TBX 5 gene mutation analysis among Tanzanian children with congenital heart diseases using high-resolution melting assays

Abstract

Early cardiac development is governed by transcription factor genes. TBX5, a T-box transcription factor gene, plays an important role in the development of the second heart field during cardiac septation by promoting cell cycle progression through the enhancement of Cdk6 and hedgehog signaling pathways. TBX5 binds to the promoter region of genes, enhancing the expression of alpha cardiac myosin heavy chain 6 (MYH6), which is a predominant isoform found in human cardiac tissue. TBX5 gene mutations are postulated to cause congenital heart diseases. A casecontrol TBX5 mutational analysis was performed to provide insight into the etiology of sporadic congenital heart diseases in our setting. We used a magnetic induction cycler (mic-PCR), which is a next-generation tool for polymerase chain reaction-high resolution melting assays, to detect mutations in children with sporadic isolated congenital heart diseases. A retrospective casecontrol study was conducted at the Jakaya Kikwete Cardiac Institute. The peripheral blood samples were collected, and DNA was extracted using the Quick-DNA Miniprep Kit. The primers were designed using Primer 3 software, validated using the program BLAST, and checked for hairpin and homo-hetero-dimerization using the IDT oligo analyzer. Real-time polymerase chain reaction (PCR)-high-resolution melting assays for screening TBX5 gene mutations were done using a magnetic induction cycler. We found two (2) TBX5 mutations in exon 5, among patients with Atrial-Ventral Septal Defects (ASVD) and Atrial-Septal Defects (ASD) and none among controls. TBX5 exon 5 is a molecular hotspot for isolated congenital heart diseases.