Hongbing Jiang

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.

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.

The axis inhibition protein 2 polymorphisms and non-syndromic orofacial clefts susceptibility in a Chinese Han population

BACKGROUND The axis inhibition protein 2 (AXIN2) is an important regulator of β-catenin degradation in the Wnt pathway, which plays a key role in craniofacial morphogenesis. The goal of this study was to investigate the potential relationship between AXIN2 polymorphisms and the risks of non-syndromic orofacial clefts (NSOC) in a Chinese Han population. METHODS Four polymorphisms of AXIN2 (rs2240307, rs11867417, rs2240308, and rs7591) were selected to perform a case-control study with 599 NSOC cases and 602 healthy individuals from a Chinese Han population. The single nucleotide polymorphisms (SNPs) were genotyped on basis of double ligation and multiplex fluorescence PCR. RESULTS Weak associations were found between these four SNPs and the risk of NSOC. Further stratified analysis showed that the overall genotype frequencies of rs2240307 were different between the cleft palate only (CPO) group and the control group (P = 0.048), and GG genotype markedly contributed to the susceptibility to CPO (OR = 3.22, 95% CI = 1.13-9.18). The similar effect was also observed on GA/AA genotype compared with GG homozygote (OR = 0.30, 95% CI = 0.11-0.84). The results of LD analysis between each pair of SNPs revealed that two SNPs (rs11867417 and rs7591) were in a LD block (r(2) > 0.8). But no statistically significant was found between cases and controls from haplotype analysis in these two loci. CONCLUSIONS The borderline results gave us a hint that rs2240307 contributed to the susceptibility to CPO in a Chinese Han population, which was conductive to improving our awareness of the causes of NSOC.

Associations between microRNA binding site SNPs in FGFs and FGFRs and the risk of non-syndromic orofacial cleft

We hypothesized that microRNA binding site single nucleotide polymorphisms (SNPs) in fibroblast growth factors (FGFs) and their receptor genes (FGFRs) may affect microRNA and mRNA interactions and are thereby associated with susceptibility of non-syndromic orofacial cleft (NSOC). Ten SNPs among the FGF and FGFR genes were selected and their associations with NSOC susceptibility were investigated in a case-control study of 602 patients with NSOC and 605 healthy controls. FGF2/rs1048201, FGF5/rs3733336 and FGF9/rs546782 showed suggestive association with NSOC susceptibility. In the combination analysis, the observed odds ratios (ORs) decreased with the number of protective alleles (rs1048201-T, rs3733336-G and rs546782-T) but were not statistically significant beyond the first comparison. Hsa-miRNA-496, hsa-miRNA-145 and hsa-miRNA-187 were predicted to be miRNAs with binding sites within/near these SNPs and were expressed in lip tissues. Decreased FGF2, FGF5 and FGF9 expression was observed in three cell lines transfected with the corresponding miRNAs. Moreover, the three SNPs could contribute to differential binding efficacy between hsa-miRNA-496 and FGF2, hsa-miRNA-145 and FGF5, hsa-miRNA-187 and FGF9 in luciferase assay. The results suggest that FGF2/rs1048201, FGF5/rs3733336 and FGF9/rs546782 are associated with the risk of NSOC and that these miRNA-FGF interactions may affect NSOC development.

The angiogenic variation of skeletal site-specific human BMSCs from same alveolar cleft patients: a comparative study

Tissue engineering strategies hold great potential for alveolar cleft reconstruction. Bone marrow stromal cells (BMSCs) from iliac crest and craniofacial regions are candidate seeding cells with site-specific characteristics and bone-repairing properties. Craniofacial BMSCs seem to possess stronger multipotency and osteogenic capabilities than BMSCs isolated from iliac crest. However, the angiogenic capabilities of these two type cell is rarely reported. We obtained human BMSCs (hBMSCs) of maxilla (M-hBMSCs) and iliac crest (I-hBMSCs) from same alveolar cleft patients to investigate the agiogenic variations using co-culture system with human umbilical vein endothelial cells (HUVECs). From in vitro comparison, M-hBMSCs allowed HUVECs to form more tube-like structures and sprouting angiogenesis by tube formation assays and 3D fibrin vasculogenic assay, respectively. By transplantation in vivo, M-hBMSCs enhanced larger size vessel like structures distributed the entire implants compared with I-hBMSCs. Western blotting was used to assess the angiogenesis related factors including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The results showed a significant higher expression of bFGF protein in M-hBMSCs and HUVECs co-culture system both in vitro and in vivo. As bFGF could promote migration and proliferation of endothelial cells, scratch wound healing and transwell migration assays were performed as well as MTT assays and cell cycle analysis. The data suggested the effect of M-hBMSCs on HUVECs was stronger than I-hBMSCs. Taken together, these results indicated that craniofacial BMSCs seemed to have greater angiogenesis capability than iliac crest BMSCs and this might be associated with the different levels of bFGF protein expression in co-culture system.

Association study between Van der Woude Syndrome causative gene GRHL3 and nonsyndromic cleft lip with or without cleft palate in a Chinese cohort

Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects worldwide and is characterized by abnormalities of the orofacial structure. Syndromic CL/P is mainly caused by Mendelian disorders such as Van der Woude Syndrome (VWS). However, >70% of CL/P cases are nonsyndromic, characterized by isolated orofacial cleft without any known syndrome. The etiology of nonsyndromic CL/P (NSCL/P) remains elusive, but it has been suggested that causative genes of syndromic CL/P might also contribute to NSCL/P. As such, the VWS causative gene IRF6 has been extensively studied in NSCL/P. Recently, GRHL3 was identified as another VWS causative gene. Thus, it may be a novel candidate gene for NSCL/P. In the present study, we genotyped 10 tag SNPs covering GRHL3 and performed association analysis with NSCL/P in 504 cases and 455 healthy controls. Our preliminary results identified rs10903078, rs4638975, and a haplotype rs10903078-rs6659209 of GRHL3 that exceeded the significance threshold (p<0.05), though none survived Bonferroni correction for multiple comparisons. As the first study between GRHL3 and NSCL/P, the contribution of this gene to NSCL/P etiology should be interpreted with caution based on existing evidence. Further, the robustness of association between GRHL3 and NSCL/P should be further validated in expanded cohorts.

TPM1 polymorphisms and nonsyndromic orofacial clefts susceptibility in a Chinese Han population.

Located at 15q22 a susceptibility region for nonsyndromic orofacial clefts (NSOC), TPM1 encodes a group of highly conserved ubiquitous actin‐binding proteins involved in the muscle contraction and cytoskeleton organization. Considering the multiple functions of TPM1 gene, we investigated the potential relationship between TPM1 polymorphisms and risk of NSOC in a Chinese Han population. Four tag single nucleotide polymorphisms (tSNPs) of TPM1 (rs11071720, rs3803499, rs12148828, and rs1972041) were selected to conduct a case‐control study with 673 NSOC patients and 705 unrelated healthy controls from a Chinese Han population. The SNPs were genotyped by the IPLEX Sequenom MassARRAY platform. SNP rs1972041GA showed a decreased risk of NSOC in heterozygotes (P = 0.038, OR = 0.77, 95%CI = [0.61, 0.99]). Further stratified analysis revealed an enhanced protective effect of the minor allele G at rs197204 on lip with cleft palate (CLP) and cleft lip with or without cleft palate (CL/P) groups under a codominant or dominant model. No association was observed between the remaining three markers (rs11071720, rs3803499, and rs12148828) and NSOC as well as its subgroups. TPM1 polymorphisms might contribute to the etiology of NSOC, and more emphasis should be placed on TPM1 during craniofacial development.

Validation of a genome-wide association study implied that SHTIN1 may involve in the pathogenesis of NSCL/P in Chinese population

Orofacial clefts are among the most common birth defects in humans worldwide. A large-scale, genome-wide association study (GWAS) in the Chinese population recently identified several genetic risk variants for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We selected 16 significant SNPs from the GWAS I stage (P < 1.00E-5) that had not been replicated to validate their association with NSCL/P in 1931 NSCL/P cases and 2258 controls. Ultimately, we identified a NSCL/P susceptibility loci (rs17095681 at 10q25.3, intron of SHTN1 and 27.2 kb downstream of VAX1, Pmeta = 3.80E-9, OR = 0.64) in Chinese Han and Hui populations. This locus was not high LD with the reported loci in 10q25.3. It was a newly identified independent locus in 10q25.3 associated with NSCL/P. These results imply that SHTIN1 may involve in the pathogenesis of NSCL/P advance our understanding of the genetic susceptibility to NSCL/P.

A functional polymorphism in the pre-miR-146a gene is associated with the risk of nonsyndromic orofacial cleft

microRNAs (miRNAs) are widely involved in craniofacial development, and genetic variants of miRNAs may be associated with the risk of nonsyndromic orofacial cleft (NSOC). Here, we systematically selected five single nucleotide polymorphisms (SNPs) of miRNAs and investigated the associations between these variants and NSOC susceptibility in a two‐stage case–control study including 1,406 NSOC patients and 1,578 controls from the Chinese population. We found that compared with the C allele, the rs2910164 G allele of pre‐miR‐146a was associated with an increased risk of NSOC (additive model: odds ratio [OR] = 1.17, 95% confidence interval [CI]: 1.06–1.30, P = 0.002), including both cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO). Bioinformatic prediction and functional assays revealed that the C allele of rs2910164 was significantly associated with inhibited HEK‐293 and HEPM cell proliferation and decreased abundance of TRAF6. Both miR‐146a and TRAF6 were expressed in the lip tissue samples of NSOC patients, and a moderate inverse correlation was observed between them. Taken together, these results demonstrated that miR‐146a/rs2910164 is associated with susceptibility to NSOC, providing novel insights into the genetic etiology and underlying biology of NSOC.

The association study of nonsyndromic cleft lip with or without cleft palate identified risk variants of the

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect due to abnormal orofacial development. Previous studies report abnormal sonic hedgehog (SHH) signalling activity during NSCL/P pathogenesis and propose several genes in the SHH pathway as candidate risk genes. As such, we focussed on GLI3, a downstream modulator of the SHH pathway. In the present study, we genotyped 34 tag SNPs covering GLI3 and performed association analysis with NSCL/P in 504 cases and 455 healthy controls. Our preliminary results identified risk variants of GLI3 that are associated with NSCL/P susceptibility in a Chinese population. In particular, rs3801161 and its haplotypes rs3801161–rs7785287 displayed significant association with NSCL/P and survived Bonferroni correction for multiple comparisons. The robustness of the association between GLI3 and NSCL/P is worth further examination in the future across different populations.