The Study On Mutation In Congenital Hypothyroidism

Congenital hypothyroidism (CH) is the most prevalent endocrine disorder in the newborn and affects 1 in 3000-4000 newborns. CH may cause severe and irreversible neurologic and developmental abnormalities when not recognized early. Many millions of newborns have now been screened and many thousands of patients with CH have been identified. Screening for CH is a major achievement of paediatrics because early diagnosis and treatment have resulted in normal development in almost all cases.The molecular cause of CH in the majority of newborns is unknown. However, in some patients the molecular basis of CH has recently been clarified. In patients with CH and a normally developed thyroid gland, the sodium iodide symporter (NIS) gene has been identified. The autosomal recessive inheritance of loss-of-function mutations of the thyroid stimulating hormone receptor (TSHR) has been identified in the patients with defective thyroid development.Objective: To investigate the mutations of the NIS and TSHR genes in the patients with CH in Tianjin area and the characteristic so inherited. Clarification of the molecular defects of thyroid development will help to explain the differences in outcome in patients with CH and to develop new diagnostic and therapeutic strategies to ensure adequate counselling and care for these patients.Methods: The total of 18 patients (11 males and 7 females ranging in age from 2 months to 22 years, mean age, 11.9+7.4 years) with confirmed diagnosis of CH, no sign of autoimmunity, were recalled in Oct. 2003. At screening TSH values in these children ranged between 44.4-175.6 mU/L, T₄ values between 0.4-30.0μg/L. Neck ultrasound, and (99)^Tc neck scan indicated that 10 of 18 patients presented thyroid gland absent, 4 with hypogenetic thyroid, 3 with enlarged thyroid while 1 with ectopic gland.The normal control group from 35 healthy individuals (18 males and 17 females ranging in age from 6 to 44 years, mean age, 26.1 + 10.6 years) randomly selected had never any kind of thyroid disease. Twelve members (6 males and 6 females ranging in age from 17 to 75 years, mean age, 52.0±8.6 years) in the family with the proband were enrolled in the study. Neck ultrasound and serum thyroid hormone measurement were detected. Antithyroperoxidase,anti-Tg, and anti-TSHR autoantibodies were evaluated by commercial kits.Total genomic DNA was extracted using TKM method from peripheral blood. Firstly, All 15 exons of NIS gene and exons 1,4,6 of TSHR gene were individually amplified by polymerase chain reaction (PCR). And the whole length of exon 10 of TSHR gene devided into 5 overlapping fragments were amplified by PCR as previously described. PCR products obtained above were subjected to single-strand conformational polymorphism (SSCP) to detect mutations and confirmed with direct sequencing. All purified PCR fragments were directly sequenced with either forward or reverse primers using the CEQ? DTCS-Quick Start kit and resolved by capillary electrophoresis on CEQ8000 Automated Sequencer (Beckman Coulter,USA). To identified inheritance of mutations in the family with proband. The relatives of a proband were enregistered and investigated.The SSCP analysis and sequences of the mutated fragment were repeated at least twice from two PCR products.Results: None mutation was identified in NIS gene. Two PCR samples in a single patient with hypoplasic gland showing electrophoretic pattern variations were further characterized by direct sequencing. Normal controls had no variations. Two variations were detected in codon 450 and codon 727, G—*A variation leading to Arg—?His in codon 450 and C—>G variation leading to Asp—?Glu in codon 727. The patient was compuond homozygous mutations for Arg450His ( CGC—?CAC, R450H) and Asp727Glu (GAC^GAG, D727E).Homozygous mutation of R450H in the second transmembrane spanning region of the TSHR was previously never described, rendering it unresponsive to TSH. To date homozygous mutation of D727E, in the C-terminal region of the intracellular domain previously described as polymorphism, had not found in patients with CH.This child, who had a gestational 36 wk and a birth weight 3 500 g, was found to have abnormal thyroid function (basal TSH, 46.7 mU/L; TT4,12 jxg/L) ,with no identified cause. Neck ultrasound and 99Tc neck scan indicated hypogenetic thyroid. Six of his 12 relatives were compound heterozygous for R450H and D727E mutations. Autoimmune thyroid disease was excluded in 11 of 12 members on the basis of clinical and biochemical parameters. Serum thyroid hormone levels of all the relatives were normal except for basal sensitive TSH (5.96-6.92hiU/L) slightly elevated in five heterozygies diagnosed as subclinical hypotyroidism. Five...