has been described in patients with hearing loss, FAM136A and DTNA being candidate gene for familiar MD.

Figure 1.

(A) Pedigree of an autosomal-dominant MD family with three affected cases. (B) Chromatogram of the variant chr2:70527974C>T from an affected individual (III.4) is compared with the sequence from a familial control (II.19). (C and E) Zoomed-in view of regions containing the variants, including the amino acid sequences of canonical isoforms and the mutated sequence caused by the variant. (D) A chromatogram of the variant chr18:32462094G>T from an affected (III.4) and control (II.19) subjects.

Figure 2.

Gene expression of FAM136A in lymphoblastoid cells, as assayed by standard quantitative PCR on cDNA from individuals harboring chr2:70527974C>T and healthy donors. The FAM136A_001 transcript has a significantly reduced Sybr Green expression in patient lymphoblasts when it was compared with controls (asterisk, P = 0.002).

Figure 3.

Immunoblotting of FAM136A and actin as a loading control from individuals harboring chr2:70527974C>T and healthy donors. The FAM136 band is located at an MW of 16 kDa. Each sample was replicated to minimize the error.

Figure 4.

Characterization of a new DTNA splice site in lymphoblastoid cells. (A) Gene expression of DTNA, as assayed by nested PCR on cDNA from individuals harboring chr18:32462094G>T (III.4) and healthy donors (II.9). (B) Chromatogram of the variant chr18:32462094G>T from an affected individual (III.4) is compared with the sequence from a familial control (II.9). (C) Zoomed-in view of regions containing the variants, including the amino acid sequences of canonical isoforms and the mutated sequence caused by the splice site which skip exon 21 (49 nucleotide) harboring the last five amino acids.

Figure 5.

Expression of FAM136A and DTNA in adult rat inner ear tissues. (A) Confocal co-localization of FAM136A (green) with mitochondrial marker COX IV (red) and Anti-β-Spectrin II (blue) as staining control, which label cytoskeletal protein and the cuticular plate of hair cells. (B) α-Dystrobrevin (DTNA, green) is expressed in the basal part of supporting cells of the rat crista. Scale bars: (A) 10 μm and (B) 20 μm.