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Functional characterisation of mutations in the SRRM2 gene associated with risk of Non-Syndromic familial Non-Medullary thyroid cancer

Main, Nathan (2017) Functional characterisation of mutations in the SRRM2 gene associated with risk of Non-Syndromic familial Non-Medullary thyroid cancer. Honours thesis, Murdoch University.

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Non-syndromic familial non-medullary thyroid cancer (FNMTC) is the most common malignancy of the endocrine system and is defined by the presence of non-medullary thyroid cancer (NMTC) in 2 or more first-degree relatives in the absence of predisposing environmental factors and a recognized thyroid cancer syndrome. There is a clear genetic basis to FNMTC, however, details of the genes involved is limited; a number of susceptibility genes have been proposed but these remain controversial.

SRRM2 is a splicing factor that promotes exonic enhancer-dependent splicing by mediating critical interactions between the spliceosomal U2 snRNP and additional splicing factors bound directly to pre-mRNA. SRRM2 is also associated with cell cycle control, where has been shown to associate with the histone H4 subtype transcriptional regulator HiNF-P and is hypothesized that the SRRM2/ HiNF-P complex may regulate histone production via a p220NPAT- dependent pathway.

SRRM2 was previously identified as a putative susceptibility loci for FNMTC, where a missense variant (S346F) was found to co-segregate with FNMTC in a well-documented FNMTC family. RNA-seq of leukocytes revealed altered splicing patterns for 1,642 exons in the FNMTC patients. The altered splicing pattern for 7 exons was experimentally verified using semi-quantitative PCR. In addition to the S346F mutation, 6 SRRM2 variants have been identified in the Western Australian FNMTC cohort: one variant co-segregated with FNMTC and was predicted by in silico methods to be pathogenic (R1805W), and the others of unknown significance.

The first aim of this study was to generate model cell systems of SRRM2 mutations previously associated with FNMTC predisposition. The second aim was to investigate whether these mutant cell systems show differences in the splicing patterns of specific pre-mRNAs through quantitative real-time PCR. The final aim was to employ flow cytometry techniques to assess the proliferative characteristics of the mutant cell systems and determine whether these mutations prevent proper regulation of the cell cycle.

The original project plan was to generate the model cell systems by performing a transient over-expression analysis in HEK293 and Nthy-ori 3-1 cell lines transfected vector constructs containing the wild type or mutant SRRM2 sequences. However, during the construction of the mutant plasmids, the transient over-expression analysis was abandoned due to challenges associated with the size of the SRRM2 ORF. The CRISPR/Cas9 system was used instead to generate model cell systems of SRRM2 mutations in the HAP1 cell line. CRISPR/Cas9 was successfully implemented to generate a mutant HAP1 cell line harboring the R1805W mutation, however, the cell line was heterozygous for the mutation and the wild type sequence.

RT-qPCR was performed to characterise the splicing pattern of the wild type and mutant cell lines to determine whether there was a difference in splicing for 7 exons previously shown to be differentially spliced in FNMTC patients heterozygous for the S346F mutation. No significant differences were identified between the wild type and mutant cell lines, suggesting that the R1805W mutation does not alter the splicing pattern of the 7 exons investigated.

Cell cycle analysis was performed to quantify the percentages of cells in the G0/G1 and G2/M phases of the cell cycle using propidium iodide staining and quantification of G0/G1 and G2/M peak fluorescence intensity. The experiments were completed successfully in this regard; however, no significant differences were observed between the wild type and mutant cell lines. The proliferation kinetics of the two cell lines were characterised with a proliferation assay using carboxyfluorescein succinimidyl ester staining and the dye dilution method, however, no significant differences were observed in the proliferation metrics quantified.

Whilst preliminary, these findings suggest that the R1805W mutation does not predispose to FNMTC through the altered splicing of the 7 exons investigated, or by altering proper regulation of the cell cycle. Further investigations are to properly interrogate the association of this this mutation to FNMTC, as well as the other mutation

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
United Nations SDGs: Goal 3: Good Health and Well-Being
Supervisor(s): Wilson, S., Ward, B., Walsh, J. and Greene, Wayne
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