Two sisters of Malaysian-Chinese origin were diagnosed with CH during infancy. The proband, III-2, was detected to have CH by neonatal screening and the diagnosis was confirmed at 10 days after birth with serum TSH of 120.0 muIU/mL (Normal range: 0.3-5.0 muIU/mL) and free T4 of 5.0 pmol/L (Normal range: 20-68 pmol/L). Her elder sister, III-1, was also detected to have CH (but not by neonatal TSH screening as it was not available), at 3 weeks of life with serum TSH of 96.5 muIU/mL and free T4 of 2.0 pmol/L when she presented with prolonged jaundice. Both sisters received thyroid replacement with L-thyroxine. Technetium-99m thyroid scintigraphy was performed when they were 3 years old when L-thyroxine was temporarily stopped for 6 weeks and the thyroid function test was repeated. The thyroid scan revealed the presence of thyroid glands despite having a rise in TSH and low free T4. In their late teenage years, they developed goiter. Thyroid ultrasonography revealed that both of them had multinodular goiter (MNG). Both sisters had elevated levels of thyroglobulin as the goiters progressed. III-2's thyroglobulin (hTG) level measured when she was 20 years old was 193 ng/mL (reference range 0-55 ng/mL), whilst her elder sister's was much higher at 2288 ng/mL. The proband and her sister (III-1) subsequently underwent total thyroidectomy at the age of 20 years and 24.5 years, respectively. Histology of their thyroid glands revealed the presence of multiple macro- and microfollicular adenomata arising in a background of dyshormonogenetic goiter. The elder sister had a small ventricular septal defect, while the proband had no other congenital anomalies and both of them had normal growth and development. Their parents (II-1 and II-2) who are biologically unrelated were all healthy and euthyroid with no congenital anomalies. However, their maternal grandmother (I-4) was reported to have goiter and had subsequently undergone thyroidectomy with the histology results unknown. Written informed consent was obtained from the parents for their blood and their children's (III-1 and III-2) blood and thyroid tissues for TPO gene analysis. This study was approved by the University of Malaya Medical Centre (UMMC) Ethical Committee (Institutional Review Board) in accordance with the ICH-GCP guideline and the Declaration of Helsinki (Reference number, 654.16).
Genomic DNA was extracted from peripheral venous blood from the proband, the affected sister, and their healthy family members using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. The TPO gene was PCR-amplified with flanking intronic primers covering all the 17 exons. A PCR reaction mixture (50 muL) containing 100-250 ng was prepared in the presence of a final concentration of 1X Taq buffer with KCl, 2.5 mM of MgCl, 200 muM of dNTP, 8% dimethyl sulfoxide (DMSO), 1 unit of Taq DNA polymerase (Fermentas, USA), and 20 pmol of each forward and reverse primers. Thirty-five cycles of amplification were carried out with standard PCR protocol at annealing temperature of 55 C for all coding exons for 30 seconds. The PCR products were purified using QIAquick PCR purification kit (Qiagen, Germany) and then sequenced using ABI Prism Gene Sequencer, Model 3100, Version 3.7 (Research Biolabs, Singapore).
The Human Splicing Finder (http://www.umd.be/HSF/) was used to analyze the existence of alternative splice sites in exon 13 of the TPO gene as a consequence of the c.2268dup mutation. The analysis was done on the whole of exon 13 (171 bp), 50 nucleotides upstream of exon 13 (3' end of intron 12), and 50 nucleotides downstream of exon 13 (5' end of intron 13).
Following thyroidectomy, tissue samples were immediately kept frozen in dry ice and then stored at -80 C. A piece of the pathologically normal tissue was taken near the colloid nodules and the abnormal tissue was taken from the thyroid lesion. Tumors were diagnosed histopathologically and no evidence of malignancy was detected. Each piece of tissue sample of about 30 mg was homogenized and total cellular RNA (tcRNA) was then extracted from the normal and lesionic areas of the thyroid tissues using RNeasy Mini (Qiagen, Germany) according to the manufacturer's instruction. The extracted tcRNA was treated with DNase (Ambion, UK) and then reverse transcribed to complimentary DNA (cDNA) using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, USA). The cDNA was then used as a template to PCR-amplify the coding exons (exons 2-17) using overlapping primers that cover the exon-exon junctions (see Supplementary Table 1 (S1) in the Supplementary Material available online at http://dx.doi.org/10.1155/2014/370538). PCR amplification and the subsequent PCR product purification and sequencing were performed as described earlier except that cDNA was used as a template instead of genomic DNA. Complementary DNA samples prepared from thyroid tissue taken from three individuals without the c.2268dup mutation were used as positive controls.
Quantitative real time PCR (qRT-PCR) was used to analyze TPO gene expression in the normal and lesion areas of the thyroid tissues of the proband and her sister. Comparison of the TPO gene expression in thyroid tissues of the proband and her sister was also made. The expression of the TPO gene was normalized to that of a housekeeping gene, the TATA box-binding protein (TBP). In addition, the ratio of TPO mRNA variants in c.2268dup mutation-free individuals was also calculated and compared to the two sisters. Total TPO mRNA transcripts were analyzed using E4F and E5R primer pair covering a region from exons 4 to 5. Primer pairs spanning exons 12 to 13 (E12/13F and E13R) were designed to target normally spliced transcripts, (TPO1, TPO2, TPO3, TPO4, TPO5, TPO2/3, and TPO2/4). Meanwhile, the E12/I12F and E13R primer pair was used to target alternatively spliced-derived transcripts with an insertion of 34 bp between exon 12 and exon 13. TPO6 that does not contain exon 12 and exon 13 was amplified using E11 and E11/15 primers. All qRT-PCR oligonucleotide sequences are listed in Supplementary Table 2 (S2). The qRT-PCR experiments were performed on a StepOnePlus system (Perkin-Elmer/Applied Biosystems). The amplification reaction was carried out using 1X SYBR Green PCR Mastermix (Applied Biosystems, USA), containing 10 ng of cDNA and 200 nM of each primer. The sample was initially denatured at 95 C for 5 min followed by 40 cycles of denaturation at 95 C for 15 sec and subsequent annealing and elongation step at 59 C for 1 min. Melting curves were performed to check the specificity of the amplicons. Each experiment was carried out in triplicate. Comparison of total or target gene mRNA expression was done by calculating the differences in the threshold cycles.
The fold change in expression between tissue samples was calculated by  where DeltaDeltaCt = (Ct  targeted  TPO  mRNA - Ct  TBP)  lesionic  area - (Ct  targeted  TPO  mRNA - Ct  TBP) normal area.
The fold change in expression between patients was calculated by  where DeltaDeltaCt = (Ct  targeted  TPO  mRNA - Ct  TBP)  proband - (Ct  targeted  TPO  mRNA - Ct  TBP) proband's sister.
Total 10 ng of cDNA sample from normal and lesionic areas of thyroid tissue (5 ng template from each area) of each patient was used as the template.
Meanwhile, the ratio of TPO mRNA variants to total TPO mRNA in each tissue types was determined by the following calculation.
For normal area of thyroid tissue,  For lesionic area of thyroid tissue,  
Microsomal protein was isolated from the frozen thyroid tissue specimens. Thirty milligram of tissue was homogenized in ice-cold phosphate buffered saline (PBS) containing Pierce Protease Inhibitors Cocktail (Thermo Scientific, USA) and subsequently centrifuged for 15 min at 3,000 xg at 4 C to remove nuclei and cytoskeletal components. The supernatant was then ultracentrifuged in the Optima L-100k (Beckman Coulter, USA) at 100,000 xg for 2 h at 4 C. The pellet was collected and then dissolved in lysis buffer (50 mM Tris-HCl, pH 7.3; 150 mM NaCl; 5 mM EDTA; 10% glycerol (w/v); 1% Triton X-100 (w/v), protease inhibitors). The mixture was ultrasonicated using Power Sonic 405 (Hwashin Technology, Korea) to improve the yield of the extracted protein.
Immunodetection analysis was performed using mouse monoclonal MoAb47 primary antibody (Abcam, UK) and WesternDot 625 Western blot kits (Invitrogen, USA) to detect the presence of TPO protein in the tissue specimens from both patients. Twenty mug of microsomal fraction extract for each sample and 300 ng of a positive control (recombinant fragment of human thyroid peroxidase from Abcam, product code: ab73765, UK) were used in this analysis. The expression of TPO in normal and lesion areas of thyroid tissue from both patients were quantitated by densitometry scanning using Image-J software (http://rsbweb.nih.gov/ij/). The expression of TPO in normal and lesionic areas of the thyroid tissues was normalized to the expression of beta actin. The total expression of TPO protein in the proband was also compared to that of her sister.
Microsomal fraction extracts prepared from patients' thyroid gland tissues were immediately used for enzymatic assay. Microsomes prepared from thyroid tissues from rats were extracted using the same method and were then used as a positive control. Twenty mug of each microsomal fraction were added to 1 mL of reaction mixture containing 40 mM guaiacol (Sigma-Aldrich, USA) and 67 mM sodium phosphate buffer, pH 7.5. The enzymatic reaction was initiated by adding final concentration of 0.25 mM H2O2. The absorbance was measured at 470 nm (OD470) at every second for 3 min at room temperature.