Genetic Analysis Of Nonsyndromic Cleft Lip With Or Without Cleft Palate (NSCLP) In The Central Chinese Population

Cleft lip with or without cleft palate (CLP) is a common birth defect with complex aetiology, which is recognized as disruptions of normal facial structure. CLP affects approximately1in700live births, with wide variability across geographic origin, race and environment. CLP has been divided into nonsyndromic cleft lip with or without cleft palate (NSCLP) and syndromic cleft lip with or without cleft palate. Syndromic cleft lip with or without cleft palate is mostly caused by the single susceptibility gene, whereas the nonsyndromic cleft lip with or without cleft palate has a complex aetiology with both genetic and environmental contributions.The latest research has identified a new region on chromosome8q24.21in human that gave extremely strong evidence of association in their European and American case-control samples. However, this association is not evident in the Asia case-control samples (Philippines). No any candidate gene of cleft lip with or without palate has been identified in this8q24.21region. It remains to be studied that whether there is association between cleft lip with or without palate and the8q24.21region in the central Chinese population, and whether there is candidate gene in this8q24.21region of cleft lip with or without palate.1. There is strong evidence of association between8q24.21region SNPs and nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population.Eight nonsyndromic cleft lip with or without cleft palate (NSCLP) families of more than three generations, twenty two core nonsyndromic cleft lip with or without cleft palate (NSCLP) families of two generations and some separate case were screened.210cases and234controls were genotyped by the SNPs chosen from HapMap. Two new SNPs, rs17241323and rs12546523in the8q24.21region were associated with nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population. Linkage disequilibrium showed that the C allele of rs17241323was associated with nonsyndromic cleft lip with or without cleft palate (NSCLP). P=0.02285,0.01<P=0.02285<0.05, OR(95%CI)=1.450(1.38～1.53); C/C heterozygous genotype P=0.006934<0.01, OR(95%CI)=1.682(1.56～1.81). The G allele of rs12546523was also associated with nonsyndromic cleft lip with or without cleft palate (NSCLP), P=0.01329,0.01<P=0.01329<0.05OR(95%CI)=1.526(1.44-1.62); G/G heterozygous genotype P=0.004261<0.01, OR(95%CI)=1.752(1.62-1.89). These results showed that there is an association between8q24.21region SNPs and nonsyndromic cleft lip with or without cleft palate (NSCLP) in the central Chinese population..2. The EST BF691581is associated with cleft palate only.To investigate the candidate genes in the8q24.21region, the genome sequence near the SNPs were screened in the centre Chinese population for rs17241323, rs12546523, rs987525. rs17241253and rsl530300by the GENESCAN software. None coding candidate genes were observed in this region. RT-PCR was used to investigate expression of ESTs in the8q24.21region in adult tissues and cell lines. The ESTs BF573425and BF691581were expressed in lip and palate tissues. Northern blot and rapid amplification of cDNA ends (RACE) were used to determine length of BF691581. The real time fluorescence quantitative PCR (Q-PCR) showed that the BF691581was highly expressed in the cleft palate only (CPO) cases. In situ hybridization showed that BF691581was expressed in the epidermal basal layer cells, which were the exclusive epidermal cells, in the palatal outer shelves and the palate epithelium of middle oral. The expressive region of the BF691581was similar with the candidate genes, fibroblast growth factor9(FGF9), interferon regulatory factor6(IRF6) and sonic hedgehog (SHH). These results suggested that BF691581may be associated with the palate morphogenesis.3. A new functional mutation in the TBX22core promoter is associated with cleft palate only.Nineteen candidate gene promoters of non-syndromic cleft lip with or without cleft palate (NSCLP) including IRF6.TBX22,MSX1,BMP4and FGF8were investigated for possible mutations. The core promoters of these candidate genes in7nonsyndromic cleft lip with or without cleft palate (NSCLP) families of more than three generations.22core nonsyndromic cleft lip with or without cleft palate (NSCLP) families of two generations and some separate case, totally210cases and234controls were screened. A G→A mutation at the-73bp upstream of TBX22transcription start site (TSS) in a3generation family, and a A→C mutation at the-32bp upstream of IRF6transcription start site (TSS) in a2generation core family were identified. The G→A mutation at the-73bp upstream of TBX22transcription start site (TSS) was a functional mutation. Important transcription factors and their binding sites in the core promoter region were tested to elucidate possible mechanisms of TBX22gene in the palate development. A potential binding site of transcriptional factor c-ETS-1in the G→A mutation was observed. The luciferase reporter assays showed this G→A mutation at the-73bp upstream of TBX22resulted in a decreased activity of up to40%. These results showed that the new functional mutation of the TBX22gene promoter may be associated with the cleft palate only.