Dataset Information

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

ABSTRACT: 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.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.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.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.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.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.

SUBMITTER: Weymouth KS

PROVIDER: S-EPMC4887369 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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

Weymouth Katelyn S KS Blanton Susan H SH Powell Tamar T Patel Chandrashekhar V CV Savill Stuart A SA Hecht Jacqueline T JT

Clinical orthopaedics and related research 20160328 7

<h4>Background</h4>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 regu ...[more]

PMID: 27020427

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Project description:Adenylylsulfate reductase (adenosine 5'-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-A resolution. Different from the alpha(2)beta(2)-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six alphabeta-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the alpha-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the alpha-helix (amino acid 289 to 299) in the alpha-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the beta-subunit wraps around the alpha-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the alpha-subunit from another alphabeta-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the alpha-subunit and Asp159 in the C terminus of the beta-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the beta-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the beta-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the beta-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the beta-subunit.

Dataset Information

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

Publications

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

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