Medicine (Austin & Northern Health) - Theses
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ItemIdentification and characterisation of genes associated with hyperglycaemia susceptibility and reduced insulin secretion( 2017)The pathogenesis of type 2 diabetes (T2D) involves a complex interplay of genetic and environmental factors which result in impaired insulin secretion in the presence of insulin resistance. A permissive genetic susceptibility is considered a predominant determinant to the onset of hyperglycaemia, however, it has a complex aetiology that is yet to be fully defined. The aim of this thesis was to identify and characterise genes associated with hyperglycaemia. A genetically diverse mouse reference population, the Collaborative Cross (CC), was utilised to identify and characterise genetic loci influencing blood glucose concentrations. Therefore, a large-scale phenotypic screening of random blood glucose, insulin sensitivity and body weight across 652 males from 53 inbred CC strains (n=3-15) and 467 females from 48 CC strains (n=3-10) was conducted. The CC mouse population exhibits an abundant diversity in blood glucose levels as well as in insulin sensitivity and body weight. Interesting to note that strains which have blood glucose levels within the top range are rather lean and sensitive to insulin-mediated glucose lowering, indicative of impaired insulin secretion. To identify the genetic loci contributing to this variation in blood glucose concentrations, an unbiased genome-wide association study (GWAS) and quantitative trait loci (QTL) mapping were performed using the random blood glucose data. Two independent loci on chromosome 7 were identified to be significantly (p <5x10e-8) associated with high blood glucose, moreover, the contributory founder haplotype was demonstrated to be the NZO-derived alleles at both loci according to a haplotype analysis. These implicated loci were then named by the genes nearby peak SNPs as the E2F8 locus (Chr7: 52.6-56.7 Mb) and Dlg2 locus (Chr7: 98.5-101.55 Mb). Potential gene candidates in both loci were determined by sequence analyses throughout all genes in the linkage disequilibrium (LD) blocks, moreover, the property of genes and variants were also taken into account during the selection process. Finally, we narrowed the number of gene candidates down to seven for each locus: Gfy, Hsd17b14, Sphk2, E2F8, Ntn5, Abcc8 and Kcnj11 were identified at the E2F8 locus; Dlg2, Ccdc90b, Andkrd42, Pcf11, Ddias, Rab30 and Prcp for the Dlg2 locus. It was important to find that, to some extent, both E2F8 and Dlg2 loci have been implicated in diabetes-related abnormalities, but the functional defects underlying these deleterious alleles have yet to be fully defined. In order to study the impact of the NZO-derived hyperglycaemia susceptibility alleles on glucose homeostasis, we conducted in vivo and in vitro examinations on the hyperglycaemic CC strains, PIPING and PUB mice, which bear the deleterious alleles from NZO mice at susceptibility loci. Compared with the C57BL/6 mice as a reference strain, the hyperglycaemia susceptible strains are severely glucose intolerant in an oral glucose tolerance test (2 g/kg glucose), due to substantial reduction in insulin secretion in response to glucose and arginine stimulation in vivo. This secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta-cell mass and pancreatic insulin content, but rather as the result of inherent pancreatic beta-cell dysfunction. On the other hand, the genetic predisposition to hyperglycaemia appears to drive the susceptible mice to become more sensitive to dietary fat-induced weight gain and beta-cell decompensation. As evidenced by the findings that PIPING and PUB mice were susceptible to high fat diet-induced weight gain, which in partly due to increased fat intake but seems independent of higher caloric intake. Furthermore, PIPING and PUB mice developed advanced glucose intolerance on high-fat diet as a result of failure to raise insulin secretion for the increased demand. These results provide evidence that the hyperglycaemia susceptibility loci, E2F8 and Dlg2 loci, have substantial influences on pancreatic beta-cell function. Further investigation on the relationship of candidate gene expression with diabetes susceptibility was determined by identifying differential gene expression in primary islets between C57BL/6 (diabetes-resistant) and NZO (diabetes-susceptible) mice. On the basis of results from genetic mapping and expression analysis, E2F8 and Dlg2 genes were prioritised as promising candidates for reduced pancreatic insulin secretion. To determine whether E2F8 and Dlg2 are involved in pancreatic insulin secretion, E2F8 and Dlg2 were knocked down using siRNA and lentiviral transduction of shRNA, respectively, in a mouse pancreatic beta-cell line, MIN6 cells. The results of insulin secretion in E2F8 knockdown cells showed that down-regulation of E2F8 resulted in reduced insulin secretion in response to glucose as well as arginine and tolbutamide but not to GLP-1 and KCl, suggesting E2F8 mediates pancreatic insulin secretion in a K+ATP -channel dependent manner. Furthermore, knockdown of Dlg2 expression in MIN6 cells led to a generalised attenuation of insulin secretion regardless of glucose or other non-glucose secretagogues. These data showed the first evidence that the expression of E2F8 and Dlg2 affect insulin secretory function in pancreatic beta-cells, which can therefore influence individual’s susceptibility to diabetes. Taken together, this thesis provides evidence of two important loci for hyperglycaemia susceptibility which are linked to impaired insulin secretory function in the pancreatic beta-cells. Importantly, our results identified a number of novel candidate genes that are likely to alter insulin secretion in the pancreatic islets. Among these, E2F8 and Dlg2 were validated and recognised as novel genes to be involved in the pathogenesis of hyperglycaemia through altering pancreatic insulin secretion. This thesis delineates the use of the CC to identify novel genes for complex traits and understand the molecular control of insulin secretion.
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ItemThe clinical genetics of focal epilepsies( 2013)This thesis examined the clinical genetics of several adult and childhood focal epilepsy syndromes. Benign childhood epilepsy with centrotemporal spikes (BECTS) is the most common epilepsy syndrome in children age under 16. Previous studies with regard to the underlying genetic influence of this syndrome have shown conflicting results (Chapter 4). We examined this issue with a relative unselected cohort and strenuous methodology (Chapter 11 & 12). Our familial study depicted a clearer picture of the underlying genetic architecture of this syndrome and its related phenotypes in the affected relatives. The spectrum of a group of childhood epilepsies ranging from Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and wave during sleep (ECSWS) on the severe end, whereas BECTS on the mild end, has emerged in the past two decades (Chapter 5). We regarded it as the “epilepsy aphasia spectrum”. The cause of most of these severe epilepsies remains uncertain. We performed a familial study of epilepsy aphasia spectrum to characterize the inheritance pattern and associated clinical phenotypes in the relatives to obtain a better understanding of its underlying genetic factors (Chapter 11 & 13). Temporal lobe epilepsy (TLE) is the most common form of adult focal epilepsies (Chapter 7). Hippocampal sclerosis (HS) is observed in half of patients with TLE. The etiology of HS was generally regarded as acquired due to early life insults, while the role of genetic factors in the pathogenesis of HS remains unclear (Chapter 8). We conducted an imaging genetic study in families where probands have TLE with HS using qualitative and quantitative MRI methods (Chapter 11&14). We observed that the unaffected relatives tend to have smaller hippocampi, particularly when there is a positive family history. These small hippocampi are likely to be inherited developmental structural variations that are vulnerable to external insults and predispose to the development of HS. They could be a potential imaging endophenotype for future molecular genetic study. Hippocampal malrotation (HIMAL) is a recently described imaging feature that has been associated with epilepsy. The relationship between HIMAL and HS as well as whether it is epileptogenic are still waiting to be clarified (Chapter 9). We sought to refine the diagnosis of HIMAL with qualitative criteria and quantitative measurements and investigated these questions with two cohorts of subjects: relatives of probands who had TLE with HS and patients with lesion negative TLE (Chapter 15). We found the HIMAL could be a developmental anomaly which predisposes to the development of HS but it is not always epileptogenic. This has important clinical implication on decision making of epilepsy surgery. These clinical genetic and imaging studies provide a clearer landscape of the genetic architecture of several focal epilepsies spanning all age groups and a better understanding of the epileptogenic role of hippocampus in TLE. This work paves the way for further molecular genetic study to discover causative genes and for imaging analysis to identify subtle structural variations that may be related to focal epilepsy.
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ItemGenetics of syncope and familial focal epilepsy( 2011)This thesis examined the clinical genetic and molecular genetic features of two major causes of transient loss of consciousness, syncope and epilepsy. To clarify if genetic factors play a role in vasovagal syncope (VVS) a twin study was performed (chapter 11). Fifty-one same-sex twin pairs where at least one had syncope were ascertained. There was a trend towards higher casewise concordance in monozygous (0.75) than dizygous (0.50) twins for syncope (p=0.06). Comparison of concordance between monozygous and dizygous twins showed significant effects for frequent syncope (0.71 vs. 0.27, p=0.018) and for syncope associated with typical vasovagal triggers (0.62 vs. 0.00, p<0.001). The family history of 19 concordant monozygous pairs was then investigated. Twelve pairs reported few or no other affected family members whereas in the remaining seven pairs multiple close relatives were affected. The findings provided strong evidence for the relevance of genetic factors to VVS. Analysis of the families suggested that most cases follow complex inheritance although some families with autosomal dominant VVS probably exist. Autosomal dominant VVS was explored in six large families (chapter 12 and 13). The largest family contained 30 affected subjects. The phenotypes of individuals in these families were remarkably similar to common VVS, although two families showed an earlier median age of onset. Within families, the triggers (eg emotion, sight of blood etc) varied considerably between affected subjects. Significant linkage to chromosome 15q26 (LOD score 3.28) was found in the largest family and excluded in two families. No mutation was found in the candidate genes SLCO3A1, ST8SIA2 and NR2F2. These families demonstrated that autosomal dominant VVS is not rare. The variability of the triggers indicated that the major dominant genes increase the susceptibility to syncope but do not predispose to syncope associated with a particular trigger. Identification of the mutated gene is likely to provide novel insights into the pathophysiology of VVS. Two forms of familial focal epilepsy, nocturnal frontal lobe epilepsy (NFLE) and familial focal epilepsy with variable foci (FFEVF) were studied. The hypothesis of digenic inheritance of NFLE was explored in seven families (chapter 14). Based on the previous identification of two loci on chromosome 8q22-24 and 15q25 in a large family with NFLE and some evidence for a shared genetic background between parasomnia and NFLE, it was hypothesised that the presence of two genes was necessary for familial NFLE, whereas the presence of only one gene may cause parasomnia. However, segregation analysis using microsatellite markers did not support this model. In chapter 15 two families with FFEVF were characterized. Previous studies had shown linkage to the known FFEVF candidate region on chromosome 22q12 in family A and suggestive linkage to chromosome 2q in family B. Both families were re-phenotyped and additional affected subjects were included in family B. Linkage to chromosome 22q12 was confirmed in family A. Using the updated phenotypic information, family B was also consistent with linkage to chromosome 22q12. Both families show a subtly different phenotype to the other published families and extend the clinical spectrum of FFEVF mapped to chromosome 22q12. These clinical genetic and molecular studies have further defined the features of inherited forms of syncope and focal epilepsy. This work represents the essential preparatory steps for identification of causal variants by massively parallel sequencing techniques, which are currently underway.
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ItemFamily studies of epilepsy with simple and complex inheritance( 2002)The epilepsies are a group of disorders where genetic factors have been long known to play an important role. Genetic research in the last decade has led to the discovery of several mutated genes in some uncommon human epilepsies with autosomal dominant inheritance. The search of genes for epilepsies with complex inheritance has however been less successful. The general aim of this thesis was to study the genetics of epilepsies with simple and complex inheritance, using families with multiple affected family members. Accurate phenotyping was a strategy I used to try and minimise the problem of genetic heterogeneity. The study involved a long interview with all available living family members to collect clinical and genealogical information. Detailed electro-clinical characterisation of each individual's epilepsy syndrome was obtained. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) was studied as a prototype of simple inheritance. Detailed clinical analysis of 5 ADNFLE families revealed some new clinical features including seizures triggered by sound or movement, and precipitated by fever, extreme tiredness or stress. In order to help distinguishing NFLE from parasomnias, I developed a motor activity scoring system in sleep (MAISS). In most cases this confirmed the clinical and or electrographic diagnosis. Molecular genetic analysis of 5 families and 11 sporadic cases by our collaborators revealed 2 new mutations in the genes coding for α4 and β2 subunits of the acetylcholine receptors (CHRNA4 and CHRNB2). A de novo CHRNA4 mutation was found in the proband and her second son in family A. This is the first de novo mutation found in epilepsy suggesting also that sporadic cases could have a genetic aetiology and such patients should be tested for the known mutations. A new mutation in CHRNB2 was found in family B, the mutation was present in family members with ADNFLE but was not found in family members with parasomnias defined by their MAISS score. The finding does not support the hypothesis of a common genetic aetiology between ADNFLE and parasomnias. Idiopathic generalized epilepsies (IGEs) are the most common and important group of epilepsies with complex inheritance. An exceptionally large family with 35 affected individuals was studied. The phenotypes included childhood absence epilepsy (CAE), febrile seizures (FS) and generalized epilepsy with febrile seizures plus (GEFS+). Molecular genetic analysis showed that affected individuals had a mutation in the gene coding for the γ2 subunit of the GABAA receptor (GABRG2). The structure of the pedigree suggested that, in this family FS were inherited in an autosomal dominant fashion and caused by GABRG2 mutation. However, clinical genetic analysis suggested that whilst this mutation contributed to the CAE and GEFS+ phenotypes, it was likely that other genes were also involved. A second linkage analysis inclusive of only subjects with CAE disclosed a number of putative loci that could contain genes contributing to the absence epilepsy phenotype. I also studied 55 small families with IGEs that were more representative of IGEs found in the community. The clinical genetic analysis of the families showed a relatively homogeneous pattern of syndrome distribution within families. The phenotypic concordance with the proband was higher in first degree relatives compared to more distant relatives. The family analysis showed that the two recognized absence epilepsy syndromes were genetically closely related whilst juvenile myoclonic epilepsy was more distinct. The GABRG2 mutation was not found in any of these families. Two families with biparental IGEs were studied. The affected offspring of the 2 families had epilepsy phenotypes with characteristics of both parents' epilepsies. Finally, of 121 consecutive patients with new-onset IGE, 28% had seizures beginning at age 20 years or later (adult-onset). Similar to the classical IGE with age of onset before 20 years (as described by the ILAE classification), patients with adult-onset IGE could also be divided in subgroups depending on the predominant seizure types including absences, tonic-clonic and myoclonic seizures. Pedigree analysis of the 121 IGE patients and the comparison of the proportion of affected relatives between classical and adult-onset IGEs patients suggested a predominant genetic aetiology in both. In some pedigrees classical and late-onset IGEs patients co-occurred suggesting that they might share genetic determinants. Therefore adult-onset IGEs should be included in studies were genes for IGEs are sought. In conclusion the data from the large family in which a GABRG2 mutation was found, from the small multiplex families including biparental families and from patients with adult-onset IGEs all underscore the genetic basis of IGEs. Moreover, the clinical analyses, with distinct identifiable subsyndromes, is more suggestive of an oligogenic model of inheritance, involving few genes, rather than a polygenic model where many genes of small effect contribute to the individual phenotype.