Deciphering the Genetic Basis of Degenerative and Developmental Eye Disorders in 50 Pakistani Consanguineous Families Using Whole-Exome Sequencing

Abstract

Degenerative and developmental eye disorders, including inherited retinal dystrophies (IRDs), anophthalmia, and congenital cataracts arise from genetic mutations, causing progressive vision loss or congenital structural abnormalities. IRDs include a group of rare, genetically, and clinically heterogeneous retinal diseases. It is caused by variations in at least 324 genes, affecting numerous retinal regions. In addition to IRDs, other developmental eye disorders such as anophthalmia and congenital cataracts also have a strong genetic basis. Autosomal recessive IRDs, anophthalmia, and congenital cataracts are common in consanguineous populations. In many endogamous populations, including those in Pakistan, a significant proportion of IRD and anophthalmia cases remain genetically undiagnosed. The present study investigated the variations in IRDs, anophthalmia, and congenital cataracts genes in 50 affected families. These unrelated consanguineous families were recruited from the different provinces of Pakistan including Punjab, Khyber Pakhtoon Khwa, Sindh, Gilgit Baltistan, and Azad Kashmir. Whole exome sequencing (WES) was conducted for the proband of each family. An in-house customized pipeline examined the data, and bioinformatics analysis predicted the pathogenic effects of identified variants. The relevant identified DNA variants of selected families were assessed in parents and healthy siblings via Sanger sequencing. WES identified 12 novel variants across 10 known IRD-associated genes. The four most frequently implicated genes were CRB1 (14.3%), GUCY2D (9.5%), AIPL1 (9.5%), and CERKL (7.1%) that together accounted for 40.4% of all molecularly diagnosed cases. Additionally, 25 reported variants in 19 known IRDs, anophthalmia, and congenital cataracts-associated genes were found. Among the identified variants, p. Trp278X, a stop-gain mutation in the AIPL1 (NM_014336) gene, was the most common causative variant detected. The most frequently observed phenotype was retinitis pigmentosa (46.5%) followed by Leber congenital amaurosis (18.6%). Furthermore, 98% of pedigrees (49 out of 50) were affected by autosomal recessive IRDs, anophthalmia and congenital cataracts. The discovery of 12 novel likely pathogenic variants in 10 IRD genes, 25 reported variants in 19 known IRDs, anophthalmia and congenital cataracts genes, atypical phenotypes, and inter and intra-familial variability underscores the genetic and phenotypic heterogeneity of developmental and degenerative eye disorders in the Pakistani population and further expands the mutational spectrum of genes associated with these ocular disorders.

Keywords: IRDs; Pakistani population; WES; anophthalmia; autosomal recessive; degenerative and developmental eye disorder; genetic analysis.

Figure 1

Summary of findings and population distribution of novel and known variations identified in various ethnic groups from Pakistan including Punjabi, Pakhtoon, Sindhi, Brushoo, and Kashmiri pedigrees.

Figure 2

(a) A Punjabi pedigree with ID (RF. MA0443) initially classified as autosomal dominant. However, genetic analysis revealed that it followed a pseudo-dominant mode of inheritance of an autosomal recessive disease and was subsequently reclassified as autosomal recessive. WES identified a novel homozygous nonsense variant, c.256G>T (p.Glu86X), in exon 5 of the RLBP1 gene in the proband. This variant was linked to autosomal recessive RP. (b) A Brushoo pedigree RF. MA0422 from Gilgit Baltistan, Province of Pakistan seggregating RP with an X-linked mode of inheritance. WES analysis identified an already reported homozygous variant c.1584_1587del, p.(Val529HisfsTer7) in the CHM (NM_000390) gene (MIM#300390), resulting in a truncation of the protein at the 3rd amino acid, seggregating with RP in this pedigree.

Figure 3

(a,b) A comparison diagram to illustrate the differences between initial clinical diagnosis and diagnosis after genetic testing (Retinitis Pigmentosa = RP; Leber congenital amaurosis = LCA; Retinal dystrophy = RD; Cone rod dystrophy = CRD; Myotonia congenita = MC; Retinal atrophy = RA; Pigmentary retinopathy = PR; Stickler syndrome type-IV = STL-IV; Stargardt = STGD; Bilateral maculopathy = BLM; Achromatopsia = ACHM; Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies syndrome = CODAS syndrome; Congenital glaucoma = CG; Congenital macular degeneration = CMD; Usher syndrome= USH; Hermansky-Pudlak Syndrome 8 = HPS8; Anophthalmia = ANOPHTH; Congenital cataract = CC; Cataract-open angle glaucoma syndrome = CC.OAGS; Retinal degeneration plus glaucoma = RDEG+GLU; Congenital open angle glaucoma = C.OAG; Septo-optic dysplasia = SOD). (c) Spectrum of IRD Genes Identified in the Pakistani Population. The X-axis represents the identified IRD genes from the current study, while the Y-axis shows the number of pedigrees harboring mutations in these genes. In our consanguineous AR population, the top four most frequently implicated genes were CRB1 (14.3%), GUCY2D (9.5%), AIPL1 (9.5%), and CERKL (7.1%).

Figure 4

(a) The pedigree RF.MA0334 represents a Punjabi RP manifesting family harboring a novel nonsense stop-gain variant c.1270C>T; p.(Arg424Ter) in exon 10 of the CNGA1 gene. The variant results in a premature stop codon at position 424 (Arg424Ter), leading to a truncated, non-functional protein. The Sanger sequencing confirmed that affected individuals (IV:2 and IV:5) were homozygous for the c.1270C>T variant and the normal sibling (IV:1) was heterozygous for the variant. (b) Kashmiri pedigree RF.MA0406 segregating RP in an autosomal recessive manner harbored a previously reported non-synonymous single nucleotide variant c.1459T>C; p. (Ser487Pro) in the CRB1 gene (NM_201253). The Sanger sequencing confirmed that both affected individuals were homozygous for the c.1459T>C variant, while the parents were heterozygous for the same variant.

Figure 5

(a) WES in patient IV:1 (the proband) of a five-generation pedigree RF.MA0537 seggregating Stickler syndrome type 4 identified a novel homozygous nonsense mutation c.851_852insCAAT in the COL9A1 gene, leading to the protein change p. (Pro285AsnfsTer20). (b) shows the Sanger sequencing results of family ID RF. MA0537. The affected individuals harbored homozygous insertion GATT. The mother was heterozygous while the unaffected brother lacked GATT insertion. (c) MetaDome health map represents STL-IV/COL9A1 residues.