Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family

Abstract

Purpose: To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa (RP).

Methods: Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome 2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the USH2A mutations with the family disease. One hundred normal controls were used without the mutations.

Results: The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound heterozygous mutations in the family of study showed both RP and hearing impairment.

Conclusions: This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.

Figure 1

Pedigree structure of the family with USH2/RP. Affected males and females are shown with filled squares and circles, respectively. Normal individuals are shown with empty symbols. The deceased individual is shown with slash. The proband (III:5) is indicated by an arrow. Results from genotyping and haplotype analysis of two markers on chromosome 1 are displayed below each symbol. The disease haplotype is shown with a black or a gray box and the normal haplotypes are shown with open boxes. The USH2A gene is located between D1S213 and D1S425. The homozygous individual (IV:1), whose haplotype is different from other patients, is indicated by an asterisk. Four patients (III:1, III:3, III:5) are affected with USH2, and patient (IV:1) displays nonsyndromic RP symptom.

Figure 2

Fundus appearance of the proband. The typical RP symptoms including attenuation of the retinal vessels, waxy pallor of the optic nerve head, and bone speckle-like pigmentation clumps in the peripheral retina were showed.

Figure 3

Audiometry results for III:5 and IV:1. A and B show the result for IV:1 who is normal, C and D show mild hearing loss for III:5.

Figure 4

Identification of two novel USH2A mutations. DNA sequence analysis for patient III1 showed the presence of compound heterozygous p.G1734R (c.5200G>C) and c.IVS32+1A>T mutations. A and B show the sequences of a normal and affected family member with mutation p.G1734R (c.5200G>C) allele, respectively. C and D show the sequences of a normal and affected family member with mutation c.IVS32+1A>T allele, respectively. E: Restriction fragment length analysis on the p.G1734R (c.5200G>C) mutation in this study. All the affected individuals (III:1, III:3, III:5, IV:1, IV:2) and the carriers (III:2, IV:3) have three bands (228 bp, 164 bp, and 64 bp), while the unaffected individuals only have two bands (164 bp and 64 bp).The patient IV1 who is homozygous for the p.G1734R (c.5200G>C) mutation displayed only one 228 bp band. F: Alignment of the amino acid sequences of laminin G-like domain in the long usherin isoform from different species. Gly1734 (G1734) is conserved during evolution. The box indicates this mutated residue in USH2A.

Figure 5

3D modeling of p.G1734R (PDB template 2JD4_B, 26% identity with Lama1). A: wild-type protein, B: mutant protein. The replacement of Gly by Arg may induce a new secondary structure that includes Leu1831, Val1832, and Val1833. The new secondary structure makes Val1833 very close to Val1756, which may change the conformation of the binding site. The structure of the wild type and the mutant USH2A protein were predicted using Swiss Pdb-Viewer 4.0.1.