Loss-of-function mutations in the PRPS1 gene cause a type of nonsyndromic X-linked sensorineural deafness, DFN2

Abstract

We report a large Chinese family with X-linked postlingual nonsyndromic hearing impairment in which the critical linkage interval spans a genetic distance of 5.41 cM and a physical distance of 15.1 Mb that overlaps the DFN2 locus. Mutation screening of the PRPS1 gene in this family and in the three previously reported DFN2 families identified four different missense mutations in PRPS1. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients. By in situ hybridization, we demonstrate expression of Prps1 in murine vestibular and cochlea hair cells, with continuous expression in hair cells and postnatal expression in the spiral ganglion. Being the second identified gene associated with X-linked nonsyndromic deafness, PRPS1 will be a good candidate gene for genetic testing for X-linked nonsyndromic hearing loss.

Figure 1

Chinese Family GZ-Z052 with X-linked Heriditary NSHL (A) Audiograms of some affected male and female subjects (red, right ear; black, left ear). IV-21, who has a history of the use of aminoglycosides, had exactly same audiogram pattern as that of IV-9 and IV-36, suggesting that his hearing impairment was not due to aminoglycoside exposure. The hearing impairment of female carriers is progressive but not completely linked with increasing age. (B) Haplotype analysis of the Chinese family GZ-Z052. The segregating haplotype associated with the NSHL is indicated by a black bar, delimited by markers DXS8020 and DXS8055 on chromosome Xq22.

Figure 2

Mapping Intervals and Mutation Analysis of the Four DFN2 Families (A) Idiogram of the X chromosome with the described DFN2 loci. The linkage intervals and flanking markers of four DFN2 families are indicated in colored fonts and bars (red, Chinese family GZ-Z052; green, British American family; black, Chinese family reported by Cui et al.; blue: American family). (B) The genomic structure of PRPS1 depicting the positions of four DFN2 mutations. (C–F) DNA sequence chromatograms showing four different missense mutations identified in affected males of four DFN2 families, compared to wild-type controls.

Figure 3

Structure of PRS-I in Complex with AMP The substrate-binding pocket of PRS-I consists of a metal ion-binding site (gray sphere), an ATP-binding site (represented by AMP moiety of ATP as sticks), and an R5P-binding site (a SO₄²⁻ ion occupies the position of the 5′ phosphate of R5P). In this figure, two different asymmetric subunits of PRS-I (aquamarine and pale yellow) form a part of the hexamer interface, and all of the structural elements in the pale yellow subunit are tagged with an apostrophe. (A) Location of Asp65′ (magenta sticks) and Lys34′ (green sticks between the β2′ strand and loop3′). (B) Location of Ala87 (green sticks in the purple β5 strand) and the ATP-binding loop (marine loop7). (C) Location of Ile290 (green sticks in the β14 strand) and of loop22 (deep blue). (D) Location of Gly306 (green sticks in the β14 strand) and the allosteric site of PRS-I (an SO₄²⁻ ion occupies the position of the β-phosphate of ADP).

Figure 4

Prps1 Expression in the Mouse Inner Ear (A) With an antisense probe, Prps1 was observed at E18.5 as robustly expressed in the cochlear hair cells (HC), the Claudius cells (CC), and the greater epithelial ridge (GER), but not in other cochlear supporting cells. (B) A sense control probe did not produce any signals. (C) At postnatal day 6, HC and CC expression of Prps1 was maintained, whereas expression in the GER was reduced. Prps1 was also expressed in the spiral ganglions (SG) at this stage. (D) A sense control probe did not produce any signal. Magnification 20×.