Genetic Spectrum and Metabolic Profiling of Congenital Hypothyroid Children in Hospital Care Settings in Bangladesh

Abstract

Congenital Hypothyroidism (CH) is the deficiency of thyroid hormones at birth and a major cause of mental retardation if left untreated. The frequency of CH is much higher in Bangladesh than that of the global incidences and late-diagnosis is a common scenario. There is limited data on genetic etiology of Congenital Hypothyroidism in Bangladesh and patients diagnosed at late-stage suffer from clinical complications even under treatment with levothyroxine drug. The present study aimed to investigate the genetic spectrum and metabolic profiling of Congenital Hypothyroid children of Bangladesh in order to correlate the clinical complications of CH with the blood acylcarnitines and amino acids levels. A total of 65 confirmed cases of Congenital Hypothyroid children under follow-up treatment were enrolled in this study. Among them 44 participants with thyroid Dyshormonogenesis were considered for TPO gene analysis; and 21 cases with thyroid Dysgenesis were studied for TSHR and PAX8 gene. Moreover, 107 healthy subjects were enrolled for metabolic profiling. Molecular analysis was performed using PCR followed by Sanger sequencing of TPO, TSHR and PAX8 genes. The effect of mutations on the 3D structure of the TPO monomer and dimer structures along with TSHR proteins were analyzed by Molecular Docking and Molecular Dynamics Simulations using Autodock vina/PyRx protocol and YASARA computational software. Finally, High Resolution Melt (HRM) curve analysis method was set up to detect the common mutations found in the TPO gene. For metabolic profiling, blood acylcarnitines and amino acids levels of patients were compared with healthy controls using LC-MS/MS technique. Analysis of TPO gene identified 12 mutations. In almost all patients, 4 mutations belonging to Exon 8 and Exon 12 (hotspot region) were commonly detected. Analysis of the effect of the three non-synonymous mutations c.1117G>T (p.Ala373Ser); c.1193G>C (p.Ser398Thr); and c.2173A>C (p.Thr725Pro) on the 3D structure of TPO protein revealed alterations in the binding affinities of the ligands (Heme, H2O2 and I-) for the wild type cases compared to mutant cases. A HRM (High Resolution Melt) curve analysis method was established to detect these 3 mutations in homozygous, heterozygous and wild type states. Analysis of TSHR gene on the other hand, found a mutation c.1523C>T (p.Ser508Leu) in one patient and 20 patients had c.2181G>C (p.Asp727Glu) mutation in Exon 10 of the gene. The effect of these two mutations on 3D structure of small molecules drug (MS437 and MS438) binding site was investigated and we found that the binding affinities of the small molecules were decreased in case of mutant TSHR protein compared to wild type TSHR protein. No mutation was detected in PAX8 gene. Profiling of acylcarnitines showed that the mean concentration of long chain acylcarnitines was significantly lower in the patients group than that of the Healthy Controls (HCs) group (with a P=0.0014). Also, the indicator ratio of β-oxidation rate, namely C0/(C16+C18) was significantly higher in the patient group than that of the HC group (P<0.0001), suggesting a low activity of CPT-I enzyme in the patients. Furthermore, the mean plasma TG level of the patients was significantly higher than that of the HC group (P=0.02) suggesting that the patients might have been suffering from fatty liver. Moreover, in terms of the analyzed amino acids profile, 9 out of 15 amino acids were significantly decreased in the patients compared to the healthy controls (P<0.0008). Serum albumin levels were also significantly (P<0.0001) lower in the patients group compared to the HCs, suggesting that there might be a decreased rate in overall protein metabolism. This study is the first of its kind that demonstrated the genetic basis of Congenital Hypothyroidism in Bangladeshi patients. The correlation between mutation and functional activity of specific proteins were also investigated using several in silico tools. In conclusion, metabolic profiling combined with mutation analysis might help to better understand the disease severity and to modify the treatment strategy of congenital hypothyroid children in Bangladesh.