Phenocopy of a heterozygous carrier of X-linked retinitis pigmentosa due to mosaicism for a RHO variant

Abstract

We describe both phenotype and pathogenesis in two male siblings with typical retinitis pigmentosa (RP) and the potentially X-linked RP (XLRP) carrier phenotype in their mother. Two affected sons, two unaffected daughters, and their mother underwent detailed ophthalmological assessments including Goldmann perimetry, color vision testing, multimodal imaging and ISCEV-standard electroretinography. Genetic testing consisted of targeted next-generation sequencing (NGS) of known XLRP genes and whole exome sequencing (WES) of known inherited retinal disease genes (RetNet-WES). Variant validation and segregation analysis were performed by Sanger sequencing. The mutational load of the RHO variant in the mother was assessed in DNA from leucocytes, buccal cells and hair follicles using targeted NGS. Both affected sons showed signs of classical RP, while the mother displayed patches of hyperautofluorescence on blue light autofluorescence imaging and regional, intraretinal, spicular pigmentation, reminiscent of a carrier phenotype of XLRP. XLRP testing was negative. RetNet-WES testing revealed RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (21% of the reads) in the mother and in a heterozygous state in both sons. Targeted NGQSS of the RHO variant in different maternal tissues showed a mutation load between 25.06% and 41.72%. We report for the first time that somatic mosaicism of RHO variant c.404G > C p.(Arg135Pro) mimics the phenotype of a female carrier of XLRP, in combination with heterozygosity for the variant in the two affected sons.

Figure 1

Pedigree of the family and segregation analysis of the RHO variant. Filled symbol: affected individual. Striped symbol: mildly affected female, mimicking carriership of XLRP. M/ + : heterozygous RHO variant c.404G > C p.(Arg135Pro). + / + : homozygous wild type. mM/ + : mosaicism for c.404G > C p.(Arg135Pro).

Figure 2

Fundus imaging. (A) Fundus pictures of both eyes of individual III.1 (age 28): note RPE alterations in retinal midperiphery and periphery. Spicular intraretinal pigment migrations in the periphery. (B) Fundus pictures of both eyes of individual II.1 (age 52): Midperipheral RPE alterations and pigment migrations, more pronounced in, but not exclusive to nasal midperiphery. (C) Blue light autofluorescent imaging of both eyes of individual III.1 (age 28): mottled hypo-autofluorescent periphery, with small hyper-autofluorescent ring around the macula, with a second hyperfluorescent ring around temporal vascular arcades. (D) Blue light autofluorescent imaging of both eyes of individual II.1 (age 52): note mosaic pattern of hyperfluorescent areas, some around the vasculature.

Figure 3

ISCEV standard full-field flash electroretinogram (ERG). (Top traces) Right eye (RE) of proband (III.1) at age 29. Scotopic ERG: complete absence of rod specific response to a dim white flash (0.01 cd.s/m2); residual response to slightly higher intensity red flash (0,3 cd.s/m2); responses to high intensity flashes (3.0 cd.s/m2 and 10.0 cd.s/m2) show significant reduction of amplitudes and major delay in peak times. Photopic ERG: response to a transient, high-intensity flash (3.0 cd.s/m2) also shows significant reduction in amplitudes and a delay of responses. This is confirmed by response to a 30 Hz high intensity (3.0 cd.s/m2) flicker stimulus. (Middle traces) RE of the mother of the proband (II.1) at age 50. Scotopic rod specific ERG: reduction of amplitude of rod specific response to a dim white flash (0.01 cd.s/m2) to about two thirds of normal; response to slightly higher intensity red flash (0.3 cd.s/m2) shows conserved response of cone-specific part (b1), with similar decrease to two thirds of normal amplitude of rod-specific response (b2); responses to high-intensity flashes (3.0 cd.s/m2 and 10.0 cd.s/m2) show similar reduction to two thirds of normal for both a- and b-waves with only minor delay of responses. Photopic ERG: response to a transient high-intensity flash (3.0 cd.s/m2) shows mild delay of b-wave (right column top trace) and mild delay to a 30 Hz high intensity (3.0 cd.s/m2) flicker stimulus (right column bottom trace). (Bottom traces) Normal reference traces of a healthy control subject.

Figure 4

Genomic testing. (Left) Whole exome sequencing reads visualized in the Integrative Genomics Viewer (IGV), showing the RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (variant allele frequency [VAF] of ~ 21%) in the mother (II.1) (top). Sanger sequencing confirmation of the mosaic variant is shown at the bottom. (Right) IGV view of the RHO variant c.404G > C p.(Arg135Pro) in a heterozygous state (VAF: ~ 47%) in the son (III.1) (top). Sanger sequencing confirmation of the heterozygous variant is shown at the bottom (III.1).

Figure 5

Mosaicism in multiple tissue types. Massively parallel sequencing reads visualized in the Integrative Genomics Viewer (IGV), showing the RHO variant c.404G > C p.(Arg135Pro) in a mosaic state in multiple tissues. (A) blood sample 1: variant allele frequency (VAF) of 31,97,279%. (B) blood sample 2: VAF of 30,4457%. (C) buccal mucosa sample 1: VAF of 40,9042%. (D) buccal mucosa sample 2: VAF of 25,0641%. (E) Sample of hair follicle: VAF of 41, 72,123%.