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This dissertation, Genotypic and Phenotypic Analysis of the Thiopurine S-methyltransferase (TPMT) Gene With Clinical Correlation by Siu-ping, Cheung, 張小屏, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Immunosuppressants (such as azathioprine and 6‐mercaptopurine) are widely used in the management of patients having rheumatic diseases, inflammatory bowel diseases, hematological malignancies and organ transplant rejections. However, the adverse effects and effectiveness of these drugs are dependent on the metabolism by the enzyme, thiopurine S‐methyltransferase (TPMT), inside the body and the activity of this enzyme is determined by its genetic polymorphism. This study mainly focused on four known mutations, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C which were detected by three sets of primers, G238C, G460A and A719G targeting exons 5, 7 & 10 of the TPMT gene.Patient blood was collected from patients who had a clinical need of knowing the TPMT level (n=202). The TPMT phenotypic status of patient was determined by measuring the enzyme activity of red blood cell lysates by Enzyme‐Linked Immunosorbent Assay (ELISA) commercially available. On the other hand, the genotype was reflected by the sequencing results generated after DNA extraction from whole blood, followed by amplification, purification and DNA sequencing by the targeted primers. The majority of patients (92%) showed normal to high TPMT enzyme activity level (>17 U) and the remaining 8% was under the category of borderline activity (between 7 U to 17 U). None of them had low or deficient activity. The mean TPMT enzyme activity of all samples was 22.9 U ranging from 7.8 U to 54.1 U. No observable difference was found between male and female. The largest group of patients was having rheumatic diseases, with enzyme activity levels from 7.8 U to 54.1 U (mean of 22.8 U) which was very close to the overall findings. Also, there was no direct relationship between the lowest white blood cell count and the TPMT activity of each patient. Low white blood cell counts were not usually associated with lower TPMT enzyme activity. From the DNA sequencing results, 62.5% of the samples (n=104) had no genetic abnormalities found, 31.7% were found to have a heterozygous allele C/T and G/A at position 474 which was known to be a silent mutation with no amino acid alteration and hence was not functionally defective. Only 4.8% had heterozygous allele A/G and T/C at position 719 and one sample was found to have heterozygous allele at both positions 719 and 474. There was no significant difference in the TPMT enzymatic activity between the samples with genetic abnormalities and those without genetic abnormalities (means of TPMT enzymatic activity were 17.3 U and 21.5 U respectively, p=0.13). And also, no apparent correlation was found among the TPMT enzymatic levels, the genetic abnormalities and the disease groups. In conclusion, the individual differences in the TPMT enzyme activity were resulted from the allelic variation at the TPMT locus, it was important to fully understand the allelic variation at the TPMT gene locus. The ghenotypic analysis could be extended to the detection of all the ten exons including their spice‐site junctions and 5' flanking promoter region of the TPMT gene by PCR single strand conformation polymorphism in future studies. DOI: 10.5353/th_b5091305 Subjects: Methyltransferases

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