Putative digenic inheritance of heterozygous <i>RP1L1</i> and <i>C2orf71</i> null mutations in syndromic retinal dystrophy

<p><i>Background</i>: Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration and can occur in non-syndromic and syndromic forms. Syndromic RP is accompanied by other symptoms such as intellectual disability, hearing loss, or congenital abnormalities. Both forms are known to exhibit complex genetic interactions that can modulate the penetrance and expressivity of the phenotype.</p> <p><i>Materials and methods</i>: In an individual with atypical RP, hearing loss, ataxia and cerebellar atrophy, whole exome sequencing was performed. The candidate pathogenic variants were tested by developing an <i>in vivo</i> zebrafish model and assaying for retinal and cerebellar integrity.</p> <p><i>Results</i>: Exome sequencing revealed a complex heterozygous protein-truncating mutation in <i>RP1L1</i>, p.[(Lys111Glnfs*27; Gln2373*)], and a heterozygous nonsense mutation in <i>C2orf71</i>, p.(Ser512*). Mutations in both genes have previously been implicated in autosomal recessive non-syndromic RP, raising the possibility of a digenic model in this family. Functional testing in a zebrafish model for two key phenotypes of the affected person showed that the combinatorial suppression of <i>rp1l1</i> and <i>c2orf71l</i> induced discrete pathology in terms of reduction of eye size with concomitant loss of rhodopsin in the photoreceptors, and disorganization of the cerebellum.</p> <p><i>Conclusions</i>: We propose that the combination of heterozygous loss-of-function mutations in these genes drives syndromic retinal dystrophy, likely through the genetic interaction of at least two loci. Haploinsufficiency at each of these loci is insufficient to induce overt pathology.</p

<i>In vivo</i> zebrafish morpholino complementation assay showing the effect of <i>SIX6</i> nonsynonymous variants.

<p>Zebrafish embryos were microinjected with a translation blocking morpholino designed to target <i>six6a</i>. Total eye size (µm²) was measured 3 days post fertilization. Compared to the uninjected controls, morphants showed a significant reduction in eye size. Zebrafish were co-injected with the morpholino and a human <i>SIX6</i> allele (Glu93Gln, Glu129Lys, Asn141His, Leu205Arg, Thr212Met, or Ser242IIe). Results of each allele were compared to the <i>SIX6</i> non-risk allele (Ref). P-values are provided below the mean of each treatment.</p

<i>SIX6</i> variants identified by sequencing POAG cases and controls.

<p>Coordinates are based on the Hg19 reference; MAF = minor allele frequency;</p><p>AA = amino acid; GERP = Genomic Evolutionary Rate Profiling.</p

OCT measurements from POAG cases homozygous for rs33912345.

<p>SD = standard deviation; OCT = optical coherence tomography.</p

Functional evaluation of <i>SIX6</i> variants on the volume of the optic nerve.

<p>Representative whole mount images of acetylated-tubulin expression in the heads of zebrafish embryos injected with a control or <i>six6a</i> morpholino, rescued by co-injection with human non-risk SIX6 transcript or a transcript containing the Leu205Arg hypomorphic variant (A). Acetylated-tubulin staining is restricted primarily to axon tracts and can be used to visualize the optic nerve. Relative to the control morphants, volumetric regions of interest (ROI) along the optic nerve in <i>six6a</i> morphants were reduced significantly. Co-injection of human variants revealed a hypomorphic (Leu205Arg, Asn141His) or benign (Glu93Gln) role of the variants on the optic nerve (B). Sample size for all injection paradigms ranged from 7–9 and p-values are plotted for each comparison (*** p<0.001; ** p<0.01). No significant changes in the volume of other axonal tracts in the head (marked by an asterisk) were detected. Standard error of the mean is shown and white scale bars = 20 um.</p

<i>In vitro</i> luciferase assay results showing the effect of <i>SIX6</i> enhancer variants.

<p><i>SIX6</i> enhancer alleles were tested using a dual luciferase assay and the ratio of the experimental luciferase: control luciferase was calculated (DLR ratio). All vectors were co-transfected with NeuroD and E47. In this context, the <i>SIX6</i> enhancer is functioning to increase expression compared to the empty vector (pGL4.23), driven by a minimal promoter. Compared to the reference enhancer (Ref), one variant (Chr14:60974449_G) significantly increases the enhancer's activity.</p

Morpholino knockdown of <i>six6a</i>.

<p>Zebrafish were microinjected with a <i>six6a</i> translation blocking morpholino. Lateral images, taken 3 days post fertilization (3 dpf), of a wild-type zebrafish (left) and a morpholino injected zebrafish (right) are shown, highlighting the small eye phenotype (dashed circle).</p

Results from the <i>in vivo</i> zebrafish <i>six6a</i> morpholino complementation assay.

<p>N = sample size; SD = standard deviation.</p