Evaluation of nine candidate genes in patients with normal tension glaucoma: a case control study

<p>Abstract</p> <p>Background</p> <p>Normal tension glaucoma is a major subtype of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. Monogenic forms following classical inheritance patterns are rare in this glaucoma subtype. Instead, multigenic inheritance is proposed for the majority of cases. The present study tested common sequence variants in candidate genes for association with normal tension glaucoma in the German population.</p> <p>Methods</p> <p>Ninety-eight SNPs were selected to tag the common genetic variation in nine genes, namely OPTN (optineurin), RDX (radixin), SNX16 (sorting nexin 16), OPA1 (optic atrophy 1), MFN1 (mitofusin 1), MFN2 (mitofusin 2), PARL (presenilin associated, rhomboid-like), SOD2 (superoxide dismutase 2, mitochondrial) and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1). These SNPs were genotyped in 285 cases and 282 fully evaluated matched controls. Statistical analyses comprised single polymorphism association as well as haplogroup based association testing.</p> <p>Results</p> <p>Results suggested that genetic variation in five of the candidate genes (RDX, SNX16, OPA1, SOD2 and CYP1B1) is unlikely to confer major risk to develop normal tension glaucoma in the German population. In contrast, we observed a trend towards association of single SNPs in OPTN, MFN1, MFN2 and PARL. The SNPs of OPTN, MFN2 and PARL were further analysed by multimarker haplotype-based association testing. We identified a risk haplotype being more frequent in patients and a vice versa situation for the complementary protective haplotype in each of the three genes.</p> <p>Conclusion</p> <p>Common variants of OPTN, PARL, MFN1 and MFN2 should be analysed in other cohorts to confirm their involvement in normal tension glaucoma.</p

Novel C8orf37 mutations cause retinitis pigmentosa in consanguineous families of Pakistani origin

Purpose: To investigate the molecular basis of retinitis pigmentosa in two consanguineous families of Pakistani origin with multiple affected members. Methods: Homozygosity mapping and Sanger sequencing of candidate genes were performed in one family while the other was analyzed with whole exome next-generation sequencing. A minigene splicing assay was used to confirm the splicing defects. Results: In family MA48, a novel homozygous nucleotide substitution in C8orf37, c.244–2A>C, that disrupted the consensus splice acceptor site of exon 3 was found. The minigene splicing assay revealed that this mutation activated a cryptic splice site within exon 3, causing a 22 bp deletion in the transcript that is predicted to lead to a frameshift followed by premature protein truncation. In family MA13, a novel homozygous null mutation in C8orf37, c.555G>A, p.W185*, was identified. Both mutations segregated with the disease phenotype as expected in a recessive manner and were absent in 8,244 unrelated individuals of South Asian origin. Conclusions: In this report, we describe C8orf37 mutations that cause retinal dystrophy in two families of Pakistani origin, contributing further data on the phenotype and the spectrum of mutations in this form of retinitis pigmentosa

Exploring functional candidate genes for genetic association in German patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma

purpose. Pseudoexfoliation (PEX) syndrome is a generalized elastic microfibrillopathy characterized by fibrillar deposits in intra- and extraocular tissues. Genetic and nongenetic factors are known to be involved in its etiopathogenesis. This study was focused on six functional candidate genes involved in PEX material deposition and the analysis of their potential association with PEX syndrome and PEX glaucoma (PEXG). methods. Fifty single-nucleotide polymorphisms (SNPs) capturing >95% of overall genetic variance observed in Europeans at loci for FBN1, LTBP2, MFAP2, TGM2, TGF-b1, and CLU were genotyped in 333 unrelated PEX-affected and 342 healthy individuals of German origin, and a genetic association study was performed. To replicate the findings, two SNPs of the CLU gene were genotyped in a further 328 unrelated German patients with PEX as well as in 209 Italian patients with PEX and 190 Italian control subjects. results. Association with PEX was observed only for the SNP rs2279590 in intron 8 of the CLU gene coding for clusterin (corrected P = 0.0347, OR = 1.34) in our first German cohort. Likewise, a frequent haplotype encompassing the associated risk allele showed nominally significant association. None of remaining SNPs or SNP haplotypes were associated with PEX. The association found was confirmed in a second German cohort (P = 0.0244) but not in the Italian cohort (P = 0.7173). In addition, the association with CLU SNP rs2279590 was more significant in German patients with PEX syndrome than in those with PEXG. conclusions. Genetic variants in the gene encoding clusterin may represent a risk factor for PEX in German patients but not in Italian patients. Variants in FBN1, LTBP2, MFAP2, TGF-b1, and TGM2 do not play a major role in the etiology of PEX syndrome, at least in German patients

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB

Optineurin Is Required for CYLD-Dependent Inhibition of TNFα-Induced NF-κB Activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP

Genome-wide transcriptomic analysis of the response to nitrogen limitation in Streptomyces coelicolor A3(2)

<p>Abstract</p> <p>Background</p> <p>The present study represents a genome-wide transcriptomic analysis of the response of the model streptomycete <it>Streptomyces coelicolor </it>A3(2) M145 to fermentor culture in Modified Evans Media limited, respectively, for nitrogen, phosphate and carbon undertaken as part of the ActinoGEN consortium to provide a publicly available reference microarray dataset.</p> <p>Findings</p> <p>A microarray dataset using samples from two replicate cultures for each nutrient limitation was generated. In this report our analysis has focused on the genes which are significantly differentially expressed, as determined by Rank Products Analysis, between samples from matched time points correlated by growth phase for the three pairs of differently limited culture datasets. With a few exceptions, genes are only significantly differentially expressed between the N6/N7 time points and their corresponding time points in the C and P-limited cultures, with the vast majority of the differentially expressed genes being more highly expressed in the N-limited cultures. Our analysis of these genes indicated expression of several members of the GlnR regulon are induced upon nitrogen limitation, as assayed for by [NH₄⁺] measurements, and we are able to identify several additional genes not present in the GlnR regulon whose expression is induced in response to nitrogen limitation. We also note SCO3327 which encodes a small protein (32 amino acid residues) unusually rich in the basic amino acids lysine (31.25%) and arginine (25%) is significantly differentially expressed in the nitrogen limited cultures. Additionally, we investigate the expression of known members of the GlnR regulon and the relationship between gene organization and expression for the SCO2486-SCO2487 and SCO5583-SCO5585 operons.</p> <p>Conclusions</p> <p>We provide a list of genes whose expression is differentially expressed in low nitrogen culture conditions, including a putative nitrogen storage protein encoded by SCO3327. Our list includes several genes whose expression patterns are similar to up-regulated members of the GlnR regulon and are induced in response to nitrogen limitation. These genes represent likely targets for future studies into the nitrogen starvation response in <it>Streptomyces coelicolor</it>.</p

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders

Genetic Heritability of Pigmentary Glaucoma and Associations With Other Eye Phenotypes

Importance: Mechanisms behind pigmentary glaucoma, a form of early-onset glaucoma that may potentially lead to severe visual impairment or blindness, are poorly understood. Objective: To calculate the single-nucleotide polymorphism (SNP) heritability of pigmentary glaucoma and identify genetic associations with the disease. Design, Setting and Participants: This genome-wide association study included affected individuals from Germany and control participants from the United Kingdom. Genome-wide information was obtained for patients with pigmentary glaucoma and control participants free of glaucoma by using the Illumina Human Omni Express Exome 8v1-2 chip and genomic imputation. The SNP heritability of pigmentary glaucoma was estimated through a restricted maximum likelihood analysis. Associations between the genetic variants and pigmentary glaucoma obtained from age, sex, and principal component-adjusted logistic regression models were compared with those of SNPs previously associated with other eye phenotypes using Pearson product-moment correlations. Data were collected from November 2008 to January 2018, and analysis was completed between April 2018 and August 2019. Main Outcomes and Measures: An estimate of SNP-explained heritability for pigmentary glaucoma; correlations of effect sizes between pigmentary glaucoma and iris pigmentation and myopia; and correlations of effect sizes between pigmentary glaucoma and other eye phenotypes. Results: A total of 227 affected individuals (mean [SD] age, 58.7 [13.3] years) and 291 control participants (mean [SD] age, 80.2 [4.9] years) were included; all were of European ancestry. The SNP heritability of pigmentary glaucoma was 0.45 (SE, 0.22; P = 6.15 × 10-10). Twelve SNPs previously reported with genome-wide significant associations with eye pigmentation were associated with pigmentary glaucoma's SNP heritability (4.9% SNP heritability; 0.022; P = 6.0 × 10-4). Pigmentary glaucoma SNP effect sizes were correlated moderately for myopia (r, 0.42 [95% CI, 0.14-0.63]; P = 4.3 × 10-3) and more strongly with those for iris pigmentation (r = -0.69 [95% CI, -0.91 to -0.20]; P = .01), although this was nonsignificant per a strict adjusted significance threshold (P < .01). Conclusions and Relevance: These findings support the conclusion that pigmentary glaucoma may have a genetic basis and be highly heritable. Variants associated with lighter eye color and myopia appear to be associated with increased risk of pigmentary glaucoma, but no shared genetic basis with primary open-angle glaucoma (or its quantitative endophenotype of cup-disc ratio) was observed