LATS Young Investigator Awards

Helton Estrela Ramos

Winner 2007


Thyroid Hypoplasia (TH) can be associated with several genetic defects, including mutations in the TSH receptor (TSHr), the PAX8 gene, the Gsα-subunit and TSHβ-subunit. The numerous cohorts of patients with Congenital Hypothyroidism (CH) studied so far have not been collected as a continuous series and, therefore, the real prevalence of different pathogenetic causes in sporadic TH is still not well defined. We have studied a continuous series of patients with CH with in situ thyroid gland and a volume ranging from normal to hypoplasic. We attempted to dissect the individual phenotypes in these patients, using a population-based approach, in an entire population screened for CH over a 16-year period. In these patients we searched for mutations in some of the most likely candidate genes: TSHr and PAX8.

Materials and Methods:
Patients: From 1990 to 2006 we screened 2.546,112 newborns from state of Parana (Brazil) by blood spot TSH measurement. 644 patients with confirmed diagnosis of CH were found and 566 are followed in our institution. Three hundred fifty three of 566 patients had the thyroid phenotype precisely defined by ultrasound and scanning. Among them, 251 patients had Thyroid Dysgenesis (TD) with Ectopy (n=133) or Athyreosis (n=82) or Hypoplasia (n=35) or Hemiagenesis (n=3). For molecular analyses we focused our attention on 35 patients with TH (26 females and 9 males). 35 patients with thyroid in situ having normal volume were included in the study. The thyroid phenotype was evaluated by history, review of medical records, physical examination, TSH measurements performed at diagnosis (TSHd) and scanning (TSHs), Thyroid Scanning and Thyroid Ultrasound. Thyroid Scanning was performed within 30 days off-therapy with levothyroxin by the age of 2 or 3 years-old. The entire PAX8 coding region and promoter were examined for mutations of genomic DNA extracted from peripheral blood lymphocytes. Each of 10 exons and promoter were amplified by PCR with a pair of primers. Individual TSHr exons were amplified and the Exon 10 was subdivided in three overlapping amplimers. PCR products were sequenced directly on ABI3100 genetic analyzer. Fifty Brazilian normal individuals were used as controls. TT4 and TSH were measured by competitive immunoassays using chemoluminescent technology. Results are expressed as mean ± SD. Data were analysed by nonparametric tests ( Mann-Whitney U-test and Kruskal-Wallis test as appropriate) for comparison between groups.

Over the period 1990-2006, 644 cases of CH were identified, giving a birth prevalence of 1 in 3953 live births. TH was comparable to Athyreosis in their inicial TSH at diagnosis, but TSHd was greater (319±47 mU/L vs 199±16 mU/L; p≤0,005) than in Ectopy. By the time of scanning, TSHs was lower (172±30 mU/L vs 247±21; p≤0,05) than in Athyreosis. There were no differences between TH and Ectopy at this time. PAX8 gene A heterozygous G>C substitution in position -569 was observed on promoter region of four patients with TH. A heterozygous A to G change at position +43 (IVS5+43A>G) was found in intron 5 of six patients with TH. Another abnormality, a G to C substitution at position +49 ( IVS6+49G>C) was observed in intron 6 of seven patients with TH. A novel heterozygous mutation in exon 3 was found in a girl with TH. It was a G>C substitution at position 155 and changed the second nucleotide of codon 52 ( Arg52Pro). The mutation leads to a lost of Bstu1 restriction site allowing independent confirmation. Genotyping of members of the family showed no mutation but the father’s DNA was not available. TSHr gene A heterozygous nonsynonymous polymorphysm in exon 1 was found in a patient with TH. It was a C>A substitution at position 234 and changed the first nucleotide of codon 52 (Pro52Thr). None mutation was found in patients with thyroid in situ and normal volume.

Estimates of the prevalence of CH in countries with screening programs are in the range of 1:3000-1:4000. Our data fall in this range, with TD being the most common aetiology. Patients with HT were as hypothyroid as Athyreosis at diagnosis but had significantly less severe form of hypothyroidism (as demonstrated by hyperthyrotropinemia at time of scanning). Only recently have mutations in thyroid-specific genes been causally associated to CH. However, information about the real frequency of these defective alleles in the population remains elusive. As an attempt to fill this gap, we have chosen to use a population-based approach. During the last 16 years, our group has identified a cohort of patients with CH who are representative of an entire population. In this study we have analyzed some of the most likely candidate genes to play important roles in CH with TH. TH is a well-known but rare congenital anomaly. Inactivating mutations in the TSHr and PAX8 genes have been found in patients with CH and TH. Two of our patients with a global TH and low iodine uptake at scanning were found to have these deffect. We found a novel mutation in PAX8 (Arg52Pro) localized in the α-helix 2 of the paired domain. Mutations on this site might affect folding and conformation of C-terminal regions of PAX8, including regions known to be crucial for transactivation activity (1). Only few PAX8 mutations have been previously found in a very large panel of patients, thus suggesting this is an infrequent event. Also in the case of TSHr mutations previously identified only in familiar groups of CH, the patients selection may have played an important role because we analyzed patients selected from an entire population, mostly sporadic cases. The C → A polymorphism leading to a Pro → Thr variation in codon 52 was previously detected in other study, in which was also found in normal subjects in a frequency of 0.062 (2). However this SNP is still particularly interesting and worthy of further investigation. No anomaly was found in the remaining patients. This might imply that other genes involved in the TSH-TSHr-GSα cascade could be affected.

1. Thyroid Transcription Factor 1 Rescues PAX8/p300 Synergism Impaired by a Natural PAX8 Paired Domain Mutation with Dominant Negative Activity. Molecular Endocrinology, 19(7):1779-1791, 2006. 2. Genetic of Specific Phenotypes of Congenital Hypothyroidism: A Population-Based Approach. THYROID, 12 (11): 945-951, 2002.