Imputation of Orofacial Clefting Data Identifies Novel Risk Loci and Sheds Light on the Genetic Background of Cleft Lip ± Cleft Palate and Cleft Palate Only.

Abstract Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signalswithin this high-density datasetare enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinementapproach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate, and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.Clefts of the lip and palate are common birth defects, and require long-term multidisciplinary management. Their etiology involves genetic factors and environmental influences and/or a combination of both, however, these interactions are poorly defined. Moreover, although clefts of the lip may or may not involve the palate, the determinants predisposing to specific subphenotypes are largely unknown. Here we demonstrate that variations in the non-coding region near the GREM1 gene show a highly significant association with a particular phenotype in which cleft lip and cleft palate co-occ

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate, and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions

INTERLEUKIN-1β LEVELS IN THE HUMAN GINGIVAL SULCUS: RATES AND FACTORS AFFECTING ITS LEVELS IN HEALTHY SUBJECTS

Background and objective:  Gingival crevicular fluid (GCF) affords a exceptional window for investigation of periodontal condition as the levels of inflammatory mediators, which consequences owing to the increased local destruction of connective tissue structural elements. This study aimed to explore the interleukin 1β (IL-1β)) levels in the human gingival sulcus in healthy normal people; and the effect of host factors as age , gender, type of tooth used in pro-inflammatory biomarkers. Methods: Eighty seven patients, 54 (62.1%) female and 33 (37.9%) male (aged 12–34 years; mean 19.58±4.4 years), participated in this study. Each subject underwent a session on professional oral hygiene and received oral hygiene instructions. Gingival crevicular fluid (GCF) sampling was conducted (baseline). GCF was collected from the Central incisor, the Lateral incisor, the Canine, the First premolar and the second premolar in this study.  Results: In total, the mean±SD of central incisor IL-1β was 32.16±4.83 pg/ml, with a mode equal to 28.01 pg/mL, the median was 32.71 pg/mL, and ranged from 20.98 to 41.25 pg/ml with the 75% interquartile range (IQR) equal to 35.94 pg/ml. For males the mean±SD of central incisor IL-1β was 31.6±5.51 pg/ml VS 32.5±4.4 pg/ml of females. For the lateral incisor, canine, first premolar, second premolar: Conclusion: This study provides the upper limit of normal values ​​for interleukin 1β (IL-1β) levels for subjects aged 12–34 years in the GCF. These upper limits of normal values ​​will guide dentists in Yemen when they consider the diagnosis of periodontal disease, as well as its role during orthodontic tooth movement where they play important role in osteocyte activities (e.g, osteoclasts and osteoblasts), and will provide useful baseline data for future studies of interventions against periodontal disease, and teeth movement by orthodontics appliances, in Yemen.                           Peer Review History: Received: 1 September 2022; Revised: 11 October; Accepted: 6 November, Available online: 15 November 2022 Academic Editor: Dr. Emmanuel O. Olorunsola, Department of Pharmaceutics &amp; Pharmaceutical Technology, University of Uyo, Nigeria, [email protected] Received file:                             Reviewer's Comments: Average Peer review marks at initial stage: 5.0/10 Average Peer review marks at publication stage: 7.0/10 Reviewers: Dr. Rima Benatoui,Laboratory of Applied Neuroendocrinology, Department of Biology, Faculty of Science, Badji Mokhtar University Annaba, Algeria.  [email protected] Dr. Gulam Mohammed Husain,, National Research Institute of Unani Medicine for Skin Disorders, Hyderabad, India, [email protected] Similar Articles:   THE EFFECT OF NANOSILVER AND CHLORHEXIDINE MOUTHWASH ON ANAEROBIC PERIODONTAL PATHOGENS COUNT

Forest plot for association of rs1258763 and nsCL/P subphenotypes.

<p>Subphenotype analyses of cleft lip and palate (nsCLP, black) and cleft lip only (nsCLO, grey) were conducted in the Ludwig 2012 meta-analysis data, the replication I and II cohorts, and in the combined analysis of the present study. Boxes represent point estimates of the relative risk for each of the four studies, with box sizes scaled according to the number of affected individuals. Lines indicate the extent of the confidence interval. These data illustrate the consistent association between rs1258763 and nsCLP across the various studies, and the presence of a narrow effect size range in the combined cohort. Please note that confidence intervals for nsCLO are larger due to the lower number of nsCLO patients. Informed by the specific expression of <i>Grem1</i> in lip and soft palate development in the mouse embryo (see below) we also analyzed the effect size of the particular soft palate subphenotype (nsCLP_soft). The arrow indicates the point estimate for rs1258763 and nsCLP_soft.</p

<i>Grem1</i> expression during mouse craniofacial development.

<p>(<b>A-E</b>) Expression of <i>Grem1</i> is visualized by X-Gal staining of heterozygous <i>Grem1</i>^<i>LacZ</i> whole mount embryos. (<b>A</b>) At E11.5, <i>Grem1</i> is expressed in the dorsal part of the lateral nasal prominence (lnp). Stippled lines demarcate the nasal pits. (<b>B</b>) At E12.5, <i>Grem1</i>-positive domains are also detectable in the merging zones (arrowheads) of medial nasal prominences (mnp) and maxillary prominences (mxp). (<b>C-G</b>) Secondary palate development. (<b>C</b>) At E13.5, <i>Grem1</i>-positive domains are observed in the forming soft palate (sp). (<b>D</b>) At E14.5, the hard palate (hp) has formed while the <i>Grem1</i>-expressing shelves of the soft palate are not yet fused. (<b>E</b>) At E15.5, the soft palate has fused and <i>Grem1</i> expression extends posterior to the pharynx (ph). Note the sharp anterior boundary of <i>Grem1</i> expression in the soft palate (arrowheads). (<b>F, G</b>) Sections of whole mount stained embryos. (<b>F</b>) Cross section at the level indicated in (D) showing that <i>Grem1</i> expression is restricted to the mesenchyme. (<b>G</b>) Cross section at the level indicated in (E) showing <i>Grem1</i> expression in the soft palate, which separates the nasopharynx (np) from the oral cavity (oc). Additional abbreviations: a, anterior; l, lateral; m, medial; mdp, mandibular prominence; p, posterior. Scale bars: 500μm.</p

Sample overview.

<p>Sample overview.</p

Regional association plot for the 15q13 region.

<p><i>P</i>-values for SNPs at 15q13 that were analyzed as part of the Ludwig 2012 meta-analysis (<i>P</i>_{nsCL/P_meta}) are plotted against their chromosomal position (hg19). Full data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005914#pgen.1005914.s004" target="_blank">S1 Dataset</a>. For each variant, color code denotes linkage disequilibrium to rs1258763, based on 1000genomes. After combination with data from replication I and II, the top variant rs1258763 (purple diamond; indicated by dotted line) reaches genome-wide significance. Plot was generated using LocusZoom [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005914#pgen.1005914.ref028" target="_blank">28</a>].</p

Regional association plots for the 15q13 region in different types of nsCL/P.

<p>In the imputed data of the Central European cohort, the 15q13 region was analyzed in the overall phenotype nsCL/P (<b>A</b>) and both subphenotypes, i.e. nsCLP (<b>B</b>) and nsCLO (<b>C</b>). For each SNP, the <i>P</i>-value is plotted against its chromosomal position (hg19). In nsCL/P and nsCLP, a highly associated cluster of SNPs in strong linkage disequilibrium is present, located between <i>GREM1</i> and <i>FMN1</i>. The lowest <i>P</i>-value was observed for rs2600520 (purple diamond). In each panel, the top genotyped variant rs1258763 is marked by an open circle. For all other variants, color code denotes linkage disequilibrium to rs2600520, based on 1000genomes. Regional association plots were generated using LocusZoom [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005914#pgen.1005914.ref028" target="_blank">28</a>].</p