Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

AbstractGenome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37°C at pH 6.0 in the presence of 3mM MgCl2. Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents

Association of OCT-Derived Drusen Measurements with AMD-Associated Genotypic SNPs in the Amish Population

Purpose: To investigate the association of optical coherence tomography (OCT)-derived drusen measures in Amish age-related macular degeneration (AMD) patients with known loci for macular degeneration. Methods: Members of the Old Order Amish community in Pennsylvania ages 50 and older were assessed for drusen area, volume and regions of retinal pigment epithelium (RPE) atrophy using a Cirrus High-Definition OCT. Measurements were obtained in the macula region within a central circle (CC) of 3 mm in diameter and a surrounding perifoveal ring (PR) of 3 to 5 mm in diameter using the Cirrus OCT RPE analysis software. Other demographic information, including age, gender and smoking status, were collected. Study subjects were further genotyped to determine their risk for the AMD-associated SNPs in the SYN3, LIPC, ARMS2, C3, CFB, CETP, CFI and CFH genes using TaqMan genotyping assays. The association of genotypes with OCT measures were assessed using linear trend p-values calculated from univariate and multivariate generalized linear models. Results: 432 eyes were included in the analysis. Multivariate analysis (adjusted by age, gender and smoking status) confirmed the known significant association between AMD and macular drusen with the number of CFH risk alleles for the drusen area (the area increased 0.12 mm2 for a risk allele increase, p < 0.01), drusen volume (the volume increased 0.01 mm3 for a risk allele increase, p ≤ 0.05) and the area of RPE atrophy (the area increased 0.43 mm2 for a risk allele increase, p = 0.003). SYN3 risk allele G is significantly associated with larger area PR (the area increased 0.09 mm2 for a risk allele increase, p = 0.03) and larger drusen volume in the central circle (the volume increased 0.01 mm3 for a risk allele increase, p = 0.04). Conclusion: Among the genotyped SNPs tested, the CFH risk genotype appears to play a major role in determining the drusen phenotype in the Amish AMD population

Evaluating TNF-α and Interleukin-2 (IL-2) Levels in African American Primary Open-Angle Glaucoma Patients

Purpose: To establish if SNPs in TNF-α and IL-2 genes are associated with Primary Open-Angle Glaucoma (POAG) in African Americans (AA). We also determined whether plasma TNF-α and IL-2 levels could serve as biomarkers for POAG in African Americans using sandwich enzyme-linked immunosorbent assay. Methods: A single SNP association analysis was performed to investigate the association between potential gene variants in TNF-α and IL-2 genes and POAG in the AA population. Plasma samples from 190 African Americans (72 from normal subjects and 118 POAG cases) were obtained for TNF- α studies and 367 samples (135 from normal subjects and 232 from POAG cases) were obtained for IL-2 studies. TNF-α levels and IL-2 levels were measured by sandwich enzyme-linked immunosorbent assays (ELISA) and analyzed to see if they reached significance in cases with POAG and endophenotypes when compared to normal subjects. Results: The SNP, rs1800630, in TNF-α gene was found to be marginally associated with POAG. SNPs in IL-2 gene were not associated with POAG in the case-control analysis. No significant difference was found between TNF-α levels and IL-2 levels in normal and POAG case subjects in our study. IL-2 levels were inversely correlated with high IOP in POAG cases. Conclusions: Although we found a marginal SNP association of TNF-α, assessing the expression levels of TNF-α and IL-2 may serve as promising biomarkers for African American POAG. Further investigation is needed to determine if POAG can be subdivided into more specified cohorts of the disease, which may affect plasma cytokine levels differently

Long-Term Effects of Gene Therapy in a Novel Mouse Model of Human MFRP-Associated Retinopathy

Patients harboring homozygous c.498_499insC mutations in MFRP demonstrate hyperopia, microphthalmia, retinitis pigmentosa, retinal pigment epithelial atrophy, variable degrees of foveal edema, and optic disc drusen. The disease phenotype is variable, however, with some patients maintaining good central vision and cone function till late in the disease. A knock-in mouse model with the c.498_499insC mutation in Mfrp (Mfrp KI/KI) was developed to understand the effects of these mutations in the retina. The model shares many of the features of human clinical disease, including reduced axial length, hyperopia, retinal degeneration, retinal pigment epithelial atrophy, and decreased electrophysiological responses. In addition, the eyes of these mice had a significantly greater refractive error (p < 0.01) when compared to age-matched wild-type control animals. Administration of recombinant adeno-associated virus-mediated Mfrp gene therapy significantly prevented thinning from retinal neurodegeneration (p < 0.005) and preserved retinal electrophysiology (p < 0.001) when treated eyes were compared to contralateral sham-treated control eyes. The Mfrp KI/KI mice will serve as a useful tool to model human disease and point to a potential gene therapeutic approach for patients with preserved vision and electrophysiological responses in MFRP-related retinopathy

A missense mutation in ASRGL1 is involved in causing autosomal recessive retinal degeneration.

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss