Substitution of a single non-coding nucleotide upstream of TMEM216 causes non-syndromic retinitis pigmentosa and is associated with reduced TMEM216 expression

Abstract

Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration.

Keywords: African ancestry; South Asian; ancestral allele; ciliopathy; equity of genetic testing; ethnic genetic diversity; gene expression; non-coding genetic variation; retinal dystrophy; retinitis pigmentosa.

Graphical abstract

Figure 1

Segregation of TMEM216 variants Segregation analysis of TMEM216 upstream sequence variants in representative pedigrees of African (A–Q) and South Asian (R–W) origin. M1, c.−69G>T; M2, g.61382891_61393975del; M3, c.35−2A>G; M4, c.−69G>A. ^∗, +, and # indicates individuals that underwent whole-genome sequencing, whole-exome sequencing, or targeted sequencing, respectively. Consanguinity was present in all six pedigrees (family A4, A5, A6, A7, A8, and A9) of South Asian origin carrying M4, while detected in only one family of African origin (family T-22) carrying M1. Additional details of pedigrees are in Table 1.

Figure 2

Homozygosity mapping of family A-4 and across families with TMEM216 c.−69G>A (A) Homozygosity in affected and unaffected members of A-4 across the genome identified an 8.25 Mb (g.55,000,000–63,258,298 [GRCh38]) region on chromosome 11 shared by three affected individuals. Red indicates variants in the region that are homozygous, blue indicates alleles that are heterozygous, and white indicates homozygous and heterozygous alleles in the same frequency. (B) Homozygosity mapping across families with TMEM216 c.−69G>A: homozygous regions identified on chromosome 11 in pedigrees A-4, A-5, A-7, and A-6 and 11 genes located within the 330 kb shared homozygous interval are shown.

Figure 3

Clinical findings for individuals with TMEM216 c.−69G>T and TMEM216 c.−69G>A variants of 72 and 24 years of age, respectively (A–D) En face pseudo color images (A and B) and green (532 nm) autofluorescence (C and D) from an Optos wide-angle fundus camera. The 72-year-old individual shows a greater amount of pigment and further reduction in autofluorescence (A and C). In the 24-year-old individual, there is typical bone-spicule pigment in the peripheral retina. This individual showed loss of autofluorescence, with retention of autofluorescence within a 10-degree area centered on the fovea (B and D). (E–H) En face infrared and OCT images of the right (E and F) and left (G and H) eyes centered on the fovea. The 72-year-old individual showed atrophy of both outer retina and RPE on OCT with some preservation of the foveal layers. In the 24-year-old individual, the region of preserved retinal anatomy on OCT imaging matches the retained autofluorescence observed in (D) (F and H).

Figure 4

The c.−69 variants downregulate TMEM216 expression in vitro and in vivo (A) Characteristics of the 931-bp region upstream of TMEM216 on chromosome 11 used in the luciferase assay (g.61,391,712–61,392,642 [GRCh38]). This 931-bp region contains cis-regulatory elements for known retinal transcription factors, such as CRX and OTX2, and two predicted promoter sequences (P1 and P2).^, (B) Relative luciferase activity for the reference and mutated constructs containing the TMEM216 c.−69G>A, c.−69G>T, and additional constructs harboring deletions of the predicted P1 and P2 promoters. Luciferase activity was normalized by a construct with no promoter sequence and the WT control. ^∗∗∗∗p < 0.0001. (C) Real-time quantitative PCR quantification of TMEM216 and the neighboring TMEM138 gene expression in blood samples from two WT control subjects and two individuals carrying the homozygous TMEM216 c.−69G>T variant (T-24 and T-23). (D) IGV representation of cDNA reads from Oxford Nanopore sequencing of leukocyte RNA. Top lane, an affected individual homozygous for c.−69G>T; middle lane, the heterozygous mother; bottom lane, a healthy WT control subject. Red vertical line represents the variant base c.−69G, which is not included in the RNA sequence. The right panel shows the relative read depths for a benign coding SNV (rs3741265, c.264G>A [p.Pro88Pro]) for which the mother is heterozygous. The A allele, in cis with the c.−69G>T mutation, is represented at a significantly lower concentration than the G allele (read depth 8,184 [A] versus 23,255 [G]). (E) RT-qPCR analysis of gene expression in hTERT-RPE1 cells. Quantitative PCR analysis of TMEM216 and TMEM138 expression across all three genotypes. Expression is relative to GAPDH housekeeping gene. One-way ANOVA results are shown (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). (F) Functional validation of TMEM216 c.−69G>A in hTERT-RPE1 cells. CRISPR-Cas9-edited hTERT-RPE1 cells with c.−69G>A variant in homozygous state showed loss of cilia or abnormal cilia (green) whereas the wild-type and heterozygous cells showed presence of cilia. The cilia were stained with acetylated tubulin (green) l and the nucleus were stained with DAPI (blue). The scale bar represents 10 μm. Two independent clones with each genotype were analyzed. (G) Reduction in percent ciliated hTERT-RPE1 cells with c.−69G>A. Percent ciliated cells is significantly low when c.−69G>A variant is present in the homozygous (3%) or heterozygous (54%) state when compared to the wild-type. Corrected p value < 0.0001 (Kruskal-Wallis non-parametric test with Dunn’s multiple comparison of the difference between all of the conditions). (H) Total TMEME216 expression in the human peripheral retina compared to all GTEx tissues. Distribution of total TMEM216 expression in combined GTEx tissues (mean expression = 11.02 ± 0.05 TPM, n = 17,382) and the human peripheral retinal samples (mean expression = 19.96 ± 0.24 TPM, n = 411), showing a significantly higher expression in the retina (Mann Whitney nonparametric test p value < 0.0001).