Vanita

A novel mutation in the DNA-binding domain of MAF at 16q23.1 associated with autosomal dominant cerulean cataract in an Indian family

Congenital cataract, a clinically and genetically highly heterogeneous eye disorder, is one of the significant causes of visual impairment or blindness in children. It is frequently inherited as an autosomal dominant trait. We investigated a three‐generation family of Indian origin with 12 members affected with cerulean cataract. Linkage analysis was carried out in this family using more than 100 microsatellite markers for the known cataract candidate gene loci. A positive two‐point lod score of 3.9 at θ = 0.000, indicative of linkage, was obtained with three microsatellite markers for chromosome 16. Multipoint and haplotype analysis narrowed the cataract locus to a 15.3 cM region between markers D16S518 and D16S511 that corresponds to the region 16q23.1. Direct sequencing of the candidate gene MAF, which lies in the critical linked region, revealed a novel heterozygous missense mutation in the basic region (BR) of the DNA‐binding domain. This sequence change was considered pathogenic as it segregated in all affected family members, neither seen in unaffected family members nor in 106 unrelated controls. The mutation also results in substitution of highly conserved lysine 297 by arginine (K297R) that affects a residue that forms a part of a predicted DNA‐interaction region of the protein. The association of microcornea with congenital cataract in some affected individuals further underlines the role of the MAF transcription factor in lens and anterior ocular development. Our findings expand the mutation spectrum of MAF in association with congenital cataract and highlight the genetic and phenotypic heterogeneity of congenital cataract.

Self-assembled vesicle and rod-like aggregates of functionalized perylene diimide: reaction-based near-IR intracellular fluorescent probe for selective detection of palladium

Herein, we report design, synthesis, and self-assembly of perylene diimide (PDI) based near-IR intracellular probe (PS-PDI). PS-PDI molecules undergo aggregation to form self-assembled nanospheres and nanorods morphology in THF : H2O (1 : 1) and DMSO : H2O (1 : 9), respectively. The nanospheres have an open hole on surface reminiscent of vesicle structure (with a diameter of internal void in the range of 20-25 nm) whereas lengths of the nanorods extended up to few μm range. The Pd0 based depropargylation leads to de-aggregation of these PS-PDI aggregates into smaller spherical aggregates as evidenced by DLS, SEM, and TEM studies. Interestingly, these aggregates of PS-PDI in solution show highly sensitive behavior in the presence of Pd0 showing absorbance changes in NIR region (λmax = 710 nm) and quenching of emission at λem 630 nm (DMSO : H2O, 1 : 9) with a limit of detection = 6.6 × 10-9 M or at λem 564 nm (THF : H2O, 1 : 1) with limit of detection = 2.1 × 10-8 M. Time and concentration dependent kinetics profiles of PS-PDI aggregates revealed an impressive rate constant value of 0.4 s-1 and 0.21 s-1 (DMSO : H2O, 1 : 9), respectively in fluorescence and UV-Vis spectroscopy using 2 × 10-5 M concentration of Pd0. PS-PDI undergoes rapid internalization into HeLa cells with low cytotoxicity and was successfully used as an intracellular imaging reagent for Pd0 in live HeLa cells. For practical applications, we exploited these nano-aggregates of PS-PDI for estimation of Pd2+ in the presence of a NaBH4-PPh3 mixture, Pd0 in drug and environmental samples, and Pd2+ in urine samples with excellent selectivity and sensitivity.

Nanomolar fluorogenic detection of Al(III) by a series of Schiff bases in an aqueous system and their application in cell imaging

Three positional isomers of a Schiff base containing -OH as end groups have been synthesized and evaluated for selective Al(III) detection due to inhibition of ESIPT, PET and activation of CHEF in 70% aqueous medium. Devoid of any conventional fluorophore, these sensors have nanomolar detection limits with high quantum yields and naked eye sensing of Al(III). Moreover, these probes have been demonstrated to enable the Al(III) detection in live human HeLa cells and rat C6 glioma cells using a confocal microscope.

A novel 7 bp deletion in PRPF31 associated with autosomal dominant retinitis pigmentosa with incomplete penetrance in an Indian family.

To localize and identify the gene linked with non-syndromic autosomal dominant retinitis pigmentosa (adRP) with high but not complete penetrance in an Indian family. A detailed family history and clinical data were recorded. A genome-wide scan by 2-point linkage analysis using nearly 400 fluorescently labeled microsatellite markers in combination with multipoint lod score and haplotype analysis was carried out. Mutation screening was performed in the candidate gene by bidirectional sequence analysis of the amplified products. A maximum 2-point lod score of 3.553 at theta = 0.0 was obtained with marker D19S572. Haplotype analysis placed the RP locus distal to marker D19S572, in close proximity to the gene for pre-mRNA processing factor 31 (PRPF31) at 19q13.42. Mutation screening in all 14 exonic regions and adjacent flanking intronic sequences of PRPF31 revealed a novel 7 bp deletion, c.59_65del7 (p.Gly20AlafsX43), in the first coding exon of PRPF31. This leads to a premature termination codon (PTC) in the next exon, 43 amino acids downstream. The observed 7 bp deletion in PRPF31 was identified in all the tested 10 affected members and in an unaffected individual, consistent with a high, but not the complete penetrance of c.59_65del7 (p.Gly20AlafsX43). This deletion was not observed in other tested six unaffected family members or in 100 ethnically matched control subjects. The present study describes mapping of a locus for non-syndromic adRP at 19q13.42 (RP11 locus) in a family of Indian origin and identifies a novel deletion, c.59_65del7, in PRPF31 within the mapped interval. Since the mutant PRPF31 is truncated relatively close to the N-terminus of the protein, haploinsufficiency rather than aberrant protein formation is likely to be the underlying mechanism of the disease. The present findings further substantiate the role of PRPF31 that encodes a component of the spliceosome complex in relation to ADRP.

Association of RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms with diabetic retinopathy in T2DM patients from north India

AIMS The present study aimed to examine the association of RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms with diabetic retinopathy (DR) in north Indian T2DM patients. METHODS In this case-control association study, 758 T2DM patients were recruited. 446 with retinal neovascularization, microneurysms and hemorrhages were considered as cases (DR) and 312 patients with T2DM and no clinical signs of retinopathy (DNR), were recruited as controls. Genotypes for RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms were generated by direct sequencing of amplified products. RESULTS Genotype distribution of p.Gly82Ser (RAGE) and p.Val16Ala (MnSOD) polymorphisms were significantly different between DR and DNR (p<0.05) whereas distribution of allele frequency did not differ significantly (p>0.05). A significantly higher frequency of homozygous Ser82 genotype in DR patients was detected compared with DNR (2.4% vs 0.64%) for p.Gly82Ser (RAGE) polymorphism whereas there was a higher frequency of homozygous Ala16 genotype for p.Val16Ala (MnSOD) polymorphism in DR patients compared with DNR (22.6% vs 19.3%). Binary logistic analyses showed an association of homozygous recessive genotype Ser82 with DR (OR: 2.63%, 95% CI: 0.16-15.88, p<0.033) for p.Gly82Ser (RAGE) polymorphism. However, we did not find a significant association of p.Val16Ala polymorphism in MnSOD with retinopathy. CONCLUSIONS The findings indicate a statistically significant association of p.Gly82Ser polymorphism in RAGE with DR in T2DM patients.

A missense mutation in CRYGD linked with autosomal dominant congenital cataract of aculeiform type.

To detect the underlying genetic defects in two autosomal dominant congenital cataract (ADCC) families, having respectively twenty and four members affected with bilateral congenital cataract. Detailed family history and clinical data were recorded. Mutation screening in twenty three candidate genes including crystallins (CRYAA, CRYAB, CRYBA1/A3, CRYBA2, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGA, CRYGB, CRYGC, CRYGD, and CRYGS), gap junctional channels; connexins (GJA8, GJA3), beaded filament chain proteins (BFSP1, BFSP2), major intrinsic protein (MIP), lens intrinsic membrane protein-2 (LIM2), transcriptional factor (MAF), and in genes encoding for membrane-associated proteins (TMEM114, CHMP4B, EPHA2) was performed by bi-directional sequence analysis of the amplified products. In family A twenty members in six generations were affected by bilateral aculeiform type cataract and in family B four affected members in three generations had granular nuclear cataract. Mutation screening in already known candidate genes by sequence analyses revealed proline to threonine substitution at codon 23 (p.Pro23Thr) in CRYGD for aculeiform type cataract in family A. The family B with four members affected by granular nuclear cataract, however, could not be linked with any of these analyzed 23 candidate genes. The present study describes identification of p.Pro23Thr mutation in CRYGD for aculeiform type cataract in an ADCC family of Indian origin. The identical mutation has previously been reported to be linked with different phenotypes; lamellar cataract, cerulean cataract, coralliform cataract, flaky silica-like nuclear cataract and fasciculiform type cataract in different ADCC families. Interestingly, a mutation of different codon, i.e., p.Arg58His in CRYGD has been reported to be linked with aculeiform cataract in four different families; two from Switzerland, one from Macedonia and in a Mexican family. The findings in present study thus expand the genetic heterogeneity for aculeiform type cataract. Further, exclusion of these twenty three known candidate genes in family B having ADCC of granular nuclear type indicates the role of some other gene apart from for crystallins, gap junction channels, beaded filaments and membrane-associated proteins, and MAF for this phenotype.

Association of aldose reductase gene (AKR1B1) polymorphism with diabetic retinopathy

AIMS Present study aimed to investigate the association of aldose reductase (AKR1B1) gene polymorphism (-106C>T; rs759853) with diabetic retinopathy (DR) in type 2 diabetes mellitus (T2DM) patients from north India. METHODS The present case-control association study recruited 926 subjects, including 487 DR patients and 439 individuals with confirmed T2DM as controls (CDR). AKR1B1 -106C>T polymorphism analysis in these 926 subjects was performed by polymerase chain reaction and direct DNA sequence analysis. Statistical analysis was performed using SPSS package. RESULTS Statistically significant differences were observed between the two analyzed groups in the age of onset of diabetes (p=0.000) and duration of diabetes (p=0.000). Genotype distribution of AKR1B1 -106C>T polymorphism differed significantly between DR and CDR groups (p=0.02), however, distribution of allele frequency did not differ significantly (p=0.19). Binary logistic regression analyses showed an association of homozygous recessive TT genotype with diabetic retinopathy (OR: 1.61%, 95% CI, 1.39-2.284, p<0.01) in comparison to wild type CC genotype. CONCLUSIONS These findings suggest a statistically significant association of AKR1B1 -106C>T polymorphism with retinopathy in North Indian patients. To our knowledge, this is the first report of association of -106C>T polymorphism in AKR1B1 in DR patients from India.

Confirmation of TTC8 as a disease gene for nonsyndromic autosomal recessive retinitis pigmentosa (RP51)

Nonsyndromic retinitis pigmentosa (RP) is genetically highly heterogeneous, with >100 disease genes identified. However, mutations in these genes explain only 60% of all RP cases. Blood samples were collected from 12 members of an autosomal recessive RP family. Whole genome homozygosity mapping and haplotype analysis placed the RP locus in this family at chromosome 14q31.3. Whole‐exome sequencing (WES) in proband revealed a mutation in TTC8, which was flagged as most likely candidate gene by bioinformatic analysis. TTC8 is mutated in Bardet–Biedl syndrome 8 (BBS8), and once reported previously in a family with nonsyndromic RP. Sequencing of amplified products of exon 13 of TTC8 validated c.1347G>C (p.Gln449His), a novel change that affects the final nucleotide of exon 13 and might deleteriously affect splicing. This mutation segregated completely with the disease in the family and was not observed in 100 ethnically matched controls from same population. This represents second report of a TTC8 mutation in nonsyndromic RP, thus confirming the identity of TTC8 as causative gene for RP51.

Differential effect of cataract-associated mutations in MAF on transactivation of MAF target genes

Three mutations in the highly conserved DNA-binding region of c-MAF (R288P, K297R, and R299S) are associated with phenotypically distinct forms of autosomal dominant congenital cataract. However, the molecular mechanisms underlying this phenotypic diversity remain unclear. In this work, we have investigated the hypothesis that differential transactivation of MAF target genes could be one factor determining the phenotypic differences. Promoter constructs were generated for four human crystallin genes with conserved half-site MAF responsive elements (MARE). MAF expression constructs were constructed with the wildtype MAF sequence and with each of the three known mutations, i.e., R288P (associated with pulverulent cataract), K297R (associated with cerulean cataract), and R299S (associated with the most severe phenotype, congenital cataract, and microcornea syndrome). Transactivation was measured using luciferase reporter assays following cotransfection in HEK cells. Responsiveness to wildtype c-MAF was established for each of the four crystallin promoter constructs. The same constructs were then investigated using c-MAF mutants corresponding to each of the three mutations. A differential response was noted for each of the tested crystallin genes. The mutation R288P significantly reduced the expression of the CRYGA and CRYBA1 constructs but had no significant effect on the other two constructs. K297R did not lead to a significant reduction in expression of any of the four constructs, although there was a tendency toward reduced expression especially for the CRYGA construct. R299S, which is associated with the most severe phenotype, congenital cataract, and microcornea syndrome, was associated with the most severe overall effect on the transactivation of the four crystallin expression constructs. Our findings suggest that differential effects of mutations on the transactivation potential of c-MAF could be a molecular correlate of the striking genotype–phenotype correlations seen in cataract forms caused by mutations in the MAF gene.

Novel EXT1 and EXT2 Mutations in Hereditary Multiple Exostoses Families of Indian Origin

BACKGROUND Hereditary multiple exostosis (HME) is an autosomal dominant bone disorder, characterized by short stature and the presence of multiple benign tumors mainly at the ends of long bones. HME is genetically heterogeneous with two known genes on 8q24 (EXT1) and 11p11 (EXT2), and a third minor locus mapped to 19p (EXT3). The majority of EXT1 and EXT2 mutations result in premature protein truncation and loss of function. MATERIALS AND METHODS We analyzed two autosomal dominant HME families of Indian origin. Linkage analysis using fluorescently labeled microsatellite markers at the candidate gene regions was performed. Mutation analysis was carried out by bidirectional sequencing of purified PCR products. RESULTS We found linkage in one family to EXT1 and in the other family to EXT2. Mutation screening in the EXT1 gene revealed a novel frameshift mutation, a single base deletion in exon 1 (c.142delC). This mutation segregated in all affected members and was absent in the unaffected family members and 60 unrelated controls. In the second family, a previously unreported stop mutation, the substitution c.817C>T, was observed in the EXT2 gene in all affected members and in none of the unaffected family members and 90 unrelated controls. CONCLUSIONS Our findings expand the mutation spectrum of EXT1 and EXT2 and highlight the genetic and phenotypic heterogeneity of HME.