Next generation sequencing identifies novel disease-associated BEST1 mutations in Bestrophinopathy patients

Abstract

Bestinopathies are a spectrum of retinal disorders associated with mutations in BEST1 including autosomal recessive bestrophinopathy (ARB) and autosomal dominant Best vitelliform macular dystrophy (BVMD). We applied whole-exome sequencing on four unrelated Indian families comprising eight affected and twelve unaffected individuals. We identified five mutations in BEST1, including p.Tyr131Cys in family A, p.Arg150Pro in family B, p.Arg47His and p.Val216Ile in family C and p.Thr91Ile in family D. Among these, p.Tyr131Cys, p.Arg150Pro and p.Val216Ile have not been previously reported. Further, the inheritance pattern of BEST1 mutations in the families confirmed the diagnosis of ARB in probands in families A, B and C, while the inheritance of heterozygous BEST1 mutation in family D (p.Thr91Ile) was suggestive of BVMD. Interestingly, the ARB families A and B carry homozygous mutations while family C was a compound heterozygote with a mutation in an alternate BEST1 transcript isoform, highlighting a role for alternate BEST1 transcripts in bestrophinopathy. In the BVMD family D, the heterozygous BEST1 mutation found in the proband was also found in the asymptomatic parent, suggesting an incomplete penetrance and/or the presence of additional genetic modifiers. Our report expands the list of pathogenic BEST1 genotypes and the associated clinical diagnosis.

Figure 1

Family A. (a) Pedigree of the family with two affected members. The proband is marked with an arrow. (b,c) Colored fundus photographs of right and left eye of proband (II.1) and affected sibling (II.2) respectively, showing focal areas of subretinal fibrosis (black arrow) with very sparse vitelliform deposit marked by white arrow. (d,e) Fundus autofluorescence images of proband (II.1) and affected sibling (II.2) respectively, showing focal dot like areas of increased autofluorescence (white arrow) corresponding to the areas of vitelliform deposit seen in fundus photography. (f) Optical coherence tomography (OCT) images of the proband showing intra retinal cystoid and schitic changes (asterisk) with minimal sub retinal fluid (white arrow). The sub retinal area is marked with the retinal layers as RPE = Retinal Pigment Epithelium, IZ = Interdigitation Zone, EZ = Ellipsoid Zone and ELM = External Limiting Membrane.

Figure 2

Family B. (a) Pedigree of the family with one affected subject (II.1; indicated by arrow). (b) Colored fundus photographs of the right and left eyes of proband demonstrating focal areas of sub-retinal fibrosis (black arrow) and extensive areas of sub-retinal vitelliform deposits across the arcade and nasal to the disk (white arrow). (c) Corresponding fundus autofluorescence (FAF) images of right and left eyes of the proband highlight the vitelliform deposits (white arrows). (d) OCT images of right and left eyes of proband showing sub retinal (white arrow) and intra retinal (white asterisk) changes with fluid accumulation.

Figure 3

Family C. (a) Pedigree of the family with four affected members. The proband is marked with an arrow. Colored fundus photographs (b, d, f and h) of the proband (II.3) (b, c), affected siblings (brother II.1 (d,e), brother II.4 (f,g), sister (II.2 (h,i) show yellowish white sub-retinal deposits (white arrows). Fundus autofluoresence (c, e, g and i) of the proband (II.3) (b,c), affected siblings (brother II.1 (d,e), brother II.4 (f,g), sister (II.2 (h,i) show hyper autoflourescence (white arrows) mainly concentrated around posterior pole. A localised area of subretinal fibrosis (black arrow; c) is observed in affected sibling II.1. (j) OCT of right and left eyes of proband show outer segment elongation (black circle) with sub retinal changes and subretinal fluid (white arrow).

Figure 4

Family D. (a) Pedigree chart of family with one affected subject indicated by black arrow. Colored fundus photographs of right and left eyes of proband (b), father (d) and sister (f). Corresponding FAF images of right and left eyes of proband (c), father (e) and sister (g). Proband (b), shows prominent yellowish vitelliform deposits in macula giving pseudohypopyon appearance, better highlighted in FAF (c). Proband’s father (d) shows a normal fundus in the right eye but left eye had focal vitelliform lesion (white arrow) better highlighted in FAF (white arrow) (e). Sister (f,g) did not show abnormal retinal features. (h) OCT of the proband shows sub-retinal deposits with sub-retinal fluid in both eyes (white arrows).

Figure 5

Identification of casual variants in bestrophinopathy families. (a) Flowchart depicting the analysis of exome data from bestrophinopathy patients and their relatives. (b) Pedigrees of four bestrophinopathy families analyzed in this study. Candidate variants identified in each family is shown. White circles - unaffected females; white squares - unaffected male; filled circles - affected females; filled squares - affected males. Number within circle/square indicates that the sample was sequenced.

Figure 6

RNA-seq analysis of BEST1 transcript isoforms. Top panel shows structure of seven human protein-coding BEST1 transcript isoforms annotated by Ensembl (release 75), and the location of two mutations p.Arg47His and p.Val216Ile. Gray boxes represent exons. Green vertical line and red vertical line indicate ORF start and stop, respectively. Bottom panel shows average per-base read coverage and junction read counts (middle).

Figure 7

BEST1 mutations depicted on (a) a cartoon of BEST1 with the conserved pfam domain, (b) the predicted topology of BEST1, (c) the structure of BEST1 (ProteinModelPortal, ID O76090), and (d) a multiple sequence alignment of BEST1 protein sequences from multiple species. ARB - Autosomal Recessive Bestrophinopathy.