Mutations in C2ORF71 cause autosomal-recessive retinitis pigmentosa

Abstract

With a worldwide prevalence of 1 in 4,000, retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration. More than 30 genes and loci have been implicated in nonsyndromic autosomal-recessive (ar) RP. Genome-wide homozygosity mapping was conducted in one Dutch and one Israeli family affected by arRP. The families were found to share a 5.9 Mb homozygous region on chromosome 2p23.1-p23.3. A missense variant in one of the genes residing in this interval, C2ORF71, has recently been reported to be associated with RP. C2ORF71, encoding a putative protein of 1,288 amino acids, was found to be specifically expressed in human retina. Furthermore, RT-PCR analysis revealed that in the mouse eye, C2orf71 is expressed as early as embryonic day 14. Mutation analysis detected a 1 bp deletion (c.946 del; p.Asn237MetfsX5) segregating with RP in the Dutch family, whereas a nonsense mutation (c.556C > T; p.Gln186X) was identified in the Israeli family. Microsatellite-marker analysis in additional Israeli families revealed cosegregation of a C2ORF71-linked haplotype in one other family, in which a 13 bp deletion (c.2756_2768 del; p.Lys919ThrfsX) was identified. Clinically, patients with mutations in C2ORF71 show signs of typical RP; these signs include poor night vision and peripheral field loss, typical retinal bone-spicule-type pigment deposits, pale appearance of the optic disk, and markedly reduced or completely extinguished electroretinograms. In conclusion, truncating mutations in C2ORF71 were identified in three unrelated families, thereby confirming the involvement of this gene in the etiology of arRP.

Figure 1

Chromosome 2 Haplotypes in Families W97-111, TB52, and TB44 Shown are three families affected by arRP. One family is of Dutch origin (W97-111 [A]), and two are of Israeli origin (TB52 [B] and TB44 [C]). Microsatellite marker and SNP analyses performed on each of the families demonstrate cosegregation of a 2p22.3-p23.3-linked haplotype with RP. Double lines indicate consanguineous unions. Filled symbols represent affected individuals, and clear symbols represent unaffected individuals. Mutation-bearing haplotypes are marked by black bars. The position of the markers, based on the human genome browser working draft hg18, is indicated between parentheses.

Figure 2

Linkage Intervals and Sequence Analysis of C2ORF71 (A) Upper panel: part of chromosome 2p showing the linkage intervals and the corresponding flanking microsatellite markers or SNPs for arRP families W97-111 and TB52. The flanking markers that determine the shared interval are indicated in bold. For family TB44, flanking markers were not determined, so the linkage interval might continue beyond the indicated markers, as is shown with the dotted line. C2ORF71 resides within the region that is shared by the three families. Lower panel: genomic structure of C2ORF71. Two exons are indicated by blue bars. The noncoding part of exon 2 is indicated with a smaller bar. (B) Sequence chromatograms for the three mutations in C2ORF71. Mutant sequences are indicated above the wild-type counterparts. In family W97-111, a 1 bp deletion (indicated in a dotted box in the wild-type sequence) that results in a frame shift and premature termination of the protein was detected. In family TB52, a nonsense mutation (indicated with an arrowhead) was detected, whereas in family TB44, a 13 bp deletion (also indicated in a dashed box in the corresponding wild-type sequence) was identified. This change also causes a shift in the reading frame and results in premature termination of the C2ORF71 protein. For all chromatograms, the corresponding amino acids are indicated above the sequence traces. The nucleotide positions of C2ORF71 are according to GenBank accession number NM_001029883.

Figure 3

Fundus Photographs of Affected Individuals from Families W97-111 and TB52 (A and B) Fundus photographs of patients II-7 (age 67) and II-8 (age 66) from family W97-111. In both patients, visual actuity has deteriorated to no light perception. The fundus photographs are somewhat hazy as a result of severe cataracts. (A) Peripheral fundus of II-7, showing widespread atrophy, severe attenuation of retinal vessels, and irregular pigmentations. (B) Posterior pole of II-8 showing central atrophy as well as midperipheral atrophy. The optic nerve head has a waxy aspect, the retinal vessels are severely attenuated, and bone-spicule pigmentations are present. (C) Fundus photographs of individual II-4 (age 50) from family TB52, showing peripheral bone-spicule-type pigment deposits, attenuation of retinal blood vessels, severe retinal atrophy (with demonstration of choroidal vessels), and pale appearance of the optic disk.

Figure 4

Expression Analysis of C2ORF71 (A) Expression analysis of C2ORF71 in different adult human tissues. RT-PCR using exon-spanning primers revealed high expression of C2ORF71 in human retina, whereas little or no expression was detected in all other tissues examined (upper panel). The expression of GUSB, encoding glucuronidase beta, was analyzed as a positive control (lower panel). (B) RT-PCR analysis of C2orf71 in the murine eye at different developmental time points: embryonic day 14 (E14), newborn (P0), and 5 months (5 mo). The analysis indicates high and relatively equal expression at all tested time points (upper panel). The Actb gene (β-actin) served as a positive control (lower panel).