Molecular analysis of PAX8 gene in unrelated patients with congenital hypothyroidism
Y Phaik-Har, F Harun, S Mat Junit
bbvci polymorphism, congenital hypothyroidism, pax8 gene, pcr-sscp
Y Phaik-Har, F Harun, S Mat Junit. Molecular analysis of PAX8 gene in unrelated patients with congenital hypothyroidism. The Internet Journal of Endocrinology. 2006 Volume 3 Number 2.
The present study was carried out to screen for potential mutations or polymorphisms in the human PAX8 gene, which may be associated with congenital hypothyroidism (CH). The study cohort consists of 44 unrelated patients with CH. PCR-based analysis of exons 2 to 9 of the PAX8 gene was performed followed by non-radioactive Single Strand Conformation Polymorphism (SSCP) detection. Two novel polymorphisms were detected and confirmed by DNA sequencing: a C>G transversion in intron 5 (Cint51G) and a phenotypically silent polymorphism in exon 9 (C372T). When analysed using Workbench web software, the C>G change creates a new restriction site, BbvCI whereas the C>T change in exon 9 creates a new BstNI restriction site. Both the BbvCI and the BstNI polymorphisms neither create nor destroy a splice site, verified using SpliceView web software at the WEBGENE page. Since both polymorphisms can be screened by restriction endonuclease digestion, normal healthy subjects were screened for the polymorphisms to obtain population frequencies. Restriction analysis performed on 133 healthy individuals revealed that a total of 25 were heterozygous and 1 was homozygous for the BbvCI polymorphism in exon 5 of the PAX 8 gene. On the other hand, BstNI polymorphism in exon 9 of the PAX8 gene was not detected in 105 healthy individuals screened. Our PCR-SSCP results did not detect any novel mutation on the PAX8 gene suggesting that mutation in PAX8 gene is not the cause of CH phenotypes in this cohort of patients.
Primary congenital hypothyroidism (CH) occurs in babies who are born without the ability to produce adequate amount of thyroid hormone, due to disorders of the thyroid gland development . CH is linked to several genetic defects including those in
Materials And Methods
Subjects, collection of samples and DNA extraction
Blood samples were obtained from 44 unrelated patients with CH, confirmed biochemically with low T4 and high TSH levels in blood, who attended the Paediatric Clinic at the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. Thyroid scanning and Technetium-99m scintigraphy revealed that 31 patients have thyroid gland of normal size and position whereas 13 patients have either no thyroid gland or thyroid gland of either abnormal in size or position. Genomic DNA was extracted from peripheral white blood cells from each individual using the Qiagen kit (Hilden, Germany) according to the manufacturer's instructions. Samples obtained were with consent and approval granted by the Ethical Committee (Institutional Review Board) of the UMMC in accordance to the ICH GCP guideline and the Declaration of Helsinki. All samples were kept at -20oC and subjected to similar treatment.
Mutation screening of the
Complete coding region of the
SSCP analysis was carried out using MDETM gels and electrophoresis was performed in the Protean II electrophoresis cell system (BioRad, USA) with a temperature-regulated water circulator. For each PCR fragment, SSCP optimisation was carried out by repeating the electrophoresis using 2 different electrophoresis temperatures (10oC and 20oC) and at 2 different gel concentrations (0.5x and 0.75x).
DNA Sequencing Analysis
Following PCR-SSCP screening, PCR fragments showing variations in SSCP banding patterns (mobility shifts) were sent for DNA sequencing. Prior to sequencing, PCR products were purified using commercially available purification kit, QIAquick (Qiagen, Germany). DNA sequencing was carried out using an automated ABI Prism Gene Sequencer (Model 377, version 2.1.1) at AMCAL, University of Malaya, Kuala Lumpur, Malaysia.
BbvC1 and BstNI PCR-RFLP assay
The sequence change identified creates a new
PCR-SSCP analysis of
Figure 1 PCR-SSCP analysis showing mobility shifts for exon 5/intron 5 (A) and exon 9 (B) of
Figure 2 Sequence analysis of
The position of the substituted nucleotide is indicated by an arrow.
Subsequent DNA sequencing analysis of exon 9 revealed that the mobility shift was due to a C>T transition at the third base of codon 372 (C372T). The C>T change creates a restriction site for
In humans, heterozygous loss-of-function mutations of
To date, 6
In addition, 1 silent mutation in
In conclusion, our PCR-SSCP results did not detect any mutation involving point mutation or minor deletion on the
This research was financially supported by IRPA grant no. 36-02-03-6005 from the Ministry of Science, Technology and the Environment of Malaysia (MOSTE) and the University of Malaya short-term research grant, Vote F0171/2003B.