Functional Assessment of Clubfoot Associated HOXA9, TPM1, and TPM2 Variants Suggests a Potential Gene Regulation Mechanism

Abstract

Background: Isolated nonsyndromic clubfoot is a common birth defect affecting 135,000 newborns worldwide each year. Although treatment has improved, substantial long-term morbidity persists. Genetic causes have been implicated in family-based studies but the genetic changes have eluded identification. Previously, using a candidate gene approach in our family-based dataset, we identified associations between clubfoot and four single nucleotide polymorphisms (SNPs) located in potential regulatory regions of genes involved in muscle development and patterning (HOXA9) and muscle function (TPM1 and TPM2) were identified.

Questions/purposes: Four SNPs, rs3801776/HOXA9, rs4075583/TPM1, rs2025126/TPM2, and rs2145925/TPM2, located in potential regulatory regions, were evaluated to determine whether they altered promoter activity.

Methods: Electrophoretic mobility shift assays were performed on these four SNPs to identify allele-specific DNA-protein interactions. SNPs showing differential banding patterns were assessed for effect on promoter activity by luciferase assay. Undifferentiated (for HOXA9) and differentiated (for TPM1 and TPM2) mouse cells were used in functional assays as a proxy for the in vivo developmental stage.

Results: Functional analyses showed that the ancestral alleles of rs3801776/HOXA9, rs4075583/TPM1, and rs2025126/TPM2 and the alternate allele of rs2145925/TPM2 created allele-specific nuclear protein interactions and caused higher promoter activity. Interestingly, while rs4075583/TPM1 showed an allele-specific nuclear protein interaction, an effect on promoter activity was observed only when rs4075583/TPM1 was expressed in the 1.7kb haplotype construct.

Conclusion: Our results show that associated promoter variants in HOXA9, TPM1, and TPM2, alter promoter expression suggesting that they have a functional role. Moreover and importantly, we show that alterations in promoter activity may be observed only in the context of the genomic architecture. Therefore, future studies focusing on proteins binding to these regulatory SNPs may provide important key insights into gene regulation in clubfoot.

Clinical relevance: Identifying the genetic risk signature for clubfoot is important to provide accurate genetic counseling for at-risk families, for development of prevention programs and new treatments.

Fig. 1A–D

Electrophoretic mobility shift assays were performed to evaluate whether the associated single nucleotide polymorphisms (SNPs) effected DNA-protein interactions. The ancestral G allele of (A) rs3801776/HOXA9 and (B) rs4075583/TPM1created DNA-protein interactions that were eliminated when the alternate A allele was present. The (C) rs2025126/TPM2 ancestral C allele created two DNA-protein interactions that were eliminated when the alternate T allele was present. The (D) rs2145925/TPM2 alternate C allele created a DNA-protein interaction that was eliminated with the ancestral T allele. The allele-specific DNA-protein interactions were confirmed through competitive assays using corresponding unlabeled probes in excess of 5×, 10× or 50×. The arrow indicates allele-specific DNA-protein complex.

Fig. 2A–D

Luciferase assays were performed to assess the effect of the DNA-protein interactions on TPM2 promoter activity for rs2025126/TPM2 and rs2145925/TPM2. The TPM2 promoter construct incorporated 460-bps upstream of the transcriptional start site and was ligated into the pGL4.10 basic luciferase vector. The ancestral and alternate allele constructs were generated by ligating the electrophoretic mobility shift assay double-stranded oligonucleotides that contained (A) rs2025126 or (B) rs2145925 in front of the TPM2 promoter construct. (C) The ancestral allele of rs2025126 that creates a DNA-protein interation caused a decrease in promoter activity. (D) The alternate allele of rs2145925 creating the DNA-protein interaction causes an increase in promoter activity.

Fig. 3A–D

Luciferase assays were performed to assess the effect of the DNA-protein interactions on TPM1 promoter activity for rs4075583 and HOXA9 promoter activity for rs3801776. (A) The HOXA9 promoter construct containing the rs3801776 is shown. (B) The rs4075583/TPM1 construct contained 500-bps upstream of the transcriptional start site for the TPM1 skeletal muscle isoform ligated into the pGL4.10 basic luciferase vector. (C) The ancestral allele-specific DNA-protein interaction for rs3801776 increased promoter activity in undifferentiated mouse muscle cells. (D) The ancestral G allele and alternate TPM1 constructs were generated by ligating the electrophoretic mobility shift assay double-stranded oligonucleotides in front of the TPM1 promoter construct (as shown in Illustration C). Although the ancestral allele for rs4075583/TPM1 creates a DNA-protein interaction (as shown in Illustration B), it did not significantly affect promoter activity in differentiated mouse muscle cells.

Fig. 4A–B

Luciferase assays were performed to evaluate whether specific TPM1 haplotypes affected TPM1 promoter activity. The 1774 bp TPM1 region containing rs4075583/TPM1 has been shown to influence expression depending on cell type and haplotype. Based in that finding, (A) constructs were designed to incorporate the four most common TPM1 haplotypes and the TPM1 promoter region. (B) The four common haplotypes produced varying degrees of promoter activity in differentiated mouse muscle cells with the least promoter activity found with haplotype 3, which contains the alternate A allele of rs40755863 which eliminates the DNA-protein interaction observed with the ancestral G allele.