A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6, GRHL3, and TBX22.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.

Genetic markers for non-syndromic orofacial clefts in populations of European ancestry: a meta-analysis.

To date, the involvement of various genetic markers in the aetiopathogenesis of non-syndromic orofacial cleft (nsOFC) has been extensively studied. In the present study, we focused on studies performed on populations of European ancestry to systematically review the available literature to define relevant genetic risk factors for nsOFC. Eligible studies were obtained by searching Ovid Medline and Ovid Embase. We gathered the genetic markers from population-based case-control studies on nsOFC, and conducted meta-analysis on the repeatedly reported markers. Whenever possible, we performed stratified analysis based on different nsOFC phenotypes, using allelic, dominant, recessive and overdominant genetic models. Effect sizes were expressed as pooled odds ratios (ORs) with 95% confidence intervals (CIs), and p ≤ 0.05 were considered statistically significant. A total of 84 studies were eligible for this systematic review, with > 700 markers included. Of these, 43 studies were included in the meta-analysis. We analysed 47 genetic variants in 30 genes/loci, which resulted in 226 forest plots. There were statistically significant associations between at least one of the nsOFC phenotypes and 19 genetic variants in 13 genes/loci. These data suggest that IRF6, GRHL3, 8q24, VAX1, TGFA, FOXE1, ABCA4, NOG, GREM1, AXIN2, DVL2, WNT3A and WNT5A have high potential as biomarkers of nsOFC in populations of European descent. Although other meta-analyses that included European samples have been performed on a limited number of genetic variants, this study represents the first meta-analysis of all genetic markers that have been studied in connection with nsOFC in populations of European ancestry.

Synthesis and Evaluation of Antioxidant Properties of 2-Substituted Quinazolin-4(3H)-ones.

Quinazolinones represent an important scaffold in medicinal chemistry with diverse biological activities. Here, two series of 2-substituted quinazolin-4(3H)-ones were synthesized and evaluated for their antioxidant properties using three different methods, namely DPPH, ABTS and TEACCUPRAC, to obtain key information about the structure-antioxidant activity relationships of a diverse set of substituents at position 2 of the main quinazolinone scaffold. Regarding the antioxidant activity, ABTS and TEACCUPRAC assays were more sensitive and gave more reliable results than the DPPH assay. To obtain antioxidant activity of 2-phenylquinazolin-4(3H)-one, the presence of at least one hydroxyl group in addition to the methoxy substituent or the second hydroxyl on the phenyl ring in the ortho or para positions is required. An additional ethylene linker between quinazolinone ring and phenolic substituent, present in the second series (compounds 25a and 25b), leads to increased antioxidant activity. Furthermore, in addition to antioxidant activity, the derivatives with two hydroxyl groups in the ortho position on the phenyl ring exhibited metal-chelating properties. Our study represents a successful use of three different antioxidant activity evaluation methods to define 2-(2,3-dihydroxyphenyl)quinazolin-4(3H)-one 21e as a potent antioxidant with promising metal-chelating properties.

Tobacco smoking induces changes in true DNA methylation, hydroxymethylation and gene expression in bronchoalveolar lavage cells.

BACKGROUND: While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). METHODS: We profiled changes in DNA methylation (5mC) and its oxidised form hydroxymethylation (5hmC) using conventional bisulphite (BS) treatment and oxidative bisulphite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. FINDINGS: We identified 1667 total 5mC + 5hmC, 1756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and non-smokers (FDR-adjusted P <.05, absolute Δß >0.15). Both 5mC DMPs and to a lesser extent 5mC + 5hmC were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated 5mC + 5hmC CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, 5mC + 5hmC (1639/1667), 5mC (1738/1756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signalling and inflammatory response of immune cells. INTERPRETATION: Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. FUND: The study was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council (ALF), the King Gustav's and Queen Victoria's Freemasons' Foundation, Knut and Alice Wallenberg Foundation, Neuro Sweden, and the Swedish MS foundation.