ABSTRACT
Objective To investigate the features and characteristics of GLIS3 gene mutation in patients with congenital hypothyroidism(CH),and to establish the theoretical basis for gene diagnosis and prenatal diagnosis of CH.Methods Genomic DNA was extracted from peripheral blood leukocytes of 50 patients with CH who were collected from February 2007 to November 2016 in Shandong Province.The exon 2 to 11 of GLIS3 were amplified with 11 pairs of sequence specific primers designed by Primer 5.0.Polymerase chain reaction and the first generation of sequencing method(Sanger sequencing) were used to detect the mutation.Comparison of the sequencing results with the GLIS3 reference sequence (National Center for Biotechnology Information Reference Sequence:NC_000009.12) helped to screen gene mutations.Results The 50 CH patients included 22 boys and 28 girls,and the sex ratio was 1.0 ∶ 1.3.The mean age was (2.5 ± 0.5) years.Six cases (12％) had thyroid gland hypoplasia,23 cases (46％) had thyroid gland agenesis and 21 cases(42％) with ectopic thyroid gland.C2507A missense mutation was found in exon 10 of GLIS3 in a thyroid gland agenesis case,which might result in proline to glutamine substitution at codon 836.One mutant (rs780019691,c.C289T) was detected which was nonsense mutation (Arg→Stop) in another thyroid gland agenesis child.Conclusions The mutation rate of GLIS3 gene is very low in CH children of Shandong province.Further studies are needed to investigate the relationship between GLIS3 genotypes and clinical phenotypes.
ABSTRACT
Objective To screen the dual oxidase maturation factor 1 (DUOXA1) gene mutations in children with congenital hypothyroidism (CH) and thyroid goiter from Shandong Province,China,and to identify the gene mutation type and characteristics of DUOXA1 gene mutations in order to provide some evidence for gene diagnosis and therapy of CH.Methods A cohort of 52 cases of CH with thyroid goiter and 100 normal controls were selected according to neonatal screening system in Shandong Province whose genomic DNA was isolated from peripheral blood leukocytes with a standard phenol chloroform method.The whole coding sequence (CDS) of DUOXA1 gene was amplified with 8 pairs of sequence specific primers by using PCR.The PCR products were directly sequenced with Sanger sequencing to detect new mutations types of DUOXA1 gene.The sequencing data were compared to the DUOXA1 gene reference sequence(National Center for Biotechnology Information:RefSeq:NG_033105.1) to see if there was any mutation.Ax2 test was done for the gene frequency of discovered single nucleotide polymorphisms (SNP).Results There was no mutation in CDS of 52 CH patients with thyroid goiter and 100 normal controls.However,a SNP (rs75981505,c.398G ＞ T) which was an missense mutation and could lead to a change of the codon from CGC to CTC,was found in 9 CH patients with thyroid goiter and 11 normal controls in the exon 7.The corresponding amino acid arginine was replaced by histidine(p.Arg133His).There was no significant difference in the SNP rate between CH patients with thyroid goiter and normal controls (17.3％ vs 11.0％,x2 =1.24,P ＞ 0.05).Conclusion DUOXA1 gene mutation rate is very low which may not be the main cause of CH patients with thyroid goiter in the population of Shandong Province.
ABSTRACT
Objective To study the thyroid hormone receptor β(TRβ)gene mutation types and characteristics in children with congenital hypothyroidism(CH)and thyroid dysgenesis(TD)from Shandong Province,and to provide theoretical basis for gene diagnosis and prenatal diagnosis. Methods Sixty cases of TD patients of which genomic DNA were isolated from peripheral blood leukocytes were selected by neonatal screening system in Shandong Province. The exon 6 to 12 of TRβ gene were amplified with 8 pairs of sequence specific primers using PCR and the first generation of sequencing method(Sanger method)to detect mutation. The sequencing results were compared with the TRβ gene reference sequence[National Center for Biotechnology Information(NCBI)Reference Sequence:NC 000003. 12]to see whether there was a mutation. Results Analysis of TRβ in 60 cases of CH patients with TD revealed no mutation was demonstrated in exons 6 - 12,but 2 single nucleotide polymorphism(SNP)( rs 3752874,c. 735C ﹥ T;rs79220627, c. 162G ﹥ A)were detected. Through the analysis,the 2 SNP were all synonymous mutations(Phe→Phe;Ser→Ser), without the change of the amino acids. Conclusions TRβ mutation rate is very low,which may not be the main mutation type in CH patients with TD in Shandong Province.