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 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.

Mutation Spectrum in TPO Gene of Bangladeshi Patients with Thyroid Dyshormonogenesis and Analysis of the Effects of Different Mutations on the Structural Features and Functions of TPO Protein through In Silico Approach.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Newborn screening in Bangladesh.

Newborn screening started in Bangladesh in 1999. The programme started as part of a regional project of the International Atomic Energy Agency (IAEA) to screen for congenital hypothyroidism (CH). In the beginning the IAEA helped the country with equipment, filter papers, reagents, training and expert services. Since 1999, 2 pilot projects to screen newborns for CH were completed. Under these projects some 30,000 newborns were screened and 16 were identified with hypothyroidism. The government of Bangladesh approved a national project in July 2006 to screen newborns in some selected areas of the country for CH. Under the project some 200,000 newborns will be screened and laboratory facilities for newborn screening will be increased. Bangladesh has a large population of about 140 million. With the current birth rate some 2 million new births take place every year. The socio-economic situation of the country is also different. Per capita income of the country is one of the lowest in the world. About 85% of babies are still delivered at home. As such newborn screening is a big challenge for Bangladesh. However, the country is trying to overcome these challenges.

Newborn screening: experience of Bangladesh.

Newborn screening for early diagnosis of many preventable diseases is already an established program in developed countries as well as in some countries in Asia. With the control of infectious and communicable diseases, the Bangladesh government is now giving more attention to the preventive aspect of health. The country is now preparing to adopt newborn screening as part of the preventive aspect of health. Among the various activities are laboratory set up, expert visits, pilot studies on CH and training of personnel. A national project to screen newborns for congenital hypothyroidism is now under active consideration. A universal newborn screening program is a highly ambitious project for a country like Bangladesh. Funds to run such a program will be the main problem. The social pattern where still more than 80% of deliveries are done at home is another constraint. However, the enthusiasm of the professionals as well as of the government is a very encouraging thing. Now it needs the support of the international communities.

Screening for congenital hypothyroidism--a new era in Bangladesh.

Neonatal screening is a new concept in Bangladesh. Currently a pilot program is ongoing where newborns are screened for congenital hypothyroidism (CH). In a country like Bangladesh, where people are still fighting childhood problems like malnutrition, diarrhea and other communicable diseases with a high infant and child mortality rate, instituting a newborn screening program is a challenge. TSH is measured on filter paper using IRMA methods. Both cord and heel prick blood are used. A cut off value of 20 mIU/l is used above which all babies are recalled for serum T4 evaluation. Approximately 2600 newborns have been screened in the pilot program. Of these, 2 newborns were detected with CH (incidence rate 1:1300). Since it is a new program in Bangladesh, effort is mainly given on popularizing the program and building necessary infra-structure. The newborn screening program has been accepted with mixed reaction in the community. Hopefully, the government is developing a positive attitude towards the program. The paediatricians and obstetricians are also interested. The program needs more publicity. Patronization by the government and other international bodies will be essential for continuation of the program.