Tayyab Husnain

Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families

Objective To identify disease-causing mutations in two consanguineous Pakistani families with fundus albipunctatus. Methods Affected individuals in both families underwent a thorough clinical examination including funduscopy and electroretinography. Blood samples were collected from all participating members and genomic DNA was extracted. Exclusion analysis was completed with microsatellite short tandem repeat markers that span all reported loci for fundus albipunctatus. Two-point logarithm of odds (LOD) scores were calculated, and coding exons and exon–intron boundaries of RLBP1 were sequenced bi-directionally. Results The ophthalmic examination of affected patients in both families was consistent with fundus albipunctatus. The alleles of markers on chromosome 15q flanking RLBP1 segregated with the disease phenotype in both families and linkage was further confirmed by two-point LOD scores. Bi-directional sequencing of RLBP1 identified a nonsense mutation (R156X) and a missense mutation (G116R) that segregated with the disease phenotype in their respective families. Conclusions These results strongly suggest that mutations in RLBP1 are responsible for fundus albipunctatus in the affected individuals of these consanguineous Pakistani families.

Association of Pathogenic Mutations in TULP1 With Retinitis Pigmentosa in Consanguineous Pakistani Families

OBJECTIVE To identify pathogenic mutations responsible for autosomal recessive retinitis pigmentosa in 5 consanguineous Pakistani families. METHODS Affected individuals in the families underwent a detailed ophthalmological examination that consisted of fundus photography and electroretinography. Blood samples were collected from all participating family members, and genomic DNA was extracted. A genome-wide linkage scan was performed, followed by exclusion analyses among our cohort of nuclear consanguineous families with microsatellite markers spanning the TULP1 locus on chromosome 6p. Two-point logarithm of odds scores were calculated, and all coding exons of TULP1 were sequenced bidirectionally. RESULTS The results of ophthalmological examinations among affected individuals in these 5 families were suggestive of retinitis pigmentosa. The genome-wide linkage scan localized the disease interval to chromosome 6p, harboring TULP1 in 1 of 5 families, and sequential analyses identified a single base pair substitution in TULP1 that results in threonine to alanine substitution (p.T380A). Subsequently, we investigated our entire cohort of families with autosomal recessive retinitis pigmentosa and identified 4 additional families with linkage to chromosome 6p, all of them harboring a single base pair substitution in TULP1 that results in lysine to arginine substitution (p.K489R). Results of single-nucleotide polymorphism haplotype analyses were suggestive of a common founder in these 4 families. CONCLUSION Pathogenic mutations in TULP1 are responsible for the autosomal recessive retinitis pigmentosa phenotype in these consanguineous Pakistani families, with a single ancestral mutation in TULP1 causing the disease phenotype in 4 of 5 families. CLINICAL RELEVANCE Clinical and molecular characterization of pathogenic mutations in TULP1 will increase our understanding of retinitis pigmentosa at a molecular level.

Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

Purpose To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family. Methods All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model. Results Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points. Conclusion A novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.

Genomic Approaches and Abiotic Stress Tolerance in Plants

Environmental threats that comprise factors related to abiotic stresses are among the major challenges to crop plants, which limit the yield and productivity. Plants respond to this threat for their endurance at the cellular and molecular level. Understanding plant responses to these stresses is imperative and thought provoking in order to combat the challenges in agricultural research. Consequently, a complex interaction between signaling molecules and pathways has been triggered. Success in breeding for better adapted varieties to abiotic stresses depends upon the combined efforts of various research domains including plant and cell physiology, molecular biology, and genetics. Genomics approaches for considering plant responses to stress have provided potent tools for in-depth dissection of tolerance mechanisms, primarily with models and latterly with other plant species. As an alternative to conventional breeding, a more competent technology is marker-assisted breeding, which has identified a number of desirable genomic regions (quantitative trait loci, QTL) of different crops under stress conditions. A large number of datasets are available through expressed sequence tags (ESTs) for species with larger but unknown genome sequences. Microarray technology is expanding rapidly and gene expression has been measured for a number of plant species, which has advanced our knowledge of the complex interaction between signaling molecules and pathways. Proteomics and metabolomics studies provide a broad representation of data related to abiotic stress outcomes and it will be supportive to improve crop breeding in the near future. Identification and isolation of abiotic stress-tolerant genes and transformation technology has made real progress in understanding how plants cope with these stresses as well as the different constituents exploited in the signaling and response pathways.

Deletion at the GCNT2 Locus Causes Autosomal Recessive Congenital Cataracts

Purpose The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree. Methods All participating individuals underwent a detailed ophthalmic examination. Each patient’s medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation. Results Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion. Conclusion Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family.

Engineered disease resistance in cotton using RNA-interference to knock down Cotton leaf curl Kokhran virus-burewala and cotton leaf curl multan betasatellite expression

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the βC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vβ, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vβ transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vβ-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vβ-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

How RNA Interference Combat Viruses in Plants

RNA mediated silencing technology has now become the tool of choice for induction of virus resistance in plants. A significant feature of this technology is the presence of doublestranded RNA (dsRNA), which is not only the product of RNA silencing but also the potent triggers of RNA interference (RNAi). Upon RNAi induction, these dsRNAs are diced into short RNA fragments termed as small interfering RNAs (siRNAs), which are hallmarks of RNAi. Considerable resistance in transgenic plants against viruses can be created by exploiting the phenomenon of RNAi. In the current chapter, generation of potato virus Y (PVY) resistant potato and sugarcane mosaic virus (SCMV) resistant sugarcane by CEMB has been quoted as an example.

Ectopia Lentis in a Consanguineous Pakistani Family and a Novel Locus on Chromosome 8q

OBJECTIVE To investigate the genetic basis and molecular characteristics of the isolated form of ectopia lentis. METHODS We ascertained a consanguineous Pakistani family with multiple individuals with ectopia lentis. All affected as well as unaffected members with isolated ectopia lentis underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was completed with 382 polymorphic microsatellite markers, and logarithm of odds (LOD) scores were calculated. RESULTS Maximum 2-point LOD scores of 5.68 and 2.88 at theta = 0 were obtained for markers D8S285 and D8S260, respectively, during the genome-wide scan. Additional microsatellite markers refined the disease locus to a 16.96-cM (14.07-Mb) interval flanked by D8S1737 proximally and D8S1117 distally. CONCLUSIONS We report on a new locus for nonsyndromic autosomal recessive ectopia lentis on chromosome 8q11.23-q13.2 in a consanguineous Pakistani family. Clinical Relevance Identification of genetic loci and genes involved in ectopia lentis will enhance our understanding of the disease at a molecular level, leading to better genetic counseling and family screening and possible future development of better treatment.