Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 45 genes. Using homozygosity mapping, we identified a ∼4 Mb homozygous region on chromosome 2p15 in patients with autosomal-recessive RP (arRP). This region partially overlaps with RP28, a previously identified arRP locus. Sequence analysis of 12 candidate genes revealed three null mutations in FAM161A in 20 families. RT-PCR analysis in 21 human tissues revealed high levels of FAM161A expression in the retina and lower levels in the brain and testis. In the human retina, we identified two alternatively spliced transcripts with an intact open reading frame, the major one lacking a highly conserved exon. During mouse embryonic development, low levels of Fam161a transcripts were detected throughout the optic cup. After birth, Fam161a expression was elevated and confined to the photoreceptor layer. FAM161A encodes a protein of unknown function that is moderately conserved in mammals. Clinical manifestations of patients with FAM161A mutations varied but were largely within the spectrum associated with arRP. On funduscopy, pallor of the optic discs and attenuation of blood vessels were common, but bone-spicule-like pigmentation was often mild or lacking. Most patients had nonrecordable electroretinographic responses and constriction of visual fields upon diagnosis. Our data suggest a pivotal role for FAM161A in photoreceptors and reveal that FAM161A loss-of-function mutations are a major cause of arRP, accounting for ∼12% of arRP families in our cohort of patients from Israel and the Palestinian territories.

Figure 1

Autozygosity Mapping Results, Chromosomal Region, Gene Structure, and Mutations Identified in FAM161A (A) The chromosomal region harboring the homozygous haplotypes at chromosome 2 (upper panel). Rulers are based on the February 2009 USCS Genome Browser build (hg19). The homozygous regions in the two previously reported RP28 Indian families are depicted as black (homozygous regions) or gray (cosegregating heterozygous regions) bars. The lower set of horizontal bars represents the homozygous region in our set of patients, as identified by whole-genome SNP analysis, depicted for each of the studied families. The family origin, family number, and number of analyzed patients per family (in parentheses) are shown on the left. Each of the three core haplotypes (A, B, C) is color-coded, with flanking homozygous regions marked by various colors indicating a deviation from the common haplotype. The region flanked by SNP markers rs13034649 and ra2555418 covering ∼4 Mb between 60.67 and 64.51 Mb was considered the shared homozygous region. A smaller homozygous region that was identified in family MOL0696 was not used to define the shared homozygous region because of its relatively small size (∼1.8 Mb; 60.67–62.44 Mb). (B) The genes located within the shared homozygous region are ordered on the basis of their genomic location, with an arrow indicating gene orientation. Twelve of the 22 genes were screened for mutations and marked in bold. The FAM161A gene is highlighted in blue. (C) Intron-exon structure of FAM161A and location of the null mutations. All seven exons are coding exons. Exon 2 contains an in-frame ATG codon that might be used for initiation of translation. Exon 4 is alternatively spliced (see Figure 3 for more details). (D) FAM161A mutations identified in patients with arRP. The chromatograms of a wild-type and a homozygous mutant individual are depicted for each of the three mutations. The nucleotide change is shown below the chromatogram, and the amino acid is shown below the second base of each codon. An arrow indicates the mutation location.

Figure 2

The Spectrum of Fundus Findings among Different Families with Mutations in FAM161A (A–I) Imaging of three pairs of siblings from families with the p.Thr452SerfsX3 mutation, showing different severity of funduscopic changes: bone-spicule-like pigmentation was observed to only a limited degree in most patients (A, C, D, F as compared to H, I), but many patients showed grayish spots extending from the arcades to the periphery. These correlated with spots of hypofluorescence on AF imaging (B, E, G). In addition, a ring of hyperfluoresence around the fovea could be observed in many cases (B, E, G). (J and K). Color fundus mosaic (J) and AF imaging of the macular area (K) in a patient with the R523X mutation. (L and M) OCT imaging shows marked thinning of the outer nuclear layer, with relative preservation under the fovea itself. Fine wrinkling of the retina can be seen in one of the scans (M), related to an epiretinal membrane.

Figure 3

RT-PCR Analysis of FAM161A (A) RT-PCR analysis of retinal cDNA (right panel) revealed two FAM161A transcripts. The major one, FAM161A_001, does not contain exon 4, and the corresponding chromatogram is depicted in the lower sequencing panel. The less common variant FAM161A_005 contains exon 4, and the corresponding chromatogram is depicted in the upper sequencing panel. (B) The structure of the two transcripts and the location of the primers used to amplify the different mRNA regions that are shown in (C). The red circle represents a possible alternative initiation codon. The black arrows above (forward) and below (reverse) the transcript scheme represent the four primer sets that were used to analyze FAM161A transcripts in different human tissues. (C) RT-PCR analysis in 21 different human tissues using the four sets of FAM161A primers (panels 1–4) and a control gene, PGM1 (panel 5). The primer names (corresponding to B) and the PCR product length are shown on the right.

Figure 4

Fam161a Is Expressed throughout the Optic Cup during Mouse Embryogenesis, and Its Expression Is Restricted to the Photoreceptor Layer in the Postnatal Mouse Retina Characterization of Fam161a and Crx expression by in situ hybridization during embryogenesis and postnatal development. Fam161a expression was detected throughout the retinal neuroblastic layer (NBL) during embryonic stages (E12.5, E14.5, E16.5; A–C), whereas Crx expression was restricted to the photoreceptor precursors (G–I). At postnatal stages, Fam161a expression became restricted to the photoreceptors located at the outer rim of the outer neuroblastic layer (ONBL) (D–F). This expression was low at P1 (D) and increased at P5 (E). At this stage, Crx was highly expressed in the ONBL and in some cells of the inner neuroblastic layer (INBL) (J–L). At P10, Fam161a was detected in the ONL similar to Crx (F). Fam161a sense probe served as a negative control and failed to produce any nonspecific staining at all stages tested (M–R). Abbreviations are as follows: OC, optic cup; NBL, neuroblastic layer; ONBL, outer neuroblastic layer; INBL, inner neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar in (A) represents 100 μm.

Figure 5

Evolutionary Analysis of FAM161A (A) A scheme representing the human protein FAM161A is depicted at the top, with identified domains highlighted in yellow (UPF0564) or dashed boxes (coiled-coil sequences). The exon boundaries are shown below. An amino acid sliding window (length of 30 amino acids) comparing the human protein sequence to selected orthologs (macaca, dog, mouse, chicken, and zebrafish) is shown. x axis: amino acid number; y axis: percentage of amino acid identity in a 30 amino acid window. The lower graph represents a summary of the data in mammalian sequences (blue) versus all five sequences (red), with a cumulative sliding window analysis of data points that are above the average percentage of amino acid identity for each studied sequence (macaca 95%, dog 72%, mouse 58%, chicken 39%, zebrafish 35%). (B) A comparison of amino acid identity levels (comparing the human protein sequence to its ortholog in each of the six species) of all 25 known arRP proteins. The results obtained for FAM161A are marked in red. Note that there is no ortholog for FAM161A in Drosophila. (C) A phylogenetic tree of FAM161A and FAM161B. Note the relatively long distances separating the orthologous FAM161A protein sequences.