Interactions between superoxide dismutase and paraoxonase polymorphic variants in nonsyndromic cleft lip with or without cleft palate in the Brazilian population

During development, oxidative stress is hypothesized to mediate embryotoxicity, which may be intensified by exposition to environmental factors and by genetic variations in the enzymes involved in protecting cells from these damaging effects, including superoxide dismutase (SOD) and paraoxonase (PON). The aim of this study was to evaluate the influence of single-nucleotide polymorphisms (SNP) in genes associated with the neutralization of oxidative stress (SOD and PON family members) in the risk of nonsyndromic oral cleft in the Brazilian population. Initially, we tested for association between 28 SNP in SOD1, SOD2, SOD3, PON1, PON2, and PON3 among 325 nonsyndromic cleft lip with or without cleft palate (NSCL±P) case-parent trios. Multiple logistic regression analyses were used to explore gene, GxG and GxE, involving factors that induce oxidative stress accumulation during pregnancy. Signals still significant after both Bonferroni correction and in permutation test were subsequently confirmed in an ancestry-structured case–control analysis with 722 NSCL±P and 866 controls from the same population. In the trio sample, transmission disequilibrium test (TDT) (allele and haplotype) and GxE analysis showed no significant associations, but multiple pairwise GxG interactions involving 10 SNP in PON1, PON2, and PON3 were detected and further examined in the case–control sample. The PON1 rs2237583 and PON2 rs17166879 yielded significant evidence of SNP-SNP interactions after adjustment for multiple tests (both Bonferroni correction and 10,000 permutation test). The C allele and the CT genotype of PON1 rs2237583 were associated with significant protective effects against NSCL±P, while rs3917490 showed a significant association only in the sample composed of patients displaying high African ancestry. Our results reveal associations between rs2237583 and rs3917490 in PON1 and GxG interactions containing rs2237583 and rs17166879 with the susceptibility of NSCL±P in the Brazilian population. Furthermore, this study underlines the recent tendency of taking into account potential GxG interactions to clarify the underlying mechanisms associated with the etiology of this common malformation. Environ. Mol. Mutagen. 60: 185–196, 2019. © 2018 Wiley Periodicals, Inc.     

2p24.2 (rs7552) is a susceptibility locus for nonsyndromic cleft lip with or without cleft palate in the Brazilian population

The population of Brazil is highly admixed, with each individual showing variable levels of Amerindian, European and African ancestry, which may interfere in the genetic susceptibility of known risk loci to nonsyndromic cleft lip with or without cleft palate (NSCL±P). Here, we investigated 5 reported genome‐wide loci for NSCL±P in an ancestry‐structured case‐control study containing 1697 Brazilian participants (831 NSCL±P and 866 healthy controls). SNPs rs7552 in 2q24.2, rs8049367 in 16p13.3, rs1880646, rs7406226, rs9891446 in 17p13, rs1588366 in 17q23.2 and rs73039426 in 19q13.11 were genotyped using TaqMan allelic discrimination assays and genomic ancestry was estimated using a panel of 40 biallelic short insertion/deletion polymorphic markers informative of the Brazilian population. Logistic regression analysis of the single‐markers revealed rs7552 in 2p24.2 as a susceptibility risk marker for NSCL±P, yielding an odds ratio (OR) of 1.71 (95% confidence interval (CI): 1.31‐2.24, P = 9 × 10^?6) in the homozygous state. Several SNP‐SNP interactions containing rs7552 reached significance after adjustment for multiple tests (both Bonferroni assumption and 1000 permutation test), with the most significant interaction involving the 3‐loci among rs7552, rs9891446 and rs73039426 (P = 6.1 × 10^?9 and p_{1000 permutation} = 0.001). Our study is the first to support the association of rs7552 in 2p24.2 with NSCL±P in the highly admixed Brazilian population.     

MSX1基因微卫星多态性与非综合征性唇腭裂的相关性研究

目的探讨肌肉片段同源盒1基因（muscle segment homeobox1,MSX1）与湖南汉族非综合征性唇腭裂（nonsydromic cleft lip and patate,NSCLP）的相关性。方法以MSX1基因内含子区的（CA）n微卫星作为遗传标记,对湖南汉族129例NSCLP患儿和108名正常儿童采用聚合酶链反应-变性聚丙烯酰胺凝胶基因分型技术进行基因分型,并经测序确定片段长度,应用病例对照研究进行相关性分析。结果湖南汉族人群MSX1基因（CA）n等位基因分布符合Hardy-Weinberg平衡,杂合率及多态信息量均为0.50;CA4等位基因频率在唇裂伴或不伴腭裂组及单纯性腭裂组高于对照组,CA4,4基因型频率在唇裂伴或不伴腭裂组和单纯性腭裂组高于对照组,差异均有统计学意义（均P〈0.05）。结论MSX1基因内（CA）n微卫星是多态性较好的遗传标记;MSX1基因可能与湖南汉族人群NSCLP发病相关。     

IRF6基因rs2235371位点多态性与非综合征性唇裂伴或不伴腭裂的相关性研究

目的 探讨干扰素调节因子6 (interferon regulatory factor 6,IRF6)基因rs2235371位点820G＞A单核苷酸多态性与非综合征性唇裂伴或不伴腭裂(nonsyndromic cleft lip with or without cleft palate,NSCL士P)的相关性.方法 收集106例患者及其父母以及129名对照及其父母的核心家庭标本,用聚合酶链反应-限制性片段长度多态性的方法进行IRF6基因rs2235371位点单核苷酸多态性检测;用人群关联研究、传递不平衡检验(transmission disequilibrium test,TDT)、单倍型相对风险率(haplotype-based haplotype relative risk,HHRR)、家系为基础的关联检验(family-based association tests,FBAT)等方法进行统计分析.结果 病例组与对照组相比,子代的基因型分布差异无统计学意义(P＞0.05),而等位基因频率比较差异有统计学意义(P＜0.05);母亲组基因型分布差异有统计学意义(P＜0.05);按唇腭裂类型分类后,单纯唇裂组子代基因型分布和等位基因的频率与对照组相比差异均有统计学意义(P＜0.05);而唇腭裂组基因型分布和等位基因频率差异均无统计学意义(P＞0.05).传递不平衡检验提示,rs2235371位点的G等位基因在NSCL±P核心家庭中存在传递不平衡(x2=5.56,P=0.024).HHRR检验x2=5.115,P=0.024,OR=1.674,95％CI:1.069～2.621;FBAT检验Z=2.218,P=0.027.结论 在中国北方人群中IRF6基因rs2235371位点突变与非综合征性唇腭裂存在关联.     

西北地区人群JAG2基因多态性与非综合征性唇腭裂的相关性研究

目的探讨JAG2基因多态性与西北地区人群非综合征性唇腭裂(nonsyndromic cleft lip with or without cleft palate,NSCLP)的相关性。方法采用病例-对照研究方法,选取NSCLP患者301例,正常对照304人,采用iMLDR™基因分型技术对JAG2基因的3个单核苷酸多态性(single nucleotide polymorphism,SNP)位点[rs741859(T/C)、rs11621316(A/G)以及rs1057744(C/T)]进行分型,比较其等位基因、基因型及所构建的单倍型在两组人群中的分布差异。结果rs741859位点等位基因C和T在NSCLP组和对照组中的分布差异有统计学意义。rs741859位点CT基因型可将NSCLP的患病风险显著降低至65%(P<0.05),将唇裂伴或不伴腭裂(cleft lip with or without cleft palate,CL/P)的患病风险降低至62%(P<0.05);而rs11621316、rs1057744处于同一连锁不平衡(linkage disequilibrium,LD)区域,连锁程度较高(r2>0.8),其基因型、等位基因在两组中的分布差异无统计学意义(P>0.05)。结论JAG2基因rs741859位点CT基因型可能是中国西北人群NSCLP的保护性基因型。     

Rs2262251 in lncRNA RP11‐462G12.2 is associated with nonsyndromic cleft lip with/without cleft palate

Nonsyndromic cleft lip with/without cleft palate (NSCL/P) is one of the most common human congenital defects. Rs2262251 (G>C) in long noncoding RNA (lncRNA) RP11‐462G12.2 is in high linkage disequilibrium with rs8049367, which was identified in our previous genome‐wide association study on NSCL/P, and is a potential causative single‐nucleotide polymorphism (SNP) for NSCL/P. To test these hypotheses, rs2262251 was evaluated in another cohort of 1,314 cases and 1,259 controls. Rs2262251 was associated with NSCL/P risk (p = .003). However, no association was detected for cleft palate only. SNP rs2262251 affected the structure and expression of lncRNA RP11‐462G12.2 in HEK293 and HEPM cells and in lip tissues from patients with NSCL/P. Overexpression of the rs2262251 G allele contributed to reducing the number of cells in the G0/G1 phase, inhibiting cell apoptosis, and promoting cell proliferation in vitro. The rs2262251 C allele regulated the expression of miR‐744‐5p and its target gene IQSEC2, both of which were expressed in human lip tissues, and showed reverse correlation during mouse lip development. Taken together, these findings suggest that rs2262251 is associated with the risk of NSCL/P and participates in a lncRNA–miRNA–mRNA regulatory axis in which miR‐744‐5p and IQSEC2 combine to control NSCL/P development.     

蛋氨酸合酶基因A275 6G位点多态性与非综合征型唇腭裂的关联性研究

目的 研究蛋氨酸合酶基因(methionine synthase,MS)A2756G位点多态性与非综合征型唇腭裂(nonsyndromic cleft lip with or without cleft palate,NSCL/P)的关联性.方法 采用PCR-限制性片段长度多态性技术检测97个NSCL/P病例组核心家庭和104个对照家庭的MS基因A2756G位点的多态性;用人群关联研究分析、病例组核心家庭的传递不平衡检测(transmission disequilibrium test,TDT)、单体型的相对危险度分析(haplotype-based haplotype relative risk,HHRR)、家庭为基础的关联研究(family-based association tests,FBAT)等统计分析.结果 子代、父亲、母亲病例组和对照组之间基因型和等位基因的分布差异均无统计学意义(P>0.05);本研究中在子代和母亲组中未检出GG基因型,AG基因型相对于AA基因型的比值比OR和95%CI分别为子代1.78(0.74～4.34)、父亲0.80(0.36～1.79)、母亲1.26(0.54～2.93),G相对于A基因的OR和95%CI分别为子代1.70(0.78～3.73)、父亲0.88(0.49～1.75)、母亲1.23(0.59～2.60),携带有突变基因G并不能增加患NSCL/P的危险.病例组核心家庭分析,TDT分析χ2=0.034,P>0.05;HHRR分析χ2=0.03,P>0.05;FBAT分析Z=0.186,P>0.05.结论 结果未显示出MS基因A2756G位点多态性和NSCL/P发生的相关性,还待进一步研究.     

Association of single nucleotide polymorphisms in TPM1 rs11071720, rs3803499, rs12148828, and rs1972041 with the risk of nonsyndromic cleft lip with or without cleft palate in a sample of the Iranian population,a preliminary report

Several lines of evidence support an association between tropomyosin 1 (TPM1) and the risk of nonsyndromic cleft lip with or without cleft palate (NSCL/P). The present study aimed to investigate the association between TPM1 polymorphisms and the risk of NSCL/P in an Iranian population. This case‐control was done on 105 NSCL/P patients and 110 unrelated healthy controls. TPM1 rs11071720, rs3803499, rs12148828, and rs1972041 polymorphisms were genotyped by the polymerase chain reaction‐restriction fragment length polymorphism method. The finding showed that rs11071720 polymorphism significantly increased the risk of NSCL/P in homozygous codominant (odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.14–5.69, p = 0.023, TT vs. CC), recessive (OR = 2.33, 95% CI = 1.06–5.18, p = 0.021, TT vs. CT + CC), and allele (OR = 1.53, 95% CI = 1.02–2.30, p = 0.030, T vs. C). The rs12148828 polymorphism was associated with protection against NSCL/P in codominant (OR = 0.27, 95% CI = 0.15–0.48, p < 0.001, TC vs. TT) and allele (OR = 0.38, 95% CI = 0.22–0.64, p < 0.001, C vs. T). Regarding rs3803499, the findings proposed that this polymorphism significantly increased the risk of NSCL/P in codominant (OR = 3.86, 95% CI = 1.19–12.56, p = 0.025, CC vs. TT) and recessive (OR = 3.74, 95% CI = 1.09–14.15, p = 0.018, CC vs. CT + TT). No significant association was practical between rs1972041 polymorphism and NSCL/P. In conclusion, the findings proposed that TPM1 polymorphisms may contribute to the etiology of NSCL/P in a sample of the Iranian population.     

亲代MTHFR基因677C/T多态性与子代非综合征性唇腭裂的相关性

目的 探讨山东地区亲代亚甲基四氢叶酸还原酶( methylenetetrahydrofolate reductase,MTHFR)基因677C/T多态性与子代发生非综合征性唇腭裂(nonsyndromic cleft lip with or without cleft palate,NSCL/P)的关联.方法 应用聚合酶链反应-限制性片段长度多态性分析技术(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)对2006年8月至2008年8月在齐鲁医院治疗的89对NSCL/P患者亲代和64对健康查体儿童亲代的MTHFR基因677C/T多态性进行检测.结果 患者母亲与正常儿童母亲的T等位基因频率分别为65.73％和46.09％,C等位基因频率分别为34.27％和53.91％,其构成比差异有统计学意义(x2=13.663,P＜0.01);携带T等位基因的母亲子代患NSCL/P的风险为未携带T等位基因的母亲子代的2.243倍(95％CI:1.408～3.572).患者的父亲与正常儿童父亲的T等位基因频率分别为62.92％和55.47％ ;C等位基因频率分别为37.08％和44.53％,其构成比差异无统计学意义(x2 =2.222,P＞0.05);病例组和对照组后代可能为纯合突变胎儿的机率分别为43％和29％(P＞0.05).结论 山东地区母亲的MTHFR基因677C/T突变对后代NSCL/P的发生有重要的影响;父亲的MTHFR基因677C/T突变则可能不是子代患NSCL/P的风险因素.     

Contribution of variants in and near the IRF6 gene to the risk of nonsyndromic cleft lip with or without cleft palate in a Malay population

Several studies have shown evidence for the contribution of interferon regulatory factor 6 (IRF6) variants to the risk of nonsyndromic oral clefts in Asians; however, this has not included the Malay population. The current study attempts to address this research gap using allele and haplotype transmission disequilibrium analyses. The results showed a strong transmission distortion for multiple haplotypes to patients with nonsyndromic cleft lip with or without cleft palate. Haplotypes carrying the 243 bp allele of D1S2136 and common alleles at the rs861019 and rs2235371 were over‐transmitted to patients. By contrast, haplotypes consisting of the 251 bp allele of D1S2136 and the rare allele at rs2235371 were more under‐transmitted. Furthermore, several variants and haplotypes showed excess maternal transmission, but none of them attained statistical significance in maternal relative risk analyses. In contrast, a significant child genotype effect was observed for several haplotypes, indicating fetal genotype could be the major genetic contribution rather than maternal genotype. The present study therefore further supports a role for IRF6 variants in clefting in this Southeast Asian population. Overall, Asian genetic backgrounds are most likely more susceptible to the haploinsufficiency of IRF6 variants. These variants may contribute to the condition either themselves, or they may be in linkage disequilibrium with other casual variants. © 2011 Wiley‐Liss, Inc.     

Biological and epidemiological evidence of interaction of infant genotypes at Rs7205289 and maternal passive smoking in cleft palate

The noncoding SNP rs7205289, located in the microRNA-140 gene has been associated with cleft palate risk. MiR-140 was found to regulate zebrafish palatal development in vivo and its expression level be reduced by environmental smoke exposure in vitro. Therefore, we sought to investigate whether the A allele of rs7205289 and maternal smoke exposure during the first trimester might contribute to cleft palate risk by regulating microRNA-140. We used in situ hybridization to explore the microRNA-140 expression pattern. A luciferase reporting system and Western blot were used to validate the target of microRNA-140. Mouse palatal mesenchymal cells (MPMC) were transfected with microRNA-140 expression vectors, or treated with cigarette smoke extract. In addition, we performed a hospital-based case-control study in 169 patients with nonsyndromic cleft palate and 306 unaffected controls. We demonstrated microRNA-140 expression in mouse palatal shelves from embryonic days 12 to 15. Pdgfrα was the target of microRNA-140 in MPMC. When these cells were transfected with the minor allele vector or exposed to cigarette smoke extract, they showed a decrease in microRNA-140 expression. Epidemiological analyses showed that infants with CA/AA genotypes and exposed to maternal passive smoking during pregnancy had evidence of synergistic interaction in contributing to cleft palate risk. We concluded that infants with CA/AA genotypes at rs7205289 and maternal passive smoking during the first trimester may synergistically contribute to cleft palate risk by decreasing microRNA-140 during palatal development.     

广东人群PVRL1的多态性与NSCL/P的相关性分析

目的: 探讨脊髓灰质炎病毒受体相关基因( PVRL1 )的多态性与广东人群中非综合征型唇腭裂(NSCL/P,MIM 119530)的相关性。方法: 应用聚合酶链式反应和直接测序的方法对71例广东人群NSCL/P患者和100个健康志愿者的 PVRL1 exon 2和exon 5的多态性进行检测;应用基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱分析的方法对100例广东人群NSCL/P患者和100个健康志愿者的 PVRL1 α亚型的334、391、1 183位点和β亚型的1082位点进行突变检测。结果: PVRL1 的exon 2和exon 5未发现有突变位点;质谱分析没有发现 PVRL1 α亚型的334、391、1 183位点和β亚型的1 082位点相关的S112A、T131A、G361V、V395M突变。结论: PVRL1 的exon 2、exon 5,α亚型334T>A、391A>T、1 183G>A,及β亚型1 082G>T的突变并不参与所研究的广东人群非综合征型唇腭裂的发生。     

Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate

The pathogenesis of cleft lip with or without cleft palate (CL/P) is complex; its onset could be due to the interaction of various genetic and environmental factors. Recently MTHFR functional polymorphisms were found to increase the risk of this common malformation; however, this finding is still debated. We investigated 110 sporadic CL/P patients, their parents and 289 unrelated controls for c.665C>T (commonly known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala) polymorphism in the MTHFR gene. Transmission disequilibrium test (TDT) showed no distortion in allele transmission. Nevertheless, association studies revealed significant differences in allele frequencies between mothers of CL/P patients and controls. This work supports the hypothesis that a lower MTHFR enzyme activity in pregnant women, mostly related to the c.665C>T variant form, is responsible for a higher risk of having CL/P affected offspring.     

经手术和语音康复后非综合征型唇腭裂儿童静息态脑功能改变的磁共振特征

目的：探讨非综合征型唇腭裂（NSCLP）患儿在外科修复术和语音治疗康复后静息状态脑功能模式。方法：选取北京儿童医院和北京口腔医院就诊的28例NSCLP儿童和28例性别和年龄相匹配的健康者（对照组）,进行3DT1和静息态功能性磁共振数据采集。使用DPARSFA软件进行预处理并计算低频振幅（ALFF）和局部一致性（ReHo）,采用双样本t检验评价组间差异（TFCE校正, P<0.05）。对于有显著性差异脑区的静息态脑功能参数与汉语语言清晰度量表（CLCDS）评分进行相关性分析。结果：NSCLP患儿左颞中回、左枕中回、右楔叶、右楔前叶、左中央后回、左顶上小叶ALFF值明显降低。与对照组相比,ReHo值降低脑区包括左侧的中央后回、中央前回、顶上小叶、缘上回和旁中央小叶。异常脑区的ALFF值和ReHo值与CLCDS评分无显著性相关。结论：经外科修复手术和语音治疗康复后的NSCLP患儿静息态自发性神经元活动及其协调性仍未达到正常水平,且以优势半球的语言相关脑区异常为主。     

Association between alleles of the transforming growth factor alpha locus and cleft lip and palate in the chilean population

Two RFLPs at the TGFA locus were studied in 39 unrelated Chilean (Caucasoid-Mongoloid) patients with non-syndromic cleft lip/palate [CL(P)] and 51 control individuals. A highly significant association between BamHI A2 allele and CL(P) was detected (χ² = 6.00; P = 0.014), while no association was found between TaqI RFLPs and clefting. No significant differences were found when comparing genotypes by type of cleft and a positive or negative family history of clefting. Our results seem to support rather definitively the association between TGFA and clefting but do not support the hypothesis that TGFA is a major causal gene of CL(P). © 1995 Wiley-Liss, Inc.     

Evidence of the involvement of the DHFR gene in nonsyndromic cleft lip with or without cleft palate

Studies aimed at evidencing genetic causes for neural tube defect (NTD) occurrence have often provided the inspiration for orofacial cleft aetiology investigations. The correlation between the two congenital malformations is provided by the similar incidence timing and the involvement of structures localized in the midline of the embryo. This connection is corroborated by the existence of a number of genes involved in both malformations. In this article, we considered the dihydrofolate reductase (DHFR) gene, previously seen implicated in NTDs, as a candidate for cleft lip with or without cleft palate (CL/P) risk. Four SNPs mapping on the DHFR gene were genotyped for 400 Italian CL/P triads, using TaqMan^® approach. The rs1677693 provided evidence of association, even if at borderline level (P value 0.049). In particular, the variant allele seems to have a protective effect OR = 0.80 (95% C.I. 0.64–0.99). Moreover, the combination of rs1677693(A)-rs1650723(G) alleles showed a significant association OR 0.64 (95% C.I. 0.47–0.86) (P value = 0.006). This represents the first attempt to demonstrate a role for DHFR in CL/P aetiology, howbeit the study of such gene deserves a deepening.