Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.

Figure 1

Schematic Representation of the RP28 Interval and Mutations in FAM161A (A) Linkage interval for RP28 on chromosome 2p is delimited by microsatellite markers D2S1337 and D2S286 and spans over 15 Mb of DNA. Black boxes represent the 188 genes screened by UHTs. (B) Structure of the two protein-coding isoforms of FAM161A, which are transcribed in the reverse direction with respect to the indicated orientation of chromosome 2, and location of the nonsense mutations detected in patients with arRP. Arrows indicate the primers used in quantitative RT-PCR experiments for detection of FAM161A mRNA expression in human tissues. The forward primer for both RT-PCR reactions lies on exon 3, whereas the reverse primer is complementary to either the 3-4 or the 3-3a exon-exon junctions, depending on the isoform analyzed. (C) Electropherograms of the c.685C>T (p.Arg229X) and c.1309A>T (p.Arg437X) mutations and the corresponding region in controls.

Figure 2

FAM161A mRNA Expression in Human and Mouse (A and B) Quantitative RT-PCR in various human tissues of the FAM161A short isoform (A) and long isoform (B). Both graphs represent mRNA expression normalized to the housekeeping gene ACTB. Note that ranges of y axes are not the same. (C) Fam161a expression in the mouse retina, as detected by quantitative RT-PCR with the use of the 18S rRNA as housekeeping gene at various developmental stages. (D) Affymetrix GeneChip expression data from wild-type versus Crx^−/− P14 retinas, as detected with the Fam161a-specific probe set 1443569_at. Error bars indicate standard errors of the mean (A and B) and standard deviations (C and D), calculated in all instances from three independent experiments.

Figure 3

CBRs around Fam161a and CBR-Driven Reporter Expression in the Murine Retina (A) Fam161a Crx-bound regions (CBR1 and CBR2, black boxes), as detected by ChIP-Seq experiments and phylogenetic conservation among mammals. (B) Constructs carrying a constitutive CMV early enhancer-chicken β actin (CAG) promoter-eGFP fusion (electroporation control) and each of two Fam161a CBRs-DsRed fusions were coelectroporated in explanted P0 mouse retinas. CBR1 was cloned immediately upstream of DsRed, whereas CBR2 (intronic enhancer) was cloned upstream of a rhodopsin minimal promoter, which by itself is not active (lower panels). Fluorescence was measured after 8 days in whole mounts (left and central panels) and cross-sections (right panels). CBR1 alone does not seem to be active, whereas CBR2 can activate transcription in a Crx-dependent manner. CBR2 activity is detected in the outer nuclear layer, but not in the inner nuclear layer. This cis-regulatory element could therefore be responsible for the photoreceptor-specific expression of Fam161a.

Figure 4

Fam161a Expression Pattern during Mouse Retinal Development and Retinal Degeneration DAB immunostaining of Fam161a in murine retinas at various developmental stages, in wild-type animals (wt) and in models of retinal degeneration. (A and B) Fam161a is minimally expressed at P0 (A), whereas at P10 it shows a marked presence in the outer nuclear layer and in the inner segments of photoreceptors (B). Note that the outer plexiform layer is also positive. (C) In adult animals (P30), Fam161a reaches a well-defined localization in the inner segment of photoreceptors, as well as in the outer plexiform layer. It is completely absent from the outer segment of photoreceptors. (D) Magnification of portions of (C), showing details of positive Fam161a staining. (E and F) Fam161a localization in retinas from the rd1 (rd) mouse at P12 and from the Rho^−/− (rho) mouse at P60. Arrows indicate nuclear or perinuclear aggregates of Fam161a within photoreceptors. Magnification: A, B, and E, 200×; C and F, 400×; D, 800×. Abbreviations: NBL, neural basal layer; OS, outer segment of photoreceptors; IS, inner segment of photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL ganglion cell layer.