Periasamy Sundaresan

Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2)

Congenital hereditary endothelial dystrophy (CHED) is a heritable, bilateral corneal dystrophy characterized by corneal opacification and nystagmus. We describe seven different mutations in the SLC4A11 gene in ten families with autosomal recessive CHED. Mutations in SLC4A11, which encodes a membrane-bound sodium-borate cotransporter, cause loss of function of the protein either by blocking its membrane targeting or nonsense-mediated decay.

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal phenotypes

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.

The mutations and potential targets of the forkhead transcription factor FOXL2

Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). This genetic disorder is characterized by eyelid and mild craniofacial abnormalities that can appear associated with premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. In this review we summarize recent data concerning FOXL2, its mutations and its potential targets. Indeed, many mutations have been described in the coding sequence of FOXL2. Among them, polyalanine expansions and premature nonsense mutations have been shown to induce protein aggregation. In the context of the ovary, FOXL2 has been suggested to be involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and inflammation processes. The elucidation of the impact of FOXL2 mutations on its function will allow a better understanding of the pathogenic mechanisms underlying the BPES phenotype.

Association analysis of nine candidate gene polymorphisms in Indian patients with type 2 diabetic retinopathy

BackgroundDiabetic retinopathy (DR) is classically defined as a microvasculopathy that primarily affects the small blood vessels of the inner retina as a complication of diabetes mellitus (DM).It is a multifactorial disease with a strong genetic component. The aim of this study is to investigate the association of a set of nine candidate genes with the development of diabetic retinopathy in a South Indian cohort who have type 2 diabetes mellitus (T2DM).MethodsSeven candidate genes (RAGE, PEDF, AKR1B1, EPO, HTRA1, ICAM and HFE) were chosen based on reported association with DR in the literature. Two more, CFH and ARMS2, were chosen based on their roles in biological pathways previously implicated in DR. Fourteen single nucleotide polymorphisms (SNPs) and one dinucleotide repeat polymorphism, previously reported to show association with DR or other related diseases, were genotyped in 345 DR and 356 diabetic patients without retinopathy (DNR). The genes which showed positive association in this screening set were tested further in additional sets of 100 DR and 90 DNR additional patients from the Aravind Eye Hospital. Those which showed association in the secondary screen were subjected to a combined analysis with the 100 DR and 100 DNR subjects previously recruited and genotyped through the Sankara Nethralaya Hospital, India. Genotypes were evaluated using a combination of direct sequencing, TaqMan SNP genotyping, RFLP analysis, and SNaPshot PCR assays. Chi-square and Fisher exact tests were used to analyze the genotype and allele frequencies.ResultsAmong the nine loci (15 polymorphisms) screened, SNP rs2070600 (G82S) in the RAGE gene, showed significant association with DR (allelic P = 0.016, dominant model P = 0.012), compared to DNR. SNP rs2070600 further showed significant association with DR in the confirmation cohort (P = 0.035, dominant model P = 0.032). Combining the two cohorts gave an allelic P < 0.003 and dominant P = 0.0013). Combined analysis with the Sankara Nethralaya cohort gave an allelic P = 0.0003 and dominant P = 0.00011 with an OR = 0.49 (0.34 - 0.70) for the minor allele. In HTRA1, rs11200638 (G>A), showed marginal significance with DR (P = 0.055) while rs10490924 in LOC387715 gave a P = 0.07. No statistical significance was observed for SNPs in the other 7 genes studied.ConclusionsThis study confirms significant association of one polymorphism only (rs2070600 in RAGE) with DR in an Indian population which had T2DM.

ABCB6 Mutations Cause Ocular Coloboma

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.

Identification of Mutations in the SLC4A11 Gene in Patients With Recessive Congenital Hereditary Endothelial Dystrophy

OBJECTIVE To identify Solute Carrier family 4 (sodium borate cotransporter) member 11 (SLC4A11) gene mutations associated with autosomal recessive congenital hereditary endothelial dystrophy (CHED2). METHODS DNA extraction from blood, polymerase chain reaction amplification, and direct sequencing of all the exons of the SLC4A11 gene were performed for 26 affected members of 20 unrelated families with CHED2. RESULTS Of 10 mutations observed, 6 were novel, 1 of which involves a complete deletion of exon 6, identified for the first time, to our knowledge, in SLC4A11. The mutations cosegregated with the disease phenotype and were absent in 200 ethnically matched control chromosomes analyzed. CONCLUSIONS This study increases the number of SLC4A11 gene mutations and confirms the role of this gene in causing CHED2. Clinical examination did not reveal any considerable variability in disease expressivity in patients carrying SLC4A11 mutations. Extensive linkage analysis may reveal the modifier genes involved in causing CHED2 in the SLC4A11 mutations unidentified in 9 families. CLINICAL RELEVANCE In India, there is a high frequency of CHED2, possibly related to consanguineous marriages. Counseling could be provided to explain the drawbacks of consanguineous marriages to assist in reducing this devastating disorder.

A novel polyalanine expansion in FOXL2: the first evidence for a recessive form of the blepharophimosis syndrome (BPES) associated with ovarian dysfunction.

The blepharophimosis syndrome (BPES) is an autosomal dominant developmental disorder in which craniofacial/eyelid malformations are associated (type I) or not (type II) with premature ovarian failure (POF). Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for both types of BPES. Heterozygous polyalanine expansions of +10 residues (FOXL2–Ala24) account for 30% of FOXL2 mutations and are fully penetrant for the eyelid phenotype. Here we describe the first homozygous FOXL2 mutation leading to a polyalanine expansion of +5 residues (FOXL2–Ala19). This novel mutation segregates in an Indian family where heterozygous mutation carriers are unaffected whereas homozygous individuals have the typical BPES phenotype, with proven POF in one female. Expression of the FOXL2–Ala19 protein in COS-7 cells revealed a significantly higher cytoplasmic retention compared to the wild-type protein. This is the first study providing genetic evidence for a recessive inheritance of BPES associated with ovarian dysfunction.

Contribution of growth differentiation factor 6-dependent cell survival to early-onset retinal dystrophies

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

EPHA2 Polymorphisms and Age-Related Cataract in India

Objective We investigated whether previously reported single nucleotide polymorphisms (SNPs) of EPHA2 in European studies are associated with cataract in India. Methods We carried out a population-based genetic association study. We enumerated randomly sampled villages in two areas of north and south India to identify people aged 40 and over. Participants attended a clinical examination including lens photography and provided a blood sample for genotyping. Lens images were graded by the Lens Opacification Classification System (LOCS III). Cataract was defined as a LOCS III grade of nuclear ≥4, cortical ≥3, posterior sub-capsular (PSC) ≥2, or dense opacities or aphakia/pseudophakia in either eye. We genotyped SNPs rs3754334, rs7543472 and rs11260867 on genomic DNA extracted from peripheral blood leukocytes using TaqMan assays in an ABI 7900 real-time PCR. We used logistic regression with robust standard errors to examine the association between cataract and the EPHA2 SNPs, adjusting for age, sex and location. Results 7418 participants had data on at least one of the SNPs investigated. Genotype frequencies of controls were in Hardy-Weinberg Equilibrium (p>0.05). There was no association of rs3754334 with cataract or type of cataract. Minor allele homozygous genotypes of rs7543472 and rs11260867 compared to the major homozygote genotype were associated with cortical cataract, Odds ratio (OR) = 1.8, 95% Confidence Interval (CI) (1.1, 3.1) p = 0.03 and 2.9 (1.2, 7.1) p = 0.01 respectively, and with PSC cataract, OR = 1.5 (1.1, 2.2) p = 0.02 and 1.8 (0.9, 3.6) p = 0.07 respectively. There was no consistent association of SNPs with nuclear cataract or a combined variable of any type of cataract including operated cataract. Conclusions Our results in the Indian population agree with previous studies of the association of EPHA2 variants with cortical cataracts. We report new findings for the association with PSC which is particularly prevalent in Indians.