The PDGF-C regulatory region SNP rs28999109 decreases promoter transcriptional activity and is associated with CL/P

Abstract

Human linkage and association studies suggest a gene(s) for nonsyndromic cleft lip with or without cleft palate (CL/P) on chromosome 4q31-q32 at or near the platelet-derived growth factor-C (PDGF-C) locus. The mouse Pdgfc(-/-) knockout shows that PDGF-C is essential for palatogenesis. To evaluate the role of PDGF-C in human clefting, we performed sequence analysis and SNP genotyping using 1048 multiplex CL/P families and 1000 case-control samples from multiple geographic origins. No coding region mutations were identified, but a novel -986 C>T SNP (rs28999109) was significantly associated with CL/P (P=0.01) in cases from Chinese families yielding evidence of linkage to 4q31-q32. Significant or near-significant association was also seen for this and several other PDGF-C SNPs in families from the United States, Spain, India, Turkey, China, and Colombia, whereas no association was seen in families from the Philippines, and Guatemala, and case-controls from Brazil. The -986T allele abolished six overlapping potential transcription regulatory motifs. Transfection assays of PDGF-C promoter reporter constructs show that the -986T allele is associated with a significant decrease (up to 80%) of PDGF-C gene promoter activity. This functional polymorphism acting on a susceptible genetic background may represent a component of human CL/P etiology.

Figure 1

Multipoint linkage analysis results for chromosome 4q in multiplex Chinese families. Depicted are the multipoint heterogeneity LOD scores. α=the estimated proportion of linked families at the peak. The black bar across cytogenetic band 4q32.1 denotes the map position of the PDGF-C locus. The vertical gray bar denotes the 1-LOD interval. Chromosomal marker locations and cytogenetic correlations are according to the Ensemble Database. The asterisk denotes the location of D4S192 (142 Mb from 4pter) reported to be significant in previous linkage and association^, reports for CL/P in Caucasian and Chilean populations.

Figure 2

Genomic structure of human PDGF-C promoter and schematic diagrams of −986C and −986T allele human PDGF-C promoter Luciferase reporter constructs. Eight-kb human PDGF-C promoter DNA double-digested with EcoRV (partial digestion) and SacI and 4 kb human PDGF-C promoter DNA double-digested with EcoRV (from the multiple cloning site of pBSKSII vector) and SacI were cloned into SmaI and SacI site of pGL2 enhancer reporter vector.

Figure 3

A C>T SNP (rs28999109) at −986 in the human PDGF-C promoter regulatory region abolishes consensus binding motifs for Sp1 (Specific protein 1); USF (upstream stimulatory factor), WT1 (Wilms tumor zinc-finger protein-1), EGR-1 (early growth response factor-1), HEB (human B-HLH factor), and ETF (epidermal growth factor receptor (EGFR)-specific transcription factor) (M=A,C; S=C,G; K=G,T; Y=C,T; R=A,G; W=A,T; N=A,C,G,T).

Figure 4

Presence of the −986T SNP (rs28999109) in the human PDGF-C promoter reduces promoter activity. Human PDGF-C Luciferase reporter constructs (2 μg) containing either the −986C or the −986T allele were cotransfected with the TK-Renilla Luciferase construct (0.2 μg) into mouse osteoblastic precursor cells (MC3T3) (a, b), mouse myoblastic cells (C2C12) (c, d), and human embryonic kidney epithelial cells (HEK293) (e, f) using Lipofectamine Plus kits. Relative activities of the 4 kb −986T allele PDGF-C promoter construct are significantly decreased (>50%) compared with that of the 4 kb −986C allele PDGF-C promoter construct in all three cell lines (a, c, e). Relative activities of 8 kb −986T allele PDGF-C promoter construct are also significantly decreased (>40%) compared with those of 8 kb −986C PDGF-C promoter construct in these cell lines (b, d, f) (^*P<0.05).

Figure 5

Effects of −1116 G>C SNP on PDGF-C promoter activity. Presence of the −986T SNP in the human PDGF-C promoter reduces promoter activity regardless of genotype at the −1116 position. Four haplotypes (−1116G −986C; −1116C −986C; −1116G −986T; −1116C −986T) were generated using the 4 kb human PDGF-C promoter reporter construct. The presence of the −986T SNP resulted in a significant decrease the PDGF-C promoter activity in MC3T3 cell line (a), 293 cell line (b), and C2C12 cell line (c) regardless of the −1116 genotype. However, the −986 T allele did not affect PDGF-C promoter activity in HeLa cell line (d) (^*P<0.05).