published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Melanoma is a malignant type of skin cancer arising from the combined effects of genetic alteration and extrinsic signaling, resulting in transformation of neural crest (NC)-derived melanocytes into metastatic melanoma. Current therapies against metastatic melanoma are merely effective with less than 5% 5-year survival rate of patients. Understanding the underlying molecular mechanism of how melanoma acquires metastatic behavior could formulate strategies for new therapeutic options. Features of metastatic melanoma resemble NC cells undergoing an epithelial-mesenchymal transition (EMT) suggesting similar regulators might be in place to control the process. Our previous studies showed that SoxE transcription factors (Sox8/9/10) play a crucial role in NC development, in particular Sox9 transactivates expression of Snail2 and co-operates with it to induce features of EMT. To examine the role of SOXE proteins in melanoma development and whether they regulate SNAIL expression, we first investigated the expression profile of SOXE and SNAIL in a human melanoma tissue array. The data showed that SOX8, SOX10, and SNAIL genes are highly expressed in metastatic melanoma whereas SOX9 and SNAIL2 transcript levels are low. Moreover, SNAIL transcript level was shown to have a positive correlation with SOX8 and SOX10 expression levels. SNAIL is well-known to be the key regulator of tumor invasiveness in various cancers. Our data raised the possibility that SOXE proteins may also regulate SNAIL expression in initiating melanoma metastatic behavior. The human metastatic melanoma cell line A375 exhibits similar SOXE and SNAIL expression profiles as the tissue array. Knockdown of SNAIL in A375 reduced its migratory ability and in vivo tumorigenecity, suggesting that SNAIL plays a crucial role in melanoma metastasis. How SNAIL transcription is regulated in melanoma has been poorly understood. Previous studies have identified a minimal enhancer region downstream of the SNAIL locus which contains YY1 and SOX consensus binding sequences. Chromatin immunoprecipitation assay revealed that SOX8 and SOX10 proteins could bind to the SNAIL 3’ minimal enhancer region specifically. Mutation of the SOX consensus binding sequence reduced the enhancer activity while mutations in both SOX and YY1 binding sites resulted in further reduction suggesting that YY1 and SOX protein binding is required and important for enhancer activity and SNAIL transcription. These findings provide a molecular basis to examine further whether metastasis of melanoma is regulated by SOXE proteins in which one of the potential mechanisms could act through regulation of SNAIL expression. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major. The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale. This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL. / published_or_final_version / Pathology / Master / Master of Medical Sciences

The thalassaemias are the commonest monogenic disorders in the world population. They occur at a particularly high frequency in Mediterranean regions and Southeast Asia, which cause a massive public health problem. In Hong Kong, the prevalence of heterozygous carriers of α or β thalassaemia mutations is approximately 8% [1]. Thalassaemia is characterized by the reduced synthesis of one or more normal globin chains. This causes globin chain imbalance and finally leads to hypochromic microcytic anaemia. Different types of thalassaemia are named according to the under-produced chains. The majority of thalassaemia can be diagnosed by basic haematologic profiles and simple phenotypic techniques. However, in some cases of thalassaemia the diagnosis are not apparent after routine laboratory investigations. To arrive at a diagnosis which is important for antenatal diagnosis and genetic counseling, it is necessary to use molecular approaches. In this study, 25 patients with microcytosis, normal phenotypic haemoglobin study results and without iron deficiency were analyzed retrospectively. This cohort of patients was suspected to have occult or masked thalassaemia. DNA was extracted from archive samples and further investigated by alpha multiplex gap polymerase chain reaction (α multiplex gap-PCR), alpha amplification refractory mutation system (α ARMS) and direct nucleotide sequencing of globin genes for the detection of possible underlying globin gene mutations. Results indicated that 60% of these cases with microcytosis were occult and silent αthalassaemia caused by deletional or non-deletional mutations. Maskedβthalassaemia due to co-existing δ thalassaemia or variants or normal Hb A2 β thalassaemia due to mild β globin gene mutations were not detected in the cohort. Forty percent of these cases of microcytosis remained unexplained, which await further molecular testing. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Waardenburg-Shah syndrome (WS4, MIM_277580) is a congenital developmental disorder characterized by pigmentary abnormalities of the skin, eyes and hair, sensorineural deafness and intestinal aganglionosis (HSCR; Hirschsprung disease). Mutations in the coding regions of EDN3, EDNRB, or SOX10 account for 65-85% of the WS4 patients. These mutations are not fully penetrant, contributing to the phenotypic variability of WS4. We screened these genes in a three-generation family (14 individuals; three members affected with HSCR only and one affected with “partial’ WS4 –iris heterochromia and HSCR-). A novel heterozygous missense mutation was identified in EDNRB. EDNRB encodes the EDNRB receptor, which is essential for the differentiation of the neural crest cells into melanocytes, enteric ganglia and Schwann cells. The mutation was present in four affected and three unaffected family members. In the EDNRB isoforms 1 and 2, the mutation results in the replacement of the translation initiation codon methionine (Met) with a valine (Val) and such replacement (M1V) would theoretically abolish the use of the translation initiation codon. However, in EDNRB isoform 3, the replacement is at Met91 (M91V) and is predicted benign. Since different EDNRB transcripts are expressed concomitantly in the still developing newborn's gut, we theorized that the intra-familial variability of the phenotype could be related to the expression ratio between benign and damaging isoforms. We examined the consequences of M1V or M91V in their respective isoforms. Constructs containing either wild-type cDNA of isoform 1 and 3 or their mutated counterpart were transiently transfected into Human Embryonic Kidney 293 cell (HEK293). Confocal and immunoblot experiments showed that EDNRB M1V generated a shortened protein (starting from Met46); the wild-type-EDNRB isoform 3 or its mutant (p.M91V) were only found in the cytosol. Although EDNRB M1V was able to generate a shorter protein, the later failed to translocate onto the cell membrane, theoretically, affecting signal-transduction. Isoform 3 did not seem to play a role as cellular receptor. We also identified a c.-248G/A rare change at the 5’-untranslated region (5’UTR) of EDN3 (EDNRB ligand) which was predicted to affect translation efficiency. The presence of this variant in affected individuals but not in healthy carriers of the EDNRB mutation, suggests that both variants are necessary for the disease manifestation. Variations within the disease phenotype may be due to each individual’s genetic background. To identify other susceptibility loci, we carried out whole-genome linkage scan in this family using a high density SNP assay. Merlin software was used for parametric and non-parametric linkage. A susceptibility locus on chromosome 4q13.3-q24 was identified by both nonparametric and parametric linkage analyses, with LOD scores of 1.204 and 1.7109 respectively. Haplotype analysis refined the region to a 27.76 cM interval, in which genes involved in neuron development reside. To conclude, the novel EDNRB M1V mutation in this family may lead to HSCR and/or WS4 when in conjunction with other genetic lesions, such as the EDN3 5’UTR rare variation and/or a not yet identified susceptibility locus on chromosome 4q13.3-q24. / published_or_final_version / Surgery / Master / Master of Philosophy