Homozygosity mapping reveals mutations of GRXCR1 as a cause of autosomal-recessive nonsyndromic hearing impairment

Abstract

We identified overlapping homozygous regions within the DFNB25 locus in two Dutch and ten Pakistani families with sensorineural autosomal-recessive nonsyndromic hearing impairment (arNSHI). Only one of the families, W98-053, was not consanguineous, and its sibship pointed toward a reduced critical region of 0.9 Mb. This region contained the GRXCR1 gene, and the orthologous mouse gene was described to be mutated in the pirouette (pi) mutant with resulting hearing loss and circling behavior. Sequence analysis of the GRXCR1 gene in hearing-impaired family members revealed splice-site mutations in two Dutch families and a missense and nonsense mutation, respectively, in two Pakistani families. The splice-site mutations are predicted to cause frameshifts and premature stop codons. In family W98-053, this could be confirmed by cDNA analysis. GRXCR1 is predicted to contain a GRX-like domain. GRX domains are involved in reversible S-glutathionylation of proteins and thereby in the modulation of activity and/or localization of these proteins. The missense mutation is located in this domain, whereas the nonsense and splice-site mutations may result in complete or partial absence of the GRX-like domain or of the complete protein. Hearing loss in patients with GRXCR1 mutations is congenital and is moderate to profound. Progression of the hearing loss was observed in family W98-053. Vestibular dysfunction was observed in some but not all affected individuals. Quantitative analysis of GRXCR1 transcripts in fetal and adult human tissues revealed a preferential expression of the gene in fetal cochlea, which may explain the nonsyndromic nature of the hearing impairment.

Figure 1

Homozygous Regions and Linkage Intervals within DFNB25 Schematic representation of chromosomal region 4p14-q12 showing the homozygous regions in families W98-053 and W07-0122 and the linkage intervals for families DEM 4265 and DEM 4349. The overlapping region contains the GRXCR1 gene and exon 1 of the ATP8A1 gene. In addition, four ESTs are localized in the region, which are not further characterized. The Mb positions and chromosome bands were determined with the UCSC Genome Browser (NCBI build 36.1; hg18 annotation tracks, March 2006).

Figure 2

Mutation Analysis of GRXCR1 in arNSHI Patients Partial sequences are shown of GRXCR1 from affected members of the different families and healthy controls. The predicted amino acid changes and the surrounding amino acids are indicated above the sequence. Mutated nucleotides are marked by an arrowhead. Reference sequences are NT_006238.10 for the nucleotide sequence and NP_001073945.1 for the amino acid sequence.

Figure 3

Alignment of the GRXCR1 Proteins of Different Species (A) Overview of the conserved domains in GRXCR1 as predicted by different software tools. The GRX-like domain is predicted to be present by all three programs. However, the exact positions differ. (B) An alignment of GRXCR1 orthologs was made with ClustalW and Jalview, and part of the GRXCR1 protein is shown. Abbreviations are as follows: Hs, Homo sapiens; Pt, Pan troglodytes; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; Dr, Danio rerio. Fully conserved amino acids are marked in dark gray, and less-conserved amino acids are in lighter colors. Accession numbers of the various protein sequences are as follows: Hs GRXCR1, NP_001073945.1; Pt LOC461192, XP_517170.2; Mm Grxcr1, NP_001018019.1; Rn Grxcr1, XP_573590.2; Gg LOC770624, XP_001233963.1; Dr LOC100150769, XP_001919567.1.

Figure 4

Splicing Effect of the c.628-9C>A Mutation Partial sequence of GRXCR1 cDNA from an affected member of family W98-053 and a healthy control. Additional nucleotides are present between exons 2 and 3, leading to a frameshift and a premature-stop codon, p.Gly210LeufsX5.

Figure 5

GRXCR1 Expression Profile Relative GRXCR1 mRNA expression determined by quantitative PCR in adult (A) and fetal (B) tissues. The relative expression values were determined with the delta delta Ct method. There is a highly preferential expression in fetal cochlea.

Figure 6

Clinical Characterization of DFNB25 Families (A–D) Representative audiograms of the right ear of affected individuals of families W98-053 and W07-0122 at different ages. Dashed lines indicate free-field measurements. (E and F) Representative audiograms of affected individuals of families DEM 4265 and DEM 4349, obtained at the ages of 19 and 26 yrs, respectively. Circles and crosses represent the right and left ear, respectively.