| Author: | Maruf, Md Abdullah Al |
| Title: | Association study and functional characterization of germline polymorphisms in myeloproliferative neoplasms |
| Advisors: | Yip, Shea Ping (HTI) Huang, Chien Ling (HTI) |
| Degree: | Ph.D. |
| Year: | 2024 |
| Subject: | Bone marrow -- Tumors Myeloproliferative disorders Single nucleotide polymorphisms Medical genetics Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Health Technology and Informatics |
| Pages: | xxv, 346 pages : color illustrations |
| Language: | English |
| Abstract: | Myeloproliferative neoplasms (MPN) are a group of hematological malignancies proliferating clonally and driven mainly by somatic mutations in the JAK2, CALR or MPL genes. However, the involvement of germline polymorphisms is not well characterized. Several germline variations at the JAK2 gene and the TERT genes have been found to be associated with MPN in populations of different ethnic origins. Previous studies have revealed that rs2736100, rs2853677 and rs7705526 of the TERT gene exhibit strong association with MPN. Besides, some other germline variants of the TET2, SH2B3, GFI1B, and LINC-PINT genes showed significant effect on MPN. The aim of this study was to identify functional SNPs in the TERT, SH2B3, GFI1B, and LINC-PINT genes in MPN in addition to establishing a unique optimization protocol for closed-tube and open-tube unlabeled probe melting analysis (UPMA) for SNP genotyping. In the first part of my study, we developed a unique optimization protocol that can be used in both closed-and open-tube formats for genotyping single-nucleotide polymorphisms (SNPs). Fluorescence-based nucleic acid detection built upon polymerase chain reaction (PCR) usually utilizes oligonucleotide probes labeled with fluorescent dyes. However, high-resolution melting analysis (HRM) of amplicons by unlabeled probe in a closed-tube format has been used for SNP genotyping. We have also applied the same method but in open-tube format for SNP genotyping. Extensive optimization is usually required for both formats for any combination of primers and unlabeled probe before an optimal combination of reaction components can be found. Therefore, we have developed a unique systematic optimization protocol wherein it is possible to find the best SNP genotyping condition from among 16 tested conditions (1:5 and 1:10 primer ratio, 0.2 µM and 0.3 µM primer concentration, 2.5 mM and 3.5 mM MgCl2, Tm and Tm - 5 annealing temperature). In the second part of my study, we genotyped 14 TERT, 6 SH2B3, 18 GFI1, B and 41 LINC-PINT germline variants in 192 Hong Kong Chinese MPN patients and 480 ethnically matched healthy control subjects by open-tube UPMA technique and iPLEX MassARRAY. Association analysis for 4 and 6 TERT SNPs under an allelic model showed significant association (P < 0.05 after correction for multiple comparisons) with MPN and JAK2V617F-positive MPN respectively. However, we have not found any significant association between the germline variants of other genes and MPN. In the third part of the study, a meta-analysis of ten potentially relevant studies was also performed after comprehensive literature search in four electronic databases (PubMed, Scopus, EMBASE and Google Scholar) to elucidate the association between a TERT SNP and MPN by increasing statistical power. Together with sensitivity analysis, heterogeneity test, cumulative meta-analysis and bias assessment, odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to assess the overall association between rs2736100 and MPN risk in a total of 3875 cases and 274993 controls. The results indicated that the rs2736100 polymorphism was associated with an increased MPN risk under five genetic models: allelic model (C vs A [reference allele]; OR: 1.55; 95% CI: 1.46–1.64; P < 1× 10-10); homozygous model (CC vs AA [reference genotype]; OR: 2.49; 95% CI: 2.20–2.82; P < 1 × 10-10); heterozygous model (CA vs AA; OR: 1.79; 95% CI: 1.60–2.01; P < 1 × 10-10); dominant model (CC+CA vs AA; OR: 2.02; 95% CI: 1.82–2.26; P < 1 × 10-10); and recessive model (CC vs CA+AA); OR: 1.67; 95% CI: 1.44–1.93; P < 1 × 10-10). In conclusion, we have demonstrated and concluded that TERT rs2736100_C is a predisposing factor for MPN in Hong Kong Chinese population and the polymorphism confers an increased risk of developing MPN. In the fourth part of the study, a high-throughput massively parallel reporter assay (MPRA) was performed to screen putative functional germline variants of MPN. The study investigated the functional activity of 615 germline variants (SNPs and indels) in JAK2 and TERT gene comprising lead SNPs from published articles and our study, and SNPs in strong linkage disequilibrium. The screening process identified 3 novel SNPs in K562 cell line. In conclusion, the overall study has explored to identify potentially associated SNPs in MPN and their functional activity on myeloid cell lines. |
| Rights: | All rights reserved |
| Access: | open access |
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