Delineating the Molecular and Phenotypic Spectrum of the CNGA3-Related Cone Photoreceptor Disorder in Pakistani Families.

Cone photoreceptor dysfunction represents a clinically heterogenous group of disorders characterized by nystagmus, photophobia, reduced central or color vision, and macular dystrophy. Here, we described the molecular findings and clinical manifestations of achromatopsia, a partial or total absence of color vision, co-segregating with three known missense variants of CNGA3 in three large consanguineous Pakistani families. Fundus examination and optical coherence tomography (OCT) imaging revealed myopia, thin retina, retinal pigment epithelial cells loss at fovea/perifovea, and macular atrophy. Combination of Sanger and whole exome sequencing revealed three known homozygous missense variants (c.827A>G, p.(Asn276Ser); c.847C>T, p.(Arg283Trp); c.1279C>T, p.(Arg427Cys)) in CNGA3, the α-subunit of the cyclic nucleotide-gated cation channel in cone photoreceptor cells. All three variants are predicted to replace evolutionary conserved amino acids, and to be pathogenic by specific in silico programs, consistent with the observed altered membrane targeting of CNGA3 in heterologous cells. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of CNGA3-related cone dystrophies.

Genetic Causes of Oculocutaneous Albinism in Pakistani Population.

Melanin pigment helps protect our body from broad wavelength solar radiation and skin cancer. Among other pigmentation disorders in humans, albinism is reported to manifest in both syndromic and nonsyndromic forms as well as with varying inheritance patterns. Oculocutaneous albinism (OCA), an autosomal recessive nonsyndromic form of albinism, presents as partial to complete loss of melanin in the skin, hair, and iris. OCA has been known to be caused by pathogenic variants in seven different genes, so far, according to all the currently published population studies. However, the detection rate of alleles causing OCA varies from 50% to 90%. One of the significant challenges of uncovering the pathological variant underlying disease etiology is inter- and intra-familial locus heterogeneity. This problem is especially pertinent in highly inbred populations. As examples of such familial locus heterogeneity, we present nine consanguineous Pakistani families with segregating OCA due to variants in one or two different known albinism-associated genes. All of the identified variants are predicted to be pathogenic, which was corroborated by several in silico algorithms and association with diverse clinical phenotypes. We report an individual affected with OCA carries heterozygous, likely pathogenic variants in TYR and OCA2, raising the question of a possible digenic inheritance. Altogether, our study highlights the significance of exome sequencing for the complete genetic diagnosis of inbred families and provides the ramifications of potential genetic interaction and digenic inheritance of variants in the TYR and OCA2 genes.

Homozygous Variant in ARL3 Causes Autosomal Recessive Cone Rod Dystrophy.

Purpose: Cone rod dystrophy (CRD) is a group of inherited retinopathies characterized by the loss of cone and rod photoreceptor cells, which results in poor vision. This study aims to clinically and genetically characterize the segregating CRD phenotype in two large, consanguineous Pakistani families. Methods: Funduscopy, optical coherence tomography (OCT), electroretinography (ERG), color vision, and visual acuity assessments were performed to evaluate the retinal structure and function of the affected individuals. Exome sequencing was performed to identify the genetic cause of CRD. Furthermore, the mutation's effect was evaluated using purified, bacterially expressed ADP-ribosylation factor-like protein 3 (ARL3) and mammalian cells. Results: Fundus photography and OCT imaging demonstrated features that were consistent with CRD, including bull's eye macular lesions, macular atrophy, and central photoreceptor thinning. ERG analysis demonstrated moderate to severe reduction primarily of photopic responses in all affected individuals, and scotopic responses show reduction in two affected individuals. The exome sequencing revealed a novel homozygous variant (c.296G>T) in ARL3, which is predicted to substitute an evolutionarily conserved arginine with isoleucine within the encoded protein GTP-binding domain (R99I). The functional studies on the bacterial and heterologous mammalian cells revealed that the arginine at position 99 is essential for the stability of ARL3. Conclusions: Our study uncovers an additional CRD gene and assigns the CRD phenotype to a variant of ARL3. The results imply that cargo transportation in photoreceptors as mediated by the ARL3 pathway is essential for cone and rod cell survival and vision in humans.

Identities and frequencies of variants in CYP1B1 causing primary congenital glaucoma in Pakistan.

Purpose: Primary congenital glaucoma (PCG) is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic causes of PCG segregating in 36 large consanguineous Pakistani families. Methods: Ophthalmic examination including fundoscopy, or slit-lamp microscopy was performed to clinically characterize the PCG phenotype. Genomic nucleotide sequences of the CYP1B1 and LTBP2 genes were analyzed with either Sanger or whole exome sequencing. In silico prediction programs were used to assess the pathogenicity of identified alleles. ClustalW alignments were performed to determine evolutionary conservation, and three-dimensional (3D) modeling was performed using HOPE and Phyre2 software. Results: Among the known loci, mutations in CYP1B1 and LTBP2 are the common causes of PCG. Therefore, we analyzed the genomic nucleotide sequences of CYP1B1 and LTBP2, and detected probable pathogenic variants cosegregating with PCG in 14 families. These included the three novel (c.542T>A, c.1436A>G, and c.1325delC) and five known (c.868dupC, c.1168C>T, c.1169G>A, c.1209InsTCATGCCACC, and c.1310C>T) variants in CYP1B1. Two of the novel variants are missense substitutions [p.(Leu181Gln), p.(Gln479Arg)], which replaced evolutionary conserved amino acids, and are predicted to be pathogenic by various in silico programs, while the third variant (c.1325delC) is predicted to cause reading frameshift and premature truncation of the protein. A single mutation, p.(Arg390His), causes PCG in six (~43%) of the 14 CYP1B1 mutations harboring families, and thus, is the most common variant in this cohort. Surprisingly, we did not find any LTBP2 pathogenic variants in the families, which further supports the genetic heterogeneity of PCG in the Pakistani population. Conclusions: In conclusion, results of the present study enhance our understanding of the genetic basis of PCG, support the notion of a genetic modifier of CYP1B1, and contribute to the development of genetic testing protocols and genetic counseling for PCG in Pakistani families.

Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Genetic Testing of Non-familial Deaf Patients for CIB2 and GJB2 Mutations: Phenotype and Genetic Counselling.

CIB2 and GJB2 genes variants contribute significantly in familial cases of prelingual recessive hearing loss (HL). This study was aimed to determine the CIB2 and GJB2 variants and associated phenotype in 150 non-familial individuals with HL. After getting informed consent, 150 non-familial deaf patients were enrolled and blood samples were obtained for DNA extraction. Pure tone air conduction audiometry was performed. Coding exons of CIB2 and GJB2 genes were Sanger sequenced. A tetra primer ARMS assay was developed for recurrent CIB2 variant. Four bi-allelic GJB2 variants, c.71G>A p.(Trp24*), c.231G>A p.(Trp77*), c.235delC p.(Leu79Cysfs3*) and c.35delG p.(Gly11Leufs24*), were found in nine hearing impaired individuals. We also found four homozygotes and five carriers of c.380G>A p. (Arg127His) variant of controversial clinical significance. CIB2 sequencing revealed single recurrent variant c.272T>C p. (Phe91Ser) segregating with HL in ten individuals. Among our patients, c.71G>A (p.Trp24*) was the most common variant, accounted for 45% of GJB2 variants. Two known GJB2 variants, c.235delC p. (Leu79Cysfs3*) and c.310del14 p. (Lys105Argfs2*), are reported here for the first time in Pakistani population. Our data further support the benign nature of c.380G>A p. (Arg127His) variant. For CIB2, c.272T>C p. (Phe91Ser) is the second common cause of HL among our sporadic cases. Phenotypically, in our patients, individuals homozygous for GJB2 variants had profound HL, whereas CIB2 homozygotes had severe to profound prelingual HL. Our results suggest that GJB2 and CIB2 are common cause of HL in different Pakistani ethnicities.

Molecular outcomes, clinical consequences, and genetic diagnosis of Oculocutaneous Albinism in Pakistani population.

Nonsyndromic oculocutaneous Albinism (nsOCA) is clinically characterized by the loss of pigmentation in the skin, hair, and iris. OCA is amongst the most common causes of vision impairment in children. To date, pathogenic variants in six genes have been identified in individuals with nsOCA. Here, we determined the identities, frequencies, and clinical consequences of OCA alleles in 94 previously unreported Pakistani families. Combination of Sanger and Exome sequencing revealed 38 alleles, including 22 novel variants, segregating with nsOCA phenotype in 80 families. Variants of TYR and OCA2 genes were the most common cause of nsOCA, occurring in 43 and 30 families, respectively. Twenty-two novel variants include nine missense, four splice site, two non-sense, one insertion and six gross deletions. In vitro studies revealed retention of OCA proteins harboring novel missense alleles in the endoplasmic reticulum (ER) of transfected cells. Exon-trapping assays with constructs containing splice site alleles revealed errors in splicing. As eight alleles account for approximately 56% (95% CI: 46.52-65.24%) of nsOCA cases, primarily enrolled from Punjab province of Pakistan, hierarchical strategies for variant detection would be feasible and cost-efficient genetic tests for OCA in families with similar origin. Thus, we developed Tetra-primer ARMS assays for rapid, reliable, reproducible and economical screening of most of these common alleles.

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population.

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α-melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole-exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in-frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist-induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild-type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss-of-function (LOF) allele, while p.Val174del displayed a partial LOF attribute.

Molecular and clinical studies of X-linked deafness among Pakistani families.

There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132 and PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild-to-profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling and molecular epidemiology of hearing loss among Pakistanis.

Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39.

A gene causing autosomal-recessive, nonsyndromic hearing loss, DFNB39, was previously mapped to an 18 Mb interval on chromosome 7q11.22-q21.12. We mapped an additional 40 consanguineous families segregating nonsyndromic hearing loss to the DFNB39 locus and refined the obligate interval to 1.2 Mb. The coding regions of all genes in this interval were sequenced, and no missense, nonsense, or frameshift mutations were found. We sequenced the noncoding sequences of genes, as well as noncoding genes, and found three mutations clustered in intron 4 and exon 5 in the hepatocyte growth factor gene (HGF). Two intron 4 deletions occur in a highly conserved sequence that is part of the 3' untranslated region of a previously undescribed short isoform of HGF. The third mutation is a silent substitution, and we demonstrate that it affects splicing in vitro. HGF is involved in a wide variety of signaling pathways in many different tissues, yet these putative regulatory mutations cause a surprisingly specific phenotype, which is nonsydromic hearing loss. Two mouse models of Hgf dysregulation, one in which an Hgf transgene is ubiquitously overexpressed and the other a conditional knockout that deletes Hgf from a limited number of tissues, including the cochlea, result in deafness. Overexpression of HGF is associated with progressive degeneration of outer hair cells in the cochlea, whereas cochlear deletion of Hgf is associated with more general dysplasia.