A comprehensive molecular genetic analysis of keratoconus patients from assam, a northeastern state of India.

INTRODUCTION: Keratoconus (KC, OMIM: 148300) is a progressive corneal ectatic disorder characterized by thinning and protrusion of cornea resulting in visual decrement. MATERIALS AND METHODS: In the current study, we recruited a total of 50 KC patients and 100 case-controls domiciles of Assam, based on preset inclusion and exclusion criteria. All the important and relevant signs and symptoms were recorded. Amsler-Krumeich's (AK) classification was followed to grade KC corneas. We screened for the novel as well as reported sequence variations in five candidate genes namely Lysyl oxidase (LOX), Visual system homeobox 1 (VSX1), MicroRNA 184 (MIR184), Superoxide dismutase 1 (SOD1), and exons 4 and 12 of Transforming growth factor beta-induced (TGFß-I). RESULTS: We report a novel double variant p.(Pro32Arg) and p.(Gln67Glu) in the LOX gene in a sporadic male patient with Grade I (OD) and Grade II (OS) of KC. A recurrent variant p.(His244Arg) in the VSX1 gene was also observed in a sporadic female patient with Grade I of KC in both eyes. These variants were absent in 100 unrelated ethnically matched case controls. DISCUSSION: Ours is the first study on molecular genetic analysis of Keratoconus patients from Assam. The novel variants p.(Pro32Arg) and p.(Gln67Glu) observed further expand the mutational spectrum of the LOX gene associated with KC. We are also the first group to report the recurrent p.(His244Arg) variant in the VSX1 gene from India. The observed variant p.(His244Arg) in the VSX1 gene could be the result of a founder effect and may be investigated further.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Mutational screening of Indian families with hereditary congenital cataract.

PURPOSE: To screen for pathogenic mutations in ten candidate genes in Indian families diagnosed with autosomal recessive and autosomal dominant cataracts. METHODS: Families with two or more affected individuals with bilateral familial congenital/developmental cataract were ophthalmically evaluated, and blood samples were obtained. Genomic DNA extracted from the blood leukocytes was screened with PCR amplification of the exons and the flanking intronic regions of various genes selected for analysis. The amplified products were subjected to single strand conformation polymorphism (SSCP) analysis. The variants in SSCP analysis were subjected to bidirectional sequencing by automated methods. RESULTS: We identified four novel sequence changes that cosegregated with the disease phenotype in each family and were absent in at least 50 ethnically matched unrelated normal controls. These changes include a homozygous missense change of c.649G>A (Val196Met) in GJA8/connexin 50 (Cx50) in a family with autosomal recessive cataract, two heterozygous missense changes, c.658C>T (Pro199Ser) in GJA8/Cx50 and c.589C>T (Pro197Ser) in GJA3/connexin 46 (Cx46) in two separate families with autosomal dominant cataract, and a silent change ( c.84G>A/p.Val28Val, predicted to result in the creation of a new potential branch point) in GJA8 one family with an autosomal dominant inheritance of cataract. Of the four novel mutations identified, three mutations, Val196Met (GJA8), Pro199Ser (GJA8), and Pro197Ser (GJA3), are predicted to be in the second extracellular domain of the respective connexin proteins. CONCLUSIONS: Our report extends the mutation spectrum of connexin genes GJA8 and GJA3 and confirms that connexin genes are among the most frequently mutated genes in hereditary cataracts. Our results suggest that connexin gene (GJA8 and GJA3) mutations occur in approximately 10% (4/40 families) of families with congenital hereditary cataracts in a population from southern India.