High-Resolution Melt Curve Analysis: An Approach for Mutation Detection in the TPO Gene of Congenital Hypothyroid Patients in Bangladesh

Thyroid Peroxidase (TPO) is known to be the major gene involved in Congenital hypothyroid patients with thyroid dyshormonogenesis. This present study aimed to establish high-resolution melting (HRM) curve analysis as a supplementary mutation detection approach of Sanger sequencing targeting commonly found mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in Bangladeshi patients. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood samples were collected, and genomic DNA was isolated for molecular techniques. Among the 36 specimens, 20 were pre-sequenced, and mutations were characterized through Sanger sequencing. The pre-sequenced specimens (n=20) were then subjected to real-time PCR-HRM curve analysis to get the appropriate HRM condition capable of differentiating heterozygous and homozygous states for the three mutations from the wild-type state. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method showed 100 percent sensitivity and specificity to distinguish wild-type alleles from homozygous or heterozygous states (c.1117G>T, c.1193G>C, and c.2173A>C) of alleles commonly found in Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene mutation. In conclusion, the established HRM-based molecular technique targeting c.1117G>T, c.1193G>C, and c.2173A>C mutations could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Ethical Review Board for Human Studies of BSMMU and the Human Participants Committee, University of Dhaka (CP-4029) on 16 May 2017. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All relevant data are within the manuscript and its Supporting Information files.