Whole Genome Sequencing Revealed Mutations in Two Independent Genes as the Underlying Cause of Retinal Degeneration in an Ashkenazi Jewish Pedigree

Abstract

Retinitis pigmentosa (RP) causes progressive photoreceptor loss resulting from mutations in over 80 genes. This study identified the genetic cause of RP in three members of a non-consanguineous pedigree. Detailed ophthalmic evaluation was performed in the three affected family members. Whole exome sequencing (WES) and whole genome sequencing (WGS) were performed in the three affected and the two unaffected family members and variants were filtered to detect rare, potentially deleterious variants segregating with disease. WES and WGS did not identify potentially pathogenic variants shared by all three affected members. However, WES identified a previously reported homozygous nonsense mutation in KIZ (c.226C>T, p.Arg76*) in two affected sisters, but not in their affected second cousin. WGS revealed a novel 1.135 kb homozygous deletion in a retina transcript of C21orf2 and a novel 30.651 kb heterozygous deletion in CACNA2D4 in the affected second cousin. The sisters with the KIZ mutation carried no copies of the C21orf2 or CACNA2D4 deletions, while the second cousin with the C21orf2 and CACNA2D4 deletions carried no copies of the KIZ mutation. This study identified two independent, homozygous mutations in genes previously reported in autosomal recessive RP in a non-consanguineous family, and demonstrated the value of WGS when WES fails to identify likely disease-causing mutations.

Keywords: genetics; retina; retinitis pigmentosa.

Figure 1

Pedigree RF.L.11.10 and segregation of mutations in KIZ and C21orf2 with recessive RD. I:1–5 represents elder siblings (three unaffected males and two unaffected females) of I:6. (-) Indicates presence of wild type allele where as V1, V2 and V3 indicate the mutant alleles. The homozygous nonsense mutation p.Arg76* in KIZ (V1) segregated with disease in one branch of the family with affected members II:2 and II:4. A 1.1Kb homozygous deletion V2 (Chr21: 45,755,728–45,756,862) in C21orf2 gene was observed in II:6 from a different branch of the pedigree RF.L.11.10. An additional 30Kb heterozygous deletion V3 (Chr12: 1,949,399–1,980,050) in CACNA2D4 gene was also observed in the affected member II:6.

Figure 2

Kinetic Perimetry. (A) Goldmann kinetic perimetry of II:2 at age 52 shows extensive mid-peripheral ring scotomas with preserved central islands; (B) Visual fields at age 60 show large central scotomas without central islands; (C,D) Goldmann kinetic perimetry of II:4 at age 47 and 51, respectively, shows progressive expansion of mid-peripheral ring scotomas and preserved central islands; (E) Goldmann kinetic perimetry of II:6 at age 56 shows mid-peripheral ring scotomas with preserved central islands. Left panels show left visual fields while right panels show right visual fields. Shaded regions indicate scotomas.

Figure 3

Retinal Imaging. (A–D) Fundus imaging of the right (A1–D1) and left (A2–D2) eye of II:2 at age 60. (A1,A2) Color fundus photos show disc pallor, retinal vascular attenuation and bone spicule pigmentary change along the arcades. (B1,B2) Fundus autofluorescence shows a ring of increased autofluorescence in the macula and nummular loss of autofluorescence along the arcades in both eyes. (C1,C2) Infrared fundus images show retinal pigment epithelium (RPE) loss along the arcades, vascular attenuation, and bone spicules in both eyes. (D1,D2) Macular horizontal spectral domain optical coherence tomography (SD-OCT) scans show loss of outer retinal layers throughout the macula with preserved outer nuclear layer (ONL), external limiting membrane (ELM), and inner segments (IS) at the fovea in both eyes. (E–G) Fundus imaging of the right (E1–G1) and left (E2–G2) eye of II:4 at age 47. (E1,E2) Color fundus photos of the right eye show pigment mottling along the arcades (E1), and photos of the left eye shows a dense epiretinal membrane with overlying vitreous opacity and pigmentary changes along the inferotemporal arcade (E2). (F1,F2) Fluorescein angiography shows staining of the optic nerve and areas of RPE atrophy in late frames. (G1,G2) SD-OCT scans show a preserved ONL, ELM, and inner segment/outer segment (IS/OS) junction band at the fovea in both eyes; the edges of the IS/OS junction band are demarcated by red arrows. (H1–H4) Infrared fundus images and SD-OCT B-scans of the right (H1,H2) and left (H3,H4) of patient II:6 at age 58. Blue lines indicate location of SD-OCT B-scans (bottom) and green lines show locations of smaller scans showing cystoid macular edema with large foveal cysts and IS/OS junction band loss; red arrows indicate the edges of the IS/OS junction band.

Figure 4

Electroretinogram (ERG). ERG responses from II:4 at age 47 show moderately reduced but measureable amplitudes in both eyes in response to (A) dim (−24 dB) scotopic flash, (B) mixed (0 db) scotopic flash, (C) single 0 dB photopic flash, and (D) 30 Hz 0 dB flicker stimuli.

Figure 5

Segregation analysis of the deletion in C21orf2. (A) Region of C21orf2 encompassing the 1,135 base pair (bp) homozygous deletion (Chr21: 45,755,728–45,756,862) observed in patient II:6 of the pedigree RF.L.11.10. A 727 bp region (Chr21: 45,755,983–45,756,710) within the 1,135 bp homozygous deletion is reported to encompass retina expressed sequence [17]. The retinal transcriptome ribonucleic acid-sequence (RNA-seq) coverage data is shown in red. In addition, this deletion also encompasses exon 3 of NM_001271442 transcript of C21orf2; (B,C) Region of C21orf2 amplified by PCR to detect the presence of wild type or mutant alleles; (D) Gel electrophoresis of the PCR products of C21orf2 deleted region in members of RF.L.10.11 family. Presence of the 345 bp PCR product in II:6 indicated the presence of the homozygous deletion in C21orf2 whereas the other members had wild type alleles.

Figure 6

Quantitative polymerase chain reaction (qPCR) analysis of the CACNA2D4 sequence with a large heterozygous deletion in pedigree RF.L.11.10. (A) Region spanning a 30 kb heterozygous deletion (Chr12: 1,949,399–1,980,050) observed in CACNA2D4 gene in patient II:6 in pedigree RF.L11.10. This deletion includes 8 exons (exon 19 to exon 26) of the CACNA2D4 gene; (B) qPCR of exons 19, 21 and 26 of CACNA2D4 indicated the presence of two wild type alleles of CACNA2D4 in II:2, II:3 and II:4; and a single copy of CACNA2D4 exon 19, 21 and 26 in II:6. The qRT-PCR was normalized to two reference genes, GPR15 and ZNF80 and Control human genomic DNA was used as calibration control (Cal) to determine the copy number of each allele present in the test samples. * p < 0.05; ** p < 0.01.