High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.
TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed...
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Public Library of Science (PLoS)
2024-01-01
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| author | Mst Noorjahan Begum Rumana Mahtarin Md Tarikul Islam Nusrat Jahan Antora Suprovath Kumar Sarker Nusrat Sultana Abu A Sajib Abul B M M K Islam Hurjahan Banu M A Hasanat Kohinoor Jahan Shyamaly Suraiya Begum Tasnia Kawsar Konika Shahinur Haque Mizanul Hasan Sadia Sultana Taufiqur Rahman Bhuiyan Kaiissar Mannoor Firdausi Qadri Sharif Akhteruzzaman |
| author_facet | Mst Noorjahan Begum Rumana Mahtarin Md Tarikul Islam Nusrat Jahan Antora Suprovath Kumar Sarker Nusrat Sultana Abu A Sajib Abul B M M K Islam Hurjahan Banu M A Hasanat Kohinoor Jahan Shyamaly Suraiya Begum Tasnia Kawsar Konika Shahinur Haque Mizanul Hasan Sadia Sultana Taufiqur Rahman Bhuiyan Kaiissar Mannoor Firdausi Qadri Sharif Akhteruzzaman |
| author_sort | Mst Noorjahan Begum |
| collection | DOAJ |
| description | TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 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 variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C 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. |
| format | Article |
| id | doaj-art-93fb1922f2634d33bc8200e38edbf08d |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-93fb1922f2634d33bc8200e38edbf08d2025-08-20T03:32:04ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01194e029357010.1371/journal.pone.0293570High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.Mst Noorjahan BegumRumana MahtarinMd Tarikul IslamNusrat Jahan AntoraSuprovath Kumar SarkerNusrat SultanaAbu A SajibAbul B M M K IslamHurjahan BanuM A HasanatKohinoor Jahan ShyamalySuraiya BegumTasnia Kawsar KonikaShahinur HaqueMizanul HasanSadia SultanaTaufiqur Rahman BhuiyanKaiissar MannoorFirdausi QadriSharif AkhteruzzamanTPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 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 variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0293570&type=printable |
| spellingShingle | Mst Noorjahan Begum Rumana Mahtarin Md Tarikul Islam Nusrat Jahan Antora Suprovath Kumar Sarker Nusrat Sultana Abu A Sajib Abul B M M K Islam Hurjahan Banu M A Hasanat Kohinoor Jahan Shyamaly Suraiya Begum Tasnia Kawsar Konika Shahinur Haque Mizanul Hasan Sadia Sultana Taufiqur Rahman Bhuiyan Kaiissar Mannoor Firdausi Qadri Sharif Akhteruzzaman High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. PLoS ONE |
| title | High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. |
| title_full | High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. |
| title_fullStr | High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. |
| title_full_unstemmed | High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. |
| title_short | High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh. |
| title_sort | high resolution melt curve analysis an approach for variant detection in the tpo gene of congenital hypothyroid patients in bangladesh |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0293570&type=printable |
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