Investigation of methylation pattern in candidate genes or regions to non-syndromic cleft lip with or without palate (CL/P NS)

As fissuras labiopalatinas não sindrômicas (FL/P NS) são malformações congênitas que afetam o lábio e/ou o palato. O padrão de herança sugerido para as FL/P NS é o de herança multifatorial. Um dos modelos genéticos mais testados é aquele em que os pacientes apresentam um acúmulo de variantes de baixo risco e frequentes na população. Entretanto, apenas essas variantes não explicam a alta herdabilidade da doença. Foi sugerido, então, que variantes raras de efeito médio e grande estejam atuando na predisposição à FL/P NS, entretanto, a identificação dessas variantes representam um grande desafio. Uma vez que apenas a contribuição genética não explica o fenótipo, tem-se sugerido que a alteração do padrão epigenético possa estar influenciando no quadro clínico dos pacientes. Alguns trabalhos observaram que há alteração de metilação no promotor de genes candidatos em pacientes com FL/P NS, dentre eles, o gene CDH1. Dessa forma, o objetivo do presente trabalho foi verificar se os genes selecionados como possíveis causas para FL/P NS têm maior probabilidade de terem alteração no padrão de metilação nos afetados de famílias multiplex do que em não-afetados, o que poderia adicionar evidências de que os genes estejam contribuindo para o fenótipo. Foram estudados o padrão de metilação da região promotora (região global e por sítio) de 16 novos genes candidatos e de 4 genes já associados às FL/Ps (ARHGAP29, SHH, TP63 e ROR2) em uma casuística brasileira de 28 afetados e 71 controles. Foi observada hipermetilação no gene TP63 (p-valor=0.025 na análise por região) e hipometilação em ROR2 (p-valores=0.045, 0.008, 0.043 e 0,0015 em 4 sítios dentro da região CpG). Ambos os genes estão envolvidos na sinalização da via Wnt que, quando desregulada, pode ser um fator de risco para FL/P. Também foi encontrada no presente trabalho uma nova região de risco candidata para FL/P NS, baseado em estudo de ligação, genômica e análise epigenética. DCHS2, o melhor candidato dessa região, tem participação na via PCP, o que torna essa região ainda mais promissora. Tais resultados sugerem que a alteração do padrão epigenético nesses genes pode estar influenciando no quadro clínico nas FL/Ps; porém esse estudo não contribuiu para uma melhor classificação da maioria dos novos genes candidatos, o que pode ter sido por limitações do tamanho amostral e metodologiaNon-syndromic cleft lip with or without cleft palate (NSCL/P) are congenital malformations that affect the lip and/or palate. Multifactorial is the most likely inheritance pattern for NSCL/P. Accumulation of low risk and frequent variants has been the most tested model in genomic studies. However, just the presence of these variants does not explain the high heritability of the disease. Rare variants of medium and large effects have been suggested as another possible model to be acting on NSCL/P predisposition, however, identification of these variants represents a great challenge. Changes in the epigenetic pattern may be influencing the clinical condition of patients in addition to genetic alterations. In this regard, methylation alterations in the promoter region of candidate genes in patients with NSCL/P, including CDH1, have been shown by some authors. Thus, the aim of the present study was to verify if novel candidate genes for NSCL/P selected from genomic studies are more likely to have changes in the methylation pattern in affected individuals from multiplex families than in non-affected ones, which could add more evidences that these genes are good FL/P candidates. We studied the methylation pattern of the promoter region (global region and by site) of 16 novel candidate genes and 4 genes that are already associated with CL/P (ARHGAP29, SHH, TP63 e ROR2) on a Brazilian cohort of 28 affected individuals and 71 controls. It was observed hypermethylation on TP63 gene (p-value=0.025 on analysis by region) and hypomethylation on ROR2 (p-values=0.045, 0.008, 0.043 and 0.0015 on 4 sites within the CpG region). Both genes are involved in Wnt signalization pathway, which when disrupted, can be a risk factor for CL/P. A new candidate risk region for NSCL/P, based on linkage, genomic and epigenetic analysis, was also found in this study. DCHS2, the best candidate gene within this region, participates in PCP pathway, which makes this candidate region even more promising. In conclusion, such results suggest that the alteration of the epigenetic pattern in these genes may be influencing the clinical condition observed in NSCL/Ps; however, our study did not contribute in adding more evidence about the pathogenicity status of the prioritized novel candidate variants and genes, which can be due to our limited sample size and methodolog

mir152 hypomethylation as a mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21–26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP

IRF6 is a Risk Factor for Nonsyndromic Cleft Lip in the Brazilian Population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5' and 3' of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P = 0.009; odds ratio (OR) for AA genotype = 1.83 [95% Confidence interval (CI), 0.64-5.31]; OR for AG genotype = 1.72 [95% CI, 1.03-2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H-2 = 0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis. (C) 2012 Wiley Periodicals, Inc.CNPqCNPqFAPESPFAPESPBrazilian Ministry of HealthBrazilian Ministry of Healt

Region 8q24 Is a Susceptibility Locus for Nonsyndromic Oral Clefting in Brazil

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate is a relatively common craniofacial defect with multifactorial inheritance. The association of the rs987525 single nucleotide variant, located in a gene desert at 8q24.21 region, has been consistently replicated in European populations. We performed a structured association approach combined with transcriptional analysis of the MYC gene to dissect the role of rs987525 in oral clefting susceptibility in the ethnically admixed Brazilian population. METHODS: We performed the association study conditioned on the individual ancestry proportions in a sample of 563 patients and 336 controls, and in an independent sample of 221 patients and 261 controls. The correlation between rs987525 genotypes and MYC transcriptional levels in orbicularis oris muscle mesenchymal stem cells was also investigated in 42 patients and 4 controls. RESULTS: We found a significant association in the larger sample (p = 0.0016; OR = 1.80 [95% confidence interval {CI}, 1.21-2.69], for heterozygous genotype, and 2.71 [95% CI, 1.47-4.96] for homozygous genotype). We did not find a significant correlation between rs987525 genotypes and MYC transcriptional levels (p = 0.14; r = -0.22, Spearman Correlation). CONCLUSIONS: We present a positive association of rs987525 in the Brazilian population for the first time, and it is likely that the European contribution to our population is driving this association. We also cannot discard a role of rs987515 in MYC regulation, because this locus behaves as an expression quantitative locus of MYC in another tissue. Birth Defects Research (Part A) 94:464-468, 2012. (C) 2012 Wiley Periodicals, Inc.FAPESP/CEPID [98/14254-2]FAPESP/CEPIDCNPq [401952/2010-S]CNPqBrazilian Ministry of HealthBrazilian Ministry of Healt

Pancreatic surgery outcomes: multicentre prospective snapshot study in 67 countries

Background: Pancreatic surgery remains associated with high morbidity rates. Although postoperative mortality appears to have improved with specialization, the outcomes reported in the literature reflect the activity of highly specialized centres. The aim of this study was to evaluate the outcomes following pancreatic surgery worldwide.Methods: This was an international, prospective, multicentre, cross-sectional snapshot study of consecutive patients undergoing pancreatic operations worldwide in a 3-month interval in 2021. The primary outcome was postoperative mortality within 90 days of surgery. Multivariable logistic regression was used to explore relationships with Human Development Index (HDI) and other parameters.Results: A total of 4223 patients from 67 countries were analysed. A complication of any severity was detected in 68.7 percent of patients (2901 of 4223). Major complication rates (Clavien-Dindo grade at least IIIa) were 24, 18, and 27 percent, and mortality rates were 10, 5, and 5 per cent in low-to-middle-, high-, and very high-HDI countries respectively. The 90-day postoperative mortality rate was 5.4 per cent (229 of 4223) overall, but was significantly higher in the low-to-middle-HDI group (adjusted OR 2.88, 95 per cent c.i. 1.80 to 4.48). The overall failure-to-rescue rate was 21 percent; however, it was 41 per cent in low-to-middle-compared with 19 per cent in very high-HDI countries.Conclusion: Excess mortality in low-to-middle-HDI countries could be attributable to failure to rescue of patients from severe complications. The authors call for a collaborative response from international and regional associations of pancreatic surgeons to address management related to death from postoperative complications to tackle the global disparities in the outcomes of pancreatic surgery (NCT04652271; ISRCTN95140761)