Yongchu Pan

Genome-wide association study identifies a new susceptibility locus for cleft lip with or without a cleft palate

Nonsyndromic cleft lip with or without a cleft palate (NSCL/P) is among the most common human congenital birth defects and imposes a substantial physical and financial burden on affected individuals. Here, we conduct a case-control-based GWAS followed by two rounds of replication; we include six independent cohorts from China to elucidate the genetic architecture of NSCL/P in Chinese populations. Using this combined analysis, we identify a new locus at 16p13.3 associated with NSCL/P: rs8049367 between CREBBP and ADCY9 (odds ratio=0.74, P=8.98 × 10(-12)). We confirm that the reported loci at 1q32.2, 10q25.3, 17p13.1 and 20q12 are also involved in NSCL/P development in Chinese populations. Our results provide additional evidence that the rs2235371-related haplotype at 1q32.2 could play a more important role than the previously identified causal variant rs642961 in Chinese populations. These findings provide information on the genetic basis and mechanisms of NSCL/P.

IRF6 polymorphisms are associated with nonsyndromic orofacial clefts in a Chinese Han population.

IRF6 plays an important role in orofacial development. In the present study, we genotyped two polymorphisms (rs642961 and rs2235371) within the IRF6 locus and estimated their associations with risk of nonsyndromic orofacial clefts (NSOC), including the subgroups, in a hospital‐based case–control study in a Chinese Han population. In the single locus analyses, we found rs642961 AG and AG/AA genotypes were associated with increased risk of NSOC, especially cleft lip with or without cleft palate (CL/P) and cleft lip with cleft palate (CLP), while significantly decreased risks were associated with rs2235371 CT and CT/TT genotypes. When examining the combined effects of these two polymorphisms and using the rs642961 A and rs2235371 C alleles as the risk alleles, we found genotypes containing 2–4 risk alleles conferred high risk to NSOC, CL/P, and CLP. Furthermore, to test whether rs642961 could modulate IRF6 expression in vivo, we surgically collected lip skin tissues within the adjacent region of lip cleft site and found rs642961 genotypes were associated with differential levels of IRF6 mRNA and protein expression in an allele‐dosage manner, providing the first evidence that rs642961 affected IRF6 expression in vivo. Taken together, these findings confirm the contribution of IRF6 genetic variants in the etiology of NSOC in a Chinese Han population.

PAX9 polymorphisms and susceptibility to sporadic tooth agenesis: a case-control study in southeast China.

Tooth agenesis is one of the most common developmental disorders in humans. The PAX9 gene, which plays an important role in odontogenesis, is associated with familial and sporadic tooth agenesis. A case-control study was performed in 102 subjects with tooth agenesis (cases) and 116 healthy controls. We genotyped four PAX9 gene polymorphisms using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The allele and genotype frequencies of the four polymorphisms were not significantly different between the controls and the subjects with tooth agenesis. Similar results were observed in a subgroup analysis of test subjects only with mandibular incisor agenesis. Further analysis showed no significant difference in the haplotype distribution between the controls and the subjects with tooth agenesis or mandibular incisor agenesis. However, we found that the AGGC haplotype was associated with a decreased risk of tooth agenesis, compared with the most common haplotype, AGCC (odds ratio, 0.14; 95% confidence interval: 0.00-0.95). These results suggest that the four PAX9 polymorphisms alone have a non-significant main effect on the risk of tooth agenesis but that the AGGC haplotype may have a protective effect associated with a decreased risk of tooth agenesis.

Polycystin-1 Mediates Mechanical Strain-Induced Osteoblastic Mechanoresponses via Potentiation of Intracellular Calcium and Akt/β-Catenin Pathway

Mechanical regulation of bone formation involves a complex biophysical process, yet the underlying mechanisms remain poorly understood. Polycystin-1 (PC1) is postulated to function as a mechanosensory molecule mediating mechanical signal transduction in renal epithelial cells. To investigate the involvement of PC1 in mechanical strain-induced signaling cascades controlling osteogenesis, PKD1 gene was stably silenced in osteoblastic cell line MC3T3-E1 by using lentivirus-mediated shRNA technology. Here, our findings showed that mechanical tensile strain sufficiently enhanced osteogenic gene expressions and osteoblastic proliferation. However, PC1 deficiency resulted in the loss of the ability to sense external mechanical stimuli thereby promoting osteoblastic osteogenesis and proliferation. The signal pathways implicated in this process were intracellular calcium and Akt/β-catenin pathway. The basal levels of intracellular calcium, phospho-Akt, phospho-GSK-3β and nuclear accumulation of active β-catenin were significantly attenuated in PC1 deficient osteoblasts. In addition, PC1 deficiency impaired mechanical strain-induced potentiation of intracellular calcium, and activation of Akt-dependent and Wnt/β-catenin pathways, which was able to be partially reversed by calcium ionophore A23187 treatment. Furthermore, applications of LiCl or A23187 in PC1 deficient osteoblasts could promote osteoblastic differentiation and proliferation under mechanical strain conditions. Therefore, our results demonstrated that osteoblasts require mechanosensory molecule PC1 to adapt to external mechanical tensile strain thereby inducing osteoblastic mechanoresponse, partially through the potentiation of intracellular calcium and downstream Akt/β-catenin signaling pathway.

Methylenetetrahydrofolate Reductase C677T and A1298C Polymorphisms and Nonsyndromic Orofacial Clefts Susceptibility in a Southern Chinese Population

Nonsyndromic orofacial clefts (NSOC) are one of the most common congenital anomalies in humans. Great efforts have been taken to unravel its genetic background. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate metabolism and two of its functional polymorphisms, MTHFR C677T and MTHFR A1298C, might be associated with NSOC susceptibility. The aim of the present study was to investigate their associations with risks of NSOC in a southern Chinese population. We found that MTHFR 677 TT and 677 CT/TT were associated with increased risk of cleft lip with or without cleft palate; meanwhile, MTHFR 1298 AC and 1298 AC/CC had protective effects against cleft lip with or without cleft palate. In further stratified analysis, we found that MTHFR 677 CT contributed to elevated risk of cleft lip only, as did MTHFR 677 CT/TT. On the contrary, MTHFR 1298 AC and 1298 AC/CC appeared to be protective against cleft lip with cleft palate. These results suggested that these two polymorphisms were involved in the development of NSOC in a southern Han Chinese population.

Replication of two novel susceptibility loci for non-syndromic orofacial clefts in a Chinese population.

OBJECTIVES Non-syndromic orofacial clefts (NSOC) are the most common developmental disorders in human beings. Recently, two genome-wide association studies in European Caucasians identified three novel NSOC susceptibility loci: rs987525 on 8q24, rs7078160 on 10q25.3, and rs223371 on 17q22. The aim of this study was to determine the association of these polymorphisms with NSOC susceptibility and its subgroups in a Chinese Han population. MATERIAL AND METHODS In this study, 199 NSOC patients and 210 healthy individuals were recruited. SNP rs987525 was not genotyped because of its low frequency in the study subjects. The other two polymorphisms (rs7078160 and rs223371) were respectively genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taqman-MGB assay. RESULTS Overall genotype distributions of rs7078160 and rs223371 polymorphisms were consistent with Hardy-Weinberg equilibrium test. The allele and genotype frequencies of the two polymorphisms were not significantly different between cases and controls. Further analysis indicated that none of the genotypes was associated with increased risk of NSOC. Similar results were also found when all cleft cases were stratified by cleft types. CONCLUSION Our findings are consistent with a lack of involvement of the rs7078160 and rs223371 polymorphisms in the development of NSOC in the Chinese Han population.

A miRNA-binding-site SNP of MSX1 is Associated with NSOC Susceptibility

MSX1 is a favorable candidate gene for susceptibility to non-syndromic orofacial clefts (NSOCs). However, the roles of MSX1 genetic variants in the development of NSOC are controversial and vary among human populations. In the present study, the roles of 4 potentially functional single-nucleotide polymorphisms (SNPs) of MSX1 (rs12532 in 3′-untranslated region [UTR], and rs3821947, rs3821949, and rs4464513 in 5′ upstream) were investigated in a case-control study of 602 NSOC cases and 605 healthy controls. The findings showed that rs12532 located within 3′-UTR of MSX1 could influence the risk of developing NSOC. Individuals who carried the variant genotype (rs12532AA genotype) showed a decreased possibility of developing NSOC (AA vs. GG: OR = 0.69, 95% CI = [0.49, 0.98]). Interestingly, similar effects were also observed on cleft lip with palate (CLP), in a stratified analysis (allelic comparison-12532A allele vs. 12532G allele, OR = 0.80, 95% CI = [0.66, 0.99]; genotypic comparison-AA vs. GG, OR = 0.58 95% CI = [0.37, 0.91]). Sequence analysis indicated that this SNP might alter the binding ability of miR-3649, confirmed by luciferase activity assay showing a lower expression level of rs12532 A allele compared with that of the G allele (p < .001 for 293A and COS7 cell lines). Furthermore, an in vivo study showed that MSX1 expression among individuals carrying the AA genotype of rs12532 was markedly lower than that in those with the GG genotype, while the inverse correlation was observed for miR-3649, thus providing a possible interaction between MSX1 and miR-3649 in the etiology of NSOC. Taken together, these findings indicate that SNPs in the miRNA-binding sites might play an important role in the development of NSOCs. Furthermore, if confirmed in subsequent studies, the polymorphisms may be considered as additional markers for the evaluation of infants’ risk of NSOCs.

Three polymorphisms in IRF6 and 8q24 are associated with nonsyndromic cleft lip with or without cleft palate: evidence from 20 studies.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common craniofacial malformation in humans. Three polymorphisms, rs2235371 and rs642961 in interferon regulatory factor 6 (IRF6), rs987525 on 8q24, have been shown to be associated with NSCL/P risk in several studies. However, the magnitudes of the association varied between studies. We therefore performed a meta‐analysis to investigate this relationship. Two authors independently extracted information on the characteristics of the eligible studies. Either a fixed‐ or a random‐effects model was used to calculate the overall combined risk estimates. Overall, 20 published case–control studies were included in the meta‐analysis. We found that rs2235371 A allele had a significantly decreased risk (OR: 0.73, 95% CI: 0.61–0.88), whereas rs642961 A allele had a significantly increased risk of NSCL/P (OR: 1.44, 95% CI: 1.30–1.59), compared with the G allele. For 8q24 rs987525, the A allele was associated with a significantly increased risk of NSCL/P, compared with the C allele (OR: 1.71, 95% CI: 1.40–2.09). Furthermore, in the stratified analysis by ethnicity and types of NSCL/P, significant associations were still observed in the subgroups of ethnicity and types. Taken together, the results suggest that the IRF6 rs2235371, rs642961, and 8q24 rs987525 polymorphisms are associated with NSCL/P risk.

Different roles of two novel susceptibility loci for nonsyndromic orofacial clefts in a Chinese Han population.

Nonsyndromic orofacial clefts (NSOC) are the most common developmental anomalies in human beings. Recently, a large‐scale genome‐wide association study identified two novel NSOC susceptibility loci: rs13041247 near MAFB and rs560426 near ABCA4. In the present study, we recruited 396 NSOC cases and 384 healthy controls to replicate their associations with risk of NSOC as well as their subgroups in a Chinese Han population. We found the overall genotype and allele frequencies of rs13041247, but not rs560426 were significantly different between cases and controls. Further logistic regression analysis showed rs13041247 CT, CC, and CT/CC were associated with decreased NSOC susceptibility, compared with rs13041247 TT wide‐type homozygote. Moreover, the apparent protection against cleft lip with or without cleft palate (CL/P), cleft lip with cleft palate (CLP), and cleft lip only (CLO) was also identified in stratified analysis. However, none of any rs560426 genotypes or alleles was associated with risk of NSOC or their subgroups. Taken together, our findings confirmed the contribution of MAFB in the etiology of NSOC in a Chinese Han population.

Association and cumulative effects of GWAS-identified genetic variants for nonsyndromic orofacial clefts in a Chinese population.

A recent genome‐wide meta‐analysis identified six new susceptible genetic variants for nonsyndromic orofacial clefts (NSOC), but it was still unknown whether these newly identified variants were associated with NSOC susceptibility in Chinese populations. In this study, we genotyped these variants in a case‐control study of 602 NSOC cases and 605 controls and found that four of these variants (rs7590268, rs7632427, rs12543318, and rs1873147) were associated with susceptibility to NSOC. We further investigated the cumulative effects of these four variants and found a dose‐dependent increase in risk with the number of variant alleles. Furthermore, an association was observed between rs7590268 and a family history of NSOC. Our results provide confirmative evidence that these risk loci contribute to NSOC susceptibility in Chinese populations. Environ. Mol. Mutagen. 54:261–267, 2013.