published_or_final_version / Surgery / Master / Master of Research in Medicine

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Heat stress inhibits spermatogenesis partly by inducing apoptosis in the testicular germ cells. Using a cryptochid rat model, we identified a temperature-related ESTs 4 (TRS4) transcript from rat testis. Trs4 mRNA is specifically expressed in the mouse and rat testis from postnatal day 21 and 28 days onwards, respectively. Trs4 protein is located mainly in the elongating spermatids and mature spermatozoa at the acrosome and tail regions. Using a yeat-2-hybrid screening, Trs4 was found to bind Gstmu1, Rslh-2 and Ddc8 proteins. To further characterize the functional role of Trs4 in spermatogenesis, and study how Trs4 interacts its binding proteins for cellular functions, we aimed (1) to screen putative ES cells with Trs4 floxed allele for knockout mice generation, (2) to generate Trs4 deletion constructs and study the cellular localization of Trs4 and its putative binding partners in transfected spermatocyte GC-2spd(s) cell line, (3) to study how heat-treatment regulates the expression of Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, with genetic factors playing critical roles in its pathogenesis. Mutations in APP, PSEN1 and PSEN2 genes are confirmed to be causative risk factors for early-onset Alzheimer’s disease (EOAD). For late-onset Alzheimer’s disease (LOAD), growing evidence suggests it is caused by multiple genetic risk factors in corporation with the environmental exposures. Although, so far, APOE is the most well recognized common genetic risk factor for LOAD, other susceptible candidate genes, such as CR1, CLU and PICALM, have recently been identified in Caucasians using genome-wide association approach. In order to have a better understanding on the genetic components of LOAD in Chinese as well as identify other potential genetic risk factors for Chinese ethnic population, we conducted a case-control study using candidate gene association approach. In view of increasing evidence on the neural protective effects of sex steroid hormones both in vivo and in vitro, we hypothesized variations on sex steroid metabolic pathway genes were associated with LOAD. Four candidate genes (ESR1, ESR2, CYP19A1, CYP11A1) were evaluated based on 462 cases and 350 non-demented controls. Apart from consistent result for APOE, polymorphisms in ESR2 and CYP11A1 were found to be significantly associated with the disease. When stratification according to gender, marginally significant associations were detected for ESR1 and ESR2 variants in men while CYP11A1 variants relevant to LOAD risk were detected exclusively in women. Additionally, genotypic and phenotypic correlation analysis revealed CYP19A1 was significantly relevant to serum 17-estradiol (E2) levels in 689 subgroup participants, especially in 400 LOAD patients of subgroup. Further gene-level analyses based on whole sample confirmed above disease association for ESR2 and CYP11A1 and pathway-level analyses highlighted the impact of sex steroid metabolic pathway on disease predisposition. The independent follow-up study for CR1, CLU and PICALM previously reported by genome-wide association study (GWAS) in Caucasians was conducted in the same Chinese cohort. Similar to the Caucasian cohort, polymorphisms in CR1 and CLU were found to be significantly different between cases and non-demented controls. However, significant disease association for PICAML was detected only in the APOE ε4 (-) subgroup of our Chinese cohort. In conclusions, genetic abnormalities were founded in Chinese LOAD patients. In addition to confirmation disease susceptibility for APOE, CR1, CLU and PICALM, we were first to report the associations between several sex steroid metabolic pathway genes and LOAD. This valuable genetic information obtained from Chinese patients may lead to the development of novel diagnostic strategies and therapeutic interventions in LOAD. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer which is well-known for its high heterogenicity and metastatic potential. Despite of the current advancement in surgical resection and the availability of targeted therapy, HCC remains a barely curable and fatal disease. We previously demonstrated that deregulation of epigenetic regulators is a common event in human HCC. Herein, we identified the frequent up-regulation of the prototype of H3K9 tri-methyltransferase SUV39H1 in clinical HCCs. SUV39H1 over-expression was also significantly associated with increased Ki67 expression and the presence of venous invasion. By using both SUV39H1 over-expression and knockdown model, we consistently demonstrated that SUV39H1 contributed to HCC tumor growth and migration. Most importantly, SUV39H1 knockdown drastically suppressed in vivo tumorigenicity and extra-hepatic metastasis of HCC cells in nude mice model. These findings evidently demonstrated the oncogenic role of SUV39H1 in HCC and implied potential therapeutic targeting of SUV39H1 for HCC treatment. Molecularly, SUV39H1 knockdown HCC cell underwent morphological changes and accompanied with increased lysosomal β-galactosidase activity and elevated p21 protein and γH2AX level. This data suggested senescence induction in SUV39H1 knockdown HCC cells. SUV39H1 has been implicated in telomere regulation and transcriptional control. However, neither telomere length nor expression of tumor suppressor genes was altered in SUV39H1 knockdown HCC cells. Interestingly, we demonstrated a novel observation that SUV39H1 may potentially methylate non-histone substrates that are yet to be identified, which may contribute to the pro-tumorigenic function of SUV39H1 in HCC. We also investigated the upstream regulation of SUV39H1 and identified miR-125b as the negative post-transcriptional regulator of SUV39H1. Ectopic expression of miR-125b abolished SUV39H1 3’UTR-coupled luciferase activity and suppressed endogenous SUV39H1 at both mRNA and protein level. Clinically, miR-125b level was found inversely correlated with SUV39H1 expression. We have previously reported the frequent under-expression of miR-125b in HCC. Collectively, our data suggested that SUV39H1 up-regulation in HCC may be the sequential outcome of miR-125b down-regulation. In conclusion, we demonstrated for the first time that SUV39H1 up-regulation contributed to HCC development and metastasis, potentially via senescence evasion. SUV39H1 elevation in HCC was attributed to the loss of its negative regulator, the tumor suppressive miR-125b. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Breast cancer is the most common cancer in females worldwide. RIP140 was one of the first proteins recognized as nuclear receptor transcriptional cofactor which interacts with several nuclear receptors. RIP140 plays a central role in metabolic tissues with multifunctional co-regulation. It is an essential protein required for energy homeostasis and mammary gland development. RIP140 has been found to be involved in development of breast cancer in response to estrogen. RIP140 is recruited by estrogen receptors in the presence of estrogen. Increasing levels of estrogen and RIP140 stimulate their transcription and regulate proliferation and differentiation of mammary glands. We hypothesize that RIP140 may be over expressed in breast cancer and may be correlated with clinicopathological features and may thus serve as a possible new prognostic marker in breast cancer. In our study, the correlation between the RIP140 expression and survival was investigated by immunohistochemistry (IHC), and analyzed by Pearson’s chi-square and Kaplan Meier analysis. Cox regression analysis was performed to examine the relationship between clinic-pathological parameters and the survival. Total of one hundred and eighteen breast cancer samples were examined for the RIP140 staining localization in breast cancer cells. Our results showed that the IHC staining of RIP140 was observed in both cytoplasm and nucleus of breast cancer cells. The ER positive staining was significantly correlated with high nuclear expression of RIP140, but not RIP140 cytoplasmic expression. Thus nuclear RIP140 expression was examined for correlation with other clinic-pathological features and patient survival. The correlation between nuclear RIP140 expression and clinic-pathological features by Pearson’s chi-square test showed that high RIP140 nuclear staining score is associated with ER positive status (p-value=0.041) and tumor stage (p-value=0.008). Kaplan Meier test shown that nuclear RIP140 expression is not significant associated with either overall survival or disease-specific survival. However, a trend of high nuclear RIP140 score was observed with poorer overall and disease-specific survival though not statistically significant. To conclude, our results suggest RIP140 is not a useful prognostic marker for breast cancer. Further investigation with larger sample size is necessary to improve the statistical significance of the test. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Background: Sudden arrhythmia death syndrome (SADS) is a genotypically and phenotypically heterogeneous condition that might produce fatal ventricular arrhythmia in otherwise healthy individuals. Congenital long QT syndrome (LQTS) is the most common type of SADS with a frequency of 1 in 2500 individuals. Up to 13 genes have been shown to be associated with LQTS and genetic testing has a role in disease diagnosis, prognostication, treatment guidance, cascade testing, and reproductive counseling. Interdisciplinary care is the standard but such service is unavailable in Hong Kong. Objectives: In this study, we aim to evaluate the clinical characteristics of a local cohort of pediatric patients with LQTS, establish the practicability of a model on interdisciplinary delivery of care for SADS, and explore the process of genetic counseling in Chinese families with LQTS from the perspective of discourse analysis. Method: Pediatric patients with LQTS and their families were recruited from the Department of Paediatric Cardiology, Queen Mary Hospital between 1 January 2011 and 31 December 2012. With informed consent, patients underwent genetic testing for 6 LQTS genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2). Clinical characteristics were documented and the process of pre-test and post-test counseling was videotaped and transcribed. Data was mapped and analyzed for discourse strategies in the focal themes of uncertainty management in risk communication. Results: 19 patients were identified, 9 were male, with the corrected QT interval (QTc) ranging from 460-619ms. Mode of presentation included syncope (n=9), ventricular tachycardia (n=2), convulsion (n=1) and as incidental finding (n=7). Pathogenic mutations were identified in 9 patients (LQT1=3, LQT2=4, LQT3=1, LQT5=1), likely pathogenic mutations in 2 (LQT2), unclassified variants in 2, and no mutation in 6. Patients with pathogenic and likely pathogenic mutations had significantly longer mean QTc than those without such mutations (p=0.046). Three mutations, all in the LQT2 genes, represented novel mutations. All 3 patients with mutations in the pore-looping forming domains of the KCNH2 (LQT2) channel had personal or family histories of malignant arrhythmia or sudden cardiac death compatible with previously reported genotype-phenotype correlation. Eight families involving 18 family members underwent cascade testing, and family mutations were identified in 10 individuals from 6 families. Autosomal dominant transmission was the likely mode of inheritance in these 6 families. Counseling sessions involved the joint input from clinical geneticist, genetic counsellor and pediatric cardiologist. Discourse analysis on 2 counseling sessions of a selected family with unclassified variants revealed increased uncertainty after genetic testing in the index patient and family members. Strategies used to mitigate uncertainty included abstraction, generalization and categorization. Conclusion: Genetic testing was crucial in the comprehensive assessment of patients with congenital LQTS, and we demonstrated a feasible model to delivery interdisciplinary care for patients with SADS in Hong Kong. The process of genetic counseling is highly complex and deserves further examination. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Biliary atresia (BA) is a rare and severe cholestatic disease in neonates characterized by an idiopathic inflammatory process affecting both intra- and extra-hepatic bile ducts, causing cholestasis and ultimately leading to obliteration of the biliary tract. Through a previous genome-wide-association-study (GWAS) on Han Chinese, we discovered association of the 10q24.2 region encompassing ADD3 and XPNPEP1 gene. But disease pathogenesis and genetic architecture of BA is still obscure. We mapped the 10q24.2 association locus with 107 single nucleotide polymorphisms (SNPs) on 339 Han Chinese patients and 401 matched controls, follow-up studies of the association signals were performed. We revealed the common risk haplotype encompassing 5 tagging-SNPs, capturing the risk-predisposing alleles in 10q24.2 [logistic regression p=5.32x10-11; odds ratio, OR:2.38; confidence interval, CI:(2.14-2.62)]. No deleterious rare variants (RVs) residing on the risk haplotype were found, dismissing the theory of “synthetic” association. Moreover, the BA-associated potential regulatory SNPs correlated ADD3 gene expression (linear regression p=0.0030). Remarkably, the risk haplotype frequency coincides with BA incidences in the general population, and, positive selection (favoring the derived alleles that arose from mutations) was evident at the ADD3 locus. Our finding suggested the complexity of BA genetic architecture and role of environmental effects in the disease. We then revisited BA GWAS dataset and annotated the association signals with expression quantitative trail loci (eQTL) information available on normal adult livers. We did not see excessive enrichment of BA associated SNPs in liver eQTLs. We speculate that the liver eQTLs currently available relate to adulthood liver function and are not necessarily involved in liver development, adaptation to oxidative stress, or inflammation changes seen in BA pathophysiology. To investigate whether rare alleles can predispose to BA, we called copy number variations (CNVs) from the GWAS Affymetrix gene chip 5.0. We obtained 86 BA private CNVs distributed among 131 BA patients were compared to the CNV profile of 11,943 database samples and 846 hypertension disease samples. Assuming that pathogenic CNVs interrupt dosage-sensitive genes, we prioritized the dosage-sensitive genes and the pathogenic CNVs by integrating multiple lines of evidence. Through gene set enrichment analysis we found that the ‘core’ genes affected by BA CNVs were members of the Calcium signaling pathway, which has been involved in the pathogenesis of polycystic liver and kidney diseases. Further we initiated the survey on rare coding variants in BA through Exome sequencing 23 BA liver genomes, while patients’ blood DNA and parental DNA would be examined in the validation stage to validate de novo mutations, including somatic mosaicism in liver. We found inherited deleterious mutations in polycystic liver and kidney disease genes in BA patients, and the role of these mutations in BA pathogenesis is being investigated. Functional validation of the BA variants identified in this study is compulsory given the overall obscurity of BA pathogenesis. Together, this study presents a comprehensive catalog of both common and rare variants implicated in BA. We hope that our findings will contribute to enriching the BA-associated genetic network. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Esophageal cancer (EC), which is geographically diverse, has only a 10.7% five-year survival rate. One of the histologic forms, esophageal squamous cell carcinoma (ESCC), in Hong Kong accounts for 81.5% of the total EC cases and its five-year survival rate is only ~14%, due to its high frequency of metastasis. In our previous studies, functional complementation study of chromosome 9 defects led to the discovery of a novel tumor suppressor gene, Deleted in Esophageal Cancer 1 (DEC1), mapping to 9q32. DEC1 was shown to reduce tumorigenicity in a mouse model and its expression was shown to be associated with lymph node metastasis, early onset of ESCC, and familial ESCC development in a tissue microarray (TMA) study. Moreover, DNAJ (Hsp40) homologue subfamily B member 6 (DNAJB6), a molecular co-chaperone protein and the focus of the current study, was identified as a DEC1-interacting protein through a yeast two-hybrid screening. The interaction was further confirmed by the GST pull-down assay and co-localization studies. Using a TMA constructed with ESCC tissues from Hong Kong, the clinical relevance of DNAJB6 expression was demonstrated. In the present study, the role of DNAJB6 in ESCC was investigated using cell line-based in vivo and in vitro studies. DNAJB6 was shown to be down-regulated in ESCC cell lines. The two isoforms of DNAJB6 have distinct subcellular localizations, with DNAJB6a mainly localized to the nucleus and DNAJB6b diffused throughout the cell. Existence of a functional nuclear localization signal peptide and a functional nuclear export signal peptide was verified in DNAJB6a and DNAJB6b, respectively. In vitro evidence of possible DNAJB6a truncation was found. In vivo subcutaneous nude mice tumorigenicity assays showed that over-expression of DNAJB6a, but not DNAJB6b, suppresses tumor growth at the primary site, while DNAJB6a silencing enhances tumor growth. The suppressive effect of DNAJB6a depends on nuclear localization of the protein and the HPD tripeptide motif in the N-terminal J domain. In vitro function studies show that DNAJB6a over-expression impairs cell proliferation by suppressing G1/S transition. AKT1 phosphorylation is down-regulated in DNAJB6a over-expressed cells, leading to up-regulation of p27KIP1 protein expression and down-regulation of cyclin E1 protein expression, the G1/S transition promoter, in an AKT1-dependent manner. DNAJB6a silencing results in the opposite effect. Over-expression of DNAJB6b, but not DNAJB6a, instead suppresses lung colonization in an experimental metastasis assay, and prolongs survival of the mice. Silencing of DNAJB6a in immortalized normal esophageal epithelial cells initially induces a senescence-like phenotype with greatly reduced proliferation possibly due to oncogenic stress from up-regulation of AKT1 phosphorylation and cyclin E1 protein expression, but promotes EMT-like molecular alterations by up-regulating STAT3 phosphorylation and TWIST1 protein expression and resumes proliferation after prolonged culture. In summary, these results suggest that DNAJB6 plays a critical role in ESCC initiation, development, and metastasis and provides valuable insight into the understanding of ESCC tumorigenesis and metastasis. This suggests its usefulness as a biomarker candidate for detecting early ESCC tumor initiation. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy