Medicine (RMH) - Theses
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ItemNo Preview AvailableRetinal drusen and genetics in glomerulonephritis( 2022)Glomerulonephritis comprises a heterogenous group of diseases that represent a common cause of impaired kidney function. Many are immune-mediated with antibodies directed against various self-antigens that form immune complexes and activate complement through the classical, alternative or lectin-binding pathways. Complement deposits are found in IgA, membranous and lupus nephropathy, anti-GBM disease, Focal and Segmental Glomerulosclerosis (FSGS) and fibrillary glomerulonephritis, and often correlate with more severe disease and a worse prognosis. The evidence for complement activation in glomerulonephritis includes glomerular complement deposits, low plasma complement levels in active disease, milder disease where complement is absent from the deposits, mild disease in animal models where serum complement levels are depleted, and the association with genetic complement variants in different forms of glomerular disease. There are also case series and reports of retinal drusen in C3 glomerulopathy, systemic lupus erythematosus, IgA and some other forms of glomerulonephritis. Retinal drusen are white-yellow deposits of oxidised lipids, immunoglobulins, complement and amyloid in Bruch’s membrane that are characteristic of age-related macular degeneration. The pathogenesis of drusen is well described with 37 genetic loci that affect multiple pathways including the complement system. Drusen have a similar composition to the glomerular immune deposits and their occurrence in glomerulonephritis is further evidence for complement activation in these conditions but also suggests that drusen represent a model for glomerular immune deposition and a biomarker for disease activity. Hence, studying drusen in glomerulonephritis may help identify further shared pathways in the pathogenesis of glomerular disease. The pathogenesis of immune-mediated glomerulonephritis is incompletely understood and treatment is still not optimal. Complement activation may represent a common mechanism of disease that can be targeted by novel therapies. Chapter 2 of this thesis examined 122 individuals with specialist-diagnosed IgA glomerulonephritis for retinal drusen from the renal clinics at two teaching hospitals. Drusen were counted using a grid overlay by two trained graders and more than ten central drusen in either eye was considered abnormal. Central drusen number was higher and abnormal drusen counts (>10) were more common in individuals with IgA nephropathy (9 +/- 27; 23, 19%) than the hospital controls (2 +/- 7; 8, 7%) (p<0.001, p=0.006). Drusen in IgA disease were more common with longer disease duration (p=0.03) and larger with stronger mesangial IgA staining (p=0.004). These findings suggest a shared pathogenesis between drusen and IgA glomerulonephritis. Chapter 3 of this thesis also examined drusen in other forms of glomerulonephritis some of which were associated with glomerular immune deposits and others which were not. These included membranous (n=8) and anti-glomerular basement membrane (GBM) glomerulonephritis (n=6) which are associated with antibodies against glomerular antigens that are also expressed in the retina; FSGS (n=49), which is associated with glomerular IgM and complement deposits; and ANCA-mediated vasculitis (n=15) and minimal change glomerulonephritis (n=6) where immunoglobulin and complement deposits are sparse. Individuals with glomerulonephritis and immune deposits (membranous nephropathy and anti-GBM disease) were compared with participants who had glomerulonephritis without immune deposits (pauci-immune ANCA-associated vasculitis and minimal change disease) and with healthy matched hospital controls. Mean central drusen number was 21 +/- 45 for individuals with immune deposits and four (29%) had abnormal drusen counts compared with a mean of 2 +/- 6 for those without immune deposits and one (5%) with abnormal counts (p=0.28, p=0.13). Membranous and anti-GBM glomerulonephritis are likely associated with drusen because the target glomerular antigens are also expressed in the retina. However, drusen were much less common in glomerulonephritis without immune deposits despite complement activation occurring in ANCA-associated vasculitis. Mean central drusen number was increased in FSGS (9 +/- 25) compared with hospital controls (3 +/- 8) (p=0.02). Abnormal drusen counts affected nine individuals with FSGS (19%) and four controls (8%) (p=0.23). Drusen were larger in FSGS (20, 41%; 10, 20%) (p=0.048). Abnormal drusen counts were seen in secondary (2/6, 33%), viral-associated (2/6, 33%) and genetic FSGS (3/10, 30%) and therefore were not specific to any aetiology. Previous studies have suggested that an unknown neo-epitope is exposed in FSGS following glomerular injury which, if present in the retina, might have contributed to drusen formation through IgM deposition and classical complement pathway activation. Thus, treatments targeting complement may still be beneficial for FSGS and other forms of glomerulonephritis regardless of the nature of the initial podocyte injury. Chapter 4 of this thesis investigated 8 families with IgA nephropathy for a causative pathogenic variant in a gene from one of five candidate lists: for IgA disease (n=102), the complement system (n=56), drusen pathogenesis (n=46), Alport syndrome (COL4A3–COL4A5) and FSGS (n=47). In addition, all variants were investigated for any pathogenic change that segregated with disease within the family and explained the phenotype. Chapter 4 investigated eight unrelated individuals with kidney-biopsy-proven IgA nephropathy and at least one other affected family member. Three of the index cases in these families had drusen (38%). Variants were prioritised based on the autosomal dominant (AD) inheritance model in all but one family who had X-linked disease. The index cases and other family members (median n=4, range n=1 to n=12) underwent Whole Exome Sequencing (Illumina NovaSeq platform at the Australian Genome Research Facility or Otogenetics) followed by variant calling and annotation using the Melbourne Bioinformatics pipeline using the Spartan supercomputer to identify predicted pathogenic variants. Variants were considered Pathogenic or Likely Pathogenic based on the ACMG criteria. Variants were examined in genes from the candidate lists and assessed for a CADD score >10 and a gnomAD minor allele frequency (MAF) <0.05, using the in-silico pathogenicity tools Polyphen2 (damaging), SIFT (deleterious) and MutationTaster (disease causing); for conservation in mice and birds; and for previous reports of pathogenicity in ClinVar. Of the 8 families, two had Gly substitutions in a collagen IV gene that segregated with disease (25%). These were p.Gly624Asp in COL4A5 (Family 10) and p.Gly395Glu in COL4A3 (Family 2). The p.Gly624Asp variant is a known hypomorphic variant (PP5, PM1, PM5, PP2, PP3) that results in X-linked Alport syndrome in later life. The p.Gly395Glu variant (PP5, PM1, PM2, PP2, PP3) results in a thinned GBM in AD Alport syndrome but not necessarily kidney failure. The association of mesangial IgA deposits and Alport syndrome is well-recognised and probably occurs because the thinned GBM predisposes to mesangial IgA deposition. Family 7 with three affected males had a Likely Pathogenic missense variant p.Pro437Leu (PM2, PP2, PP3, PP5) in HNF1B which segregated with the disease. This gene is associated with Renal Cysts And Diabetes syndrome suggesting that genes causing other kidney diseases may explain other familial forms of IgA glomerulonephritis. In this family two of the three affected individuals underwent retinal imaging and both had multiple retinal drusen. The index case in Family 1 had IgA glomerulonephritis and drusen. He was demonstrated to have a pathogenic nonsense variant in C9 (p.Cys54Ter) (PVS1, PP5, PM2, PP3). C9 is a late component of the complement pathway. This variant was found in four affected individuals and one unaffected family member consistent with incomplete penetrance. This variant causes partial C9 deficiency and predisposes to severe infections. C3, C4 and C9 deficiency have been described in IgA glomerulonephritis. This finding contrasts with the observation that complement activation is increased in IgA disease and suggests that the complement deficiency predisposes to more severe infection and increased IgA production. Hence, as for lupus nephritis, IgA disease in some may involve a genetic complement deficiency. These studies suggest that familial IgA nephropathy may be caused by IgA deposits which occur in 16% of the otherwise normal population being found together with another genetic kidney disease, as for the COL4A3–COL4A5 genes, or with genes that predispose to autoimmune disease, such as the Complement deficiencies. No pathogenic variants were found in the candidate list for FSGS or in the IgA list in this cohort. Chapter 5 investigated the genetic aetiology of fibrillary glomerulonephritis in a young woman from a consanguineous family with retinal drusen. The fibrils were confirmed to comprise DNAJB9 and were intermediate in diameter between those seen in renal amyloid and immunotactoid glomerulopathy. Fibrillary glomerulonephritis is typically a disease of middle-aged-elderly women who go on to develop kidney failure within two years. It is usually not familial but there are occasional reports of families where the disease occurs in successive generations and inheritance is presumed to be AD. The only gene implicated to date is MEFV in an individual who also had Familial Mediterranean Fever. The pathogenesis is presumed to be similar to that of amyloid because of the physical resemblance of their fibrils. The index case with fibrillary glomerulonephritis was investigated for a homozygous pathogenic variant that was absent from her three normal brothers. DNA was examined from all siblings and both parents. Two strategies were used to identify the pathogenic variant. One was to identify homozygous variants present only in the affected individual and not in her unaffected brothers; the second was to identify homozygous variants present in the affected individual and one other sibling, assuming the variant was of reduced penetrance. The same strategy was used as described above for IgA glomerulonephritis. The first strategy identified 31 homozygous variants but only two were Pathogenic or Likely Pathogenic. The second strategy identified seven homozygous variants but none was Pathogenic. A candidate variant was identified and may have influenced the deposition of DNAJB9 in the kidney. Thus, this thesis has described retinal drusen in different forms of glomerulonephritis. It found that drusen occurred commonly in IgA glomerulonephritis and were found more often with longer disease duration and were larger with stronger mesangial IgA staining. It also found that drusen occurred commonly in other forms of glomerulonephritis even when it appeared to be secondary to structural kidney damage (FSGS). Since drusen in macular degeneration depend on complement activation these observations suggest that treatments targeting complement may be useful in different forms of glomerulonephritis. These studies suggest that familial IgA glomerulonephritis occurs commonly because IgA is deposited in other genetic kidney diseases. The results also demonstrate overlap between the different kidney diseases and that it is not sufficient to examine a family with genetic kidney disease for variants in only one gene panel. Interestingly pathogenic variants in the complement pathway and drusen susceptibility genes did not appear to be common in these families. Finally, a new gene candidate was identified in autosomal recessive fibrillary glomerulonephritis with drusen. Future studies of drusen in glomerulonephritis should use a more sensitive method of drusen detection and correlate drusen with disease activity on kidney biopsy. Drusen may represent a biomarker for glomerulonephritis and in predicting the likelihood of kidney failure or response to treatment. Complement-targeted therapies should be investigated for efficacy for these forms of glomerulonephritis. More families with IgA glomerulonephritis should be investigated for pathogenic variants in genes previously described in genetic kidney disease. The role of COL4A3–COL4A5 variants in predisposing to IgA nephropathy needs to be determined. The association with complement deficiency, particularly C9, needs to be investigated. While complement pathway defects were not common, they may contribute to disease in a subset of individuals with IgA nephropathy. Lastly, more individuals with hereditary fibrillary glomerulonephritis need to be investigated for a genetic variant, particularly children or others with familial disease. Laboratory studies are needed to confirm the role of our gene candidate in the deposition of DNAJB9.
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ItemAntiepileptic drug teratogenicity: a human and laboratory translational study( 2018)Antiepileptic drug (AED) associated teratogenicity has been well documented in the literature. The risk of physical birth defects during the first trimester of pregnancy is increased threefold for most AEDs and over ten-fold for the most teratogenic AED, valproate. Despite this risk, women require long term treatment to stop or reduce the occurrence of seizures and the consequent harm to both mother and foetus, including the possibility of sudden unexpected death in epilepsy. The mechanism resulting in this teratogenicity, and in particular why some women are more susceptible to have children with AED induced birth defects is incompletely elucidated. In recent years there has been emerging evidence that AEDs may be interacting with genomic factors to result in birth defects. These genomic factors may be susceptibility alleles in the mother or father, de novo mutations in the child or epigenetic factors such as alterations in DNA methylation in the mother or child. The studies reported in this thesis aim to a) develop an animal model of valproate induced defects that closely mimics a human clinical setting and can be used to better understand the pathogenesis of AED induced defects, and b) identify genomic markers of AED induced defects using whole genome analysis of human samples and determining if having epilepsy is a contributing factor to the onset of these defects. For aim a) the development of the animal model entailed using an epileptic strain of rats, Genetic Absence Epilepsy Rats from Strasbourg, determining a dose at which dietary valproate is therapeutic, mating the rats and conducting a morphological assessment of both internal and external defects. For aim b) human samples were collected and subjected to whole genome analysis, including whole exome sequencing and DNA methylation scans. Additionally, birth defect rates for non-epileptic women in the Australian Pregnancy Register were also separately quantified. The human samples for investigations were collected from participants and their families enrolled in the Register. Using both human and animal models this study aimed to generate new knowledge, which could ultimately lead to a pharmacogenomic approach to the selection of AEDs for women who wish to become pregnant. This would allow women to make more informed decisions, reduce the risk of having a baby with a birth defect and potentially assist in the formation of new AEDs with lower teratogenic risk.
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ItemLynch syndrome in the Asian populations( 2016)Lynch syndrome is an autosomal dominant genetic disorder. Mutation carriers are at significant risk of developing colorectal and a variety of extra-colonic cancers, often at a younger age compared to sporadic cancers. The current guidelines on detection and management of Lynch syndrome are based upon studies in Caucasian populations with Lynch syndrome. The applicability of these guidelines to Asian populations is not known, as the prevalence and risks associated with mutations in mismatch repair genes may be different compared to Caucasian populations. The aims of this thesis were: 1. To assess the applicability of the current guidelines for the screening of Lynch syndrome and cancer surveillance strategies in the Asian populations (Chapter 2). 2. To study the phenotype of Lynch syndrome in Asian populations (Chapter 3). 3. To describe the mutation profile of novel pathogenic Asian variants and the associated phenotype of Lynch syndrome (Chapter 4). 4. To examine the applicability of next generation sequencing technology in microsatellite instability testing (Chapter 5). Chapter 2 describes the performance of seven predictive models (PREMM1,2,6, MMRpro, MMRpredict, Wijnen, Myriad, Amsterdam Plus, and Amsterdam Alternative), which were derived from European/Caucasian populations, on the screening of Lynch syndrome in the Asian populations with colorectal cancer. There was no evidence that these models will perform poorly in Asian families with performance similar to that reported for Caucasian families. However, many mutation carriers were not detected when these models were tested on Asian families simulated under the one-child policy (China). In addition, similar inferior performance was observed when these models were applied to families with a low penetrant gene associated with Lynch syndrome (e.g.: PMS2). These findings provide strong evidence that these models are applicable to Asian families where the size is not restricted. Their superiority over existing clinical guidelines indicates that case detection of Lynch syndrome can be maximised while minimizing false positive results when these models are applied instead of simply the Bethesda Guidelines or Amsterdam Criteria. However, alternative methods for assessing who should be tested for mutations need to be developed apart from family history in settings where small families are common. All models in this study may be deficient for the families with a PMS2 mutation, and alternative methods should be explored. Chapter 3 examines the phenotype of Lynch syndrome in Asian populations. To date, many conflicting results are observed about phenotype of Lynch syndrome in the Asian populations amongst the published studies. While the majority of Asian studies indicated that Lynch syndrome phenotype in Asian and Caucasian populations share many similarities, other studies have disagreed. In Chapter 3, no differences in the risk of cancers related to Lynch syndrome were observed when the phenotype of Asian and Caucasian mutation carriers were compared. These findings suggest that Lynch syndrome in the Asian and Caucasian populations are indeed quite similar, and raise the possibility of a similar carcinogenesis pathway that is not affected by ethnicity. Consequently, Asian mutation carriers with Lynch syndrome should be managed according to the current cancer surveillance programs, which are based on data of Lynch syndrome in Caucasian populations, until an ethnicity-specific program becomes available. The findings of this chapter also explain why the predictive models, which were developed using European/Caucasian populations, performed well in Asian populations as demonstrated in Chapter 2. The discrepancy of Lynch syndrome phenotypes reported amongst the Asian studies can be explained by their recruitment strategies. Many of these studies derived their phenotypic data from families that only fulfilled clinical guidelines without germline testing, and many sporadic cancers may have been included in their analyses. In contrast, all the families with Lynch syndrome in the present study have been confirmed to carry a pathogenic mismatch repair (MMR) gene mutation. Chapter 4 describes the phenotype of Lynch syndrome in Asian families with a novel pathogenic mutation. These mutations are yet to be described in the literature pertaining to Lynch syndrome in Caucasian populations. The reported phenotypic manifestation of Lynch syndrome in the Asian populations varies widely amongst the available studies, with some studies concluding that the cancer risk of Asian and Caucasian populations with Lynch syndrome is similar while other studies disagree. It remains uncertain if these novel variants confer a different cancer risk compared with Caucasian populations with Lynch syndrome. In Chapter 4, Asian families with a novel pathogenic MMR gene mutation were demonstrated to share many phenotypic manifestations as described in the Lynch syndrome literature based on Caucasian populations. This strengthens the hypothesis that Lynch syndrome mutation carriers of different ethnicities progress through similar carcinogenesis pathway despite the presence of novel mutations. These findings also strengthen the conclusions derived from the study of Lynch syndrome phenotype in the Asian populations in Chapter 3, and explain why the MMR predictive models, that were developed and validated in the Caucasian populations, performed well in Asian populations with colorectal cancer as described in Chapter 2. Chapter 5 describes the testing of microsatellite instability (MSI) using next generation sequencing (NGS) technology. The current family history-based screening strategies are inadequate in the detection of Lynch syndrome in small Asian families and families harbouring a gene with reduced penetrance (e.g.: PMS2) as described in Chapter 2. An alternative screening strategy would be to implement universal MSI testing, regardless of age of diagnosis or family history. The current MSI testing platform is based on multiplex polymerase chain reaction (PCR) technology, which may not meet the demand if MSI testing of cancers related to Lynch syndrome becomes universal. In chapter 5, NGS technology has been demonstrated to be a feasible platform for MSI testing. The main advantage of NGS over the traditional multiplex PCR-based method is that it allows simultaneous testing of large batches of cancer samples, thus improving the efficiency of testing. In addition, compared to other NGS-based MSI testing approaches, the method described in the present study obviates the need for genome-wide alignment in data analysis. Therefore, complex data processing pipelines are not required and data analysis can be performed using standard computing resources. In conclusion, this thesis describes the characteristics of various cancers related to Lynch syndrome and the screening of Lynch syndrome using the prediction models in the Asian populations. The data suggests that Asian and Caucasian populations with Lynch syndrome share many clinical characteristics, and the current Caucasian-based clinical guidelines and screening models are applicable to the Asian populations. NGS technology is applicable to MSI testing, and this would be a promising platform if universal colorectal cancer screening for Lynch syndrome becomes the standard of practice.
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemHLA genetics of autoimmune Graves’ disease and the effects of lifestyle factors on the prevalence of Vitamin D deficiency in Vietnamese living in Melbourne( 2011)Background: There has been an increasingly high attendance rate of Vietnamese Graves’ disease (GD) patients at Western Hospital and local clinics in Melbourne. This proposes interplay between genetic and environmental factors, causing increased susceptibility to GD, which could be specific to Vietnamese. Evidence of HLA genetic susceptibility was established in Asian and Caucasian populations worldwide, but it is not defined in Vietnamese because HLA studies have not been carried out on this ethnic group. A possible environmental factor in GD is Vitamin D deficiency, which was found to be highly prevalent in the Vietnamese population of our previous study in 2008. Aims: This study aims to investigate HLA genetic factors associated with GD in Vietnamese and lifestyle factors associated with the prevalence of Vitamin D deficiency in Vietnamese living in Melbourne. Methods: There were 201 Vietnamese participants from recruitment, including 61 GD patients and 140 control participants. Prior to participation, all participants were subjected to a physical examination, where their medical and family history was obtained. Following the process of providing written consent for participation, all participants were asked to complete a Medical Questionnaire, Food Frequency Questionnaire and Sun Exposure Questionnaire, and have their height and weight measured for BMI calculation. Serology samples were collected from participants at the end of participation for HLA genotyping, and tested using Vitamin D assays and TRAb assays. Results: Associations were found between GD and two HLA-DRB1 alleles in Vietnamese. DR7 has a protective effect against GD in controls (OR 0.204, 95% CI 0.044-0.946, p-value 0.042) and DR9 has a predisposal effect to GD in cases (OR 2.247, 95% CI 1.156-4.366, p-value 0.017). There was no evidence of association between Vitamin D deficiency (<75 nmol/L) and GD. However, Vitamin D deficiency and insufficiency was determined to be as high as 85.6% (172 out of 201) in Vietnamese living in Melbourne. Mean serum 25(OH)D level in cases was 53.49 nmol/L (SD 20.73) and controls was 50.46 nmol/L (SD 23.60). Mean daily total Vitamin D intake per participant excluding supplements (381.174 IU) is lower than Recommended Daily Intake (400 IU/day) for adults and adolescents. Mean daily total calcium intake per participant (429.123 mg) is much lower than Recommended Daily Intake (1,000 mg/day) for adult men and women. Significant associations exist between Vitamin D deficiency and age, and two lifestyle factors including sun exposure and sun avoidance. Conclusion: For the first time, HLA genetic profile in association with GD was reported in a Vietnamese population, adding to the existing knowledge of HLA genetics in Asians. Better understanding of the genetic background for GD could potentially lead to improved diagnosis and treatment methods, as well as being useful for developing primary prevention strategies in GD. This research study also confirmed the high prevalence of Vitamin D deficiency in Vietnamese living in Melbourne and identified associated lifestyle factors for this condition.
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ItemThe U12-dependent spliceosome is essential for regulating gene expression during zebrafish development( 2010)Removal of introns from pre-mRNA is an essential step in generating mature mRNA. The majority of introns are removed by the major, or U2-type, spliceosome, while the minor, or U12-type, spliceosome catalyses the removal of a small set of introns with characteristic features that are highly conserved in metazoans and plants. A novel zebrafish mutant, caliban (cal), with specific U12-type splicing defects, was used to study the biochemistry of the U12-type spliceosome and the role of U12-type introns in the regulation of gene expression. Greater than 99% of vertebrate introns are U2-type introns. However, a second class of U12-type introns exists that is defined by highly conserved consensus splice sites. U12-type introns represent about 0.5% of all introns in vertebrate genomes and usually occur alone alongside U2-type introns in pre-mRNA. U12-type introns are removed by a separate spliceosome that shares many components with the U2-type spliceosome, but contains several unique small nuclear RNAs (snRNAs) and spliceosomal proteins. Several lines of evidence suggest a function for U12-type splicing in the regulation of gene expression. These include the high evolutionary conservation of U12-type introns, their enrichment in certain functional gene groups and the demonstration that their excision can be rate-limiting in the generation of mature mRNAs. Despite these observations, little is known about the role and possible regulatory function of U12-type splicing in vivo. cal is a zebrafish development mutant with abnormalities in the intestinal epithelium, which is poorly polarised and unfolded compared to wildtype (wt). Mutant embryos also show a reduction in size of the liver and the pancreas and display a morphologically abnormal lens in the context of a smaller eye. The genetic lesion in cal was mapped to rnpc3, encoding the zebrafish orthologue of the human U11/U12 snRNP 65KDa protein, a specific component of the U12-type spliceosome. It was shown that cal embryos specifically retain U12-type introns compared to wildtype. Biochemical analyses of U12-type spliceosomal small nuclear ribonucleoproteins (snRNPs) demonstrate abnormal formation of the U11/U12 di-snRNP, which represents the first step in U12-type spliceosome assembly. However, it was also demonstrated that larger spliceosomal particles form and accumulate in cal embryos in the absence of Rnpc3/65K, suggesting a potential novel role for Rnpc3/65K in U12-type spliceosome disassembly and recycling. Whole transcriptome analysis of cal and wt embryos by microarrays and RNA sequencing demonstrated retention of U12-type introns on a global scale as well as a set of about 700 differentially expressed genes between cal and wt at two different developmental time points. Further analysis of gene expression data has led to the emergence of a model in which cal embryos are sustained through the first 48-72hpf by maternally deposited rnpc3 mRNA and Rnpc3/65K protein. After this time, defective U12-type splicing induces a cell cycle arrest in the endoderm-derived tissues of the liver, pancreas and intestine, which are highly proliferative between 72 and 108hpf. These results are leading to further studies with a focus on the role of U12-type splicing in human cancer. It was found that several prominent human tumour suppressor genes such as PTEN, LKB1 and PROX1, contain U12-type introns, and we propose a model by which an intermediate reduction of U12-type splicing efficiency can be tumourigenic by reducing the activity of particular tumour suppressor genes in a dose-dependent fashion. To test this hypothesis, conditional Rnpc3 knockout mouse models are currently being generated on a range of different colorectal cancer-susceptible backgrounds.