Identification of CRYM as a candidate responsible for nonsyndromic deafness, through cDNA microarray analysis of human cochlear and vestibular tissues

Abstract

Through cDNA microarray analysis of gene expression in human cochlea and vestibule, we detected strong expression of mu-crystallin (CRYM; also known as "NADP-regulated thyroid hormone-binding protein") only in these inner-ear tissues. In a subsequent search for mutations of CRYM, among 192 patients with nonsyndromic deafness, we identified two mutations at the C-terminus; one was a de novo change (X315Y) in a patient with unaffected parents, and the other was a missense mutation (K314T) that segregated dominantly in the proband's family. When the mutated proteins were expressed in COS-7 cells, their subcellular localizations were different from that of the normal protein: the X315Y mutant showed vacuolated distribution in the cytoplasm, and the K314T mutant localized in perinuclear areas, whereas normal protein was distributed homogeneously in the cytoplasm. Aberrant intracellular localization of the mutated proteins might cause dysfunction of the CRYM product and result in hearing impairment. In situ hybridization analysis using mouse tissues indicated its expression in the lateral region of the spiral ligament and the fibrocytes of the spiral limbus, implying its possible involvement in the potassium-ion recycling system. Our results strongly implicate CRYM in normal auditory function and identify it as one of the genes that can be responsible for nonsyndromic deafness.

Figure 1

Analysis and confirmation of cDNA microarray results. A, Signals of CRYM in the microarray (white arrows). aRNA from the human inner ear (cochlear and vestibular tissues) was labeled with Cy3-dCTP (red); mixed aRNA from 29 other tissues yielded green signals from Cy5-dCTP (green). B, Confirmation of microarray data by semiquantitative RT-PCR of CRYM with one-round amplified RNAs.

Figure 2

Pedigrees and electrophoregrams showing mutations in the CRYM gene in two families. Corresponding normal sequences are shown in the left-hand panels. A, In family 1, only patient II-2 shows a heterozygous A→T substitution, changing the stop codon to a Tyr residue and bringing about an extension of the protein by five amino acids at the C-terminal. B, In family 12, the patient (II-1) and her affected mother (I-2) are both heterozygous for a Lys314Thr mutation.

Figure 3

Fluorescent images of COS-7 cells expressing HA-tagged CRYM proteins. Nuclei are counterstained with DAPI (blue). Cells representative of six independent experiments are shown at a magnification of 60×. Subsequently, the numbers of cells were counted in 8–10 randomly selected fields (60×) using fluorescence microscopy (Nikon) at 24 h, 48 h, 72 h, and 96 h, respectively. A, Wild-type (WT) CRYM protein (green) localizes to cytoplasm diffusely throughout the time course of the experiments. B, The X315Y mutant with an extended C-terminal tail leads to vacuolated cytoplasmic distribution. The K314T mutant protein is localized predominantly in the perinuclear area.

Figure 4

In situ hybridization analysis of Crym expression. The mouse cochlea reveals distinct labeling by the Crym antisense probe (arrows). Crym mRNA expression in the fibrocytes of spiral ligaments (SLg) and the fibrocytes of spiral limbus (SLm) in all turns of the mouse cochlea. Control hybridization was performed with a sense probe on consecutive tissue sections.