As doenças autoimunes da tireoide (DAIT) representadas, principalmente pela doença de Graves (DG) e tireoidite de Hashimoto (TH), apresentam causas multifatoriais, incluindo fatores genéticos e ambientais. Diversos genes estão envolvidos, entre eles CTLA4, PTPN22 e mais recentemente IFIH1, principalmente quando associados a diabetes mellitus tipo 1 (DM1). OBJETIVOS: Determinar a frequência alélica e genotípica dos polimorfismos: -318C/T, A49G e CT60 do CTLA4, R620W do PTPN22 e A946T do IFIH1 em pacientes pediátricos portadores de DG, TH e DM1 associado a TH e em uma população controle normal, e determinar a associação com características clínicas e laboratoriais. MATERIAL E MÉTODOS: Foram estudados 142 pacientes menores de 21 anos ao diagnóstico. Os dados clínicos e laboratoriais foram obtidos em prontuário. A genotipagem foi realizada por PCR em tempo real de todos os polimorfismos. Dados clínicos e laboratoriais como sexo, idade de início, bócio, anticorpos anti-GAD, IA2 e IAA e níveis de TRAb e anti-TPO foram analisados. RESULTADOS: Sessenta e dois pacientes foram diagnosticados com DG, com idade média ao diagnóstico (IMD) de 10,8 ± 4,4 anos, sendo 43 do sexo feminino; TH esteve presente em 44 pacientes, sendo 37 meninas, com IMD de 10,3 (± 2,9 anos); e 36 pacientes com DM1 associado a TH, sendo 21 meninas, com IMD de 6,2 (± 4,0 anos) no momento do diagnóstico de DM1 e de 11,6 (± 4,6 anos) ao diagnóstico de TH. O grupo controle foi constituído por 81 indivíduos sem diabetes, função tireoidiana normal e ausência de anticorpos antitireoidianos. Todos os polimorfismos estavam em equilíbrio de Hardy-Weinberg. O polimorfismo -318C/T não esteve associado com nenhum dos grupos. O alelo de risco G do polimorfismo A49G foi mais frequente em pacientes com TH (p=0,047) e o genótipo patogênico (AG e GG) foi mais frequente em pacientes com DG (p=0,049). O alelo de risco G do polimorfismo CT60 foi mais frequente apenas em pacientes com DG (p=0,035). O alelo de risco T do polimorfismo R620W foi mais frequente em pacientes com DM1 associado a TH (p=0,043). O alelo de risco T do polimorfismo A946T foi mais frequente em pacientes com DM1 associado a TH (p=0,009), assim como o genótipo patogênico (CT e TT) quando comparado ao grupo controle (p=0,007). Quando agrupamos todas as DAIT, observamos associação com A49G (p=0,024) e R620W (p=0,047). Quando agrupamos apenas pacientes com TH, encontramos diferença no A49G (p=0,018) e no A946T (p=0,041). O polimorfismo CT60 foi associado com menor duração da terapia medicamentosa no grupo DG (p=0,004), mas não com os níveis de TRAb ou presença de bócio. No DM1 com HT, o alelo de risco do A49G foi mais frequentemente encontrado no sexo masculino (p=0,04); R620W foi relacionado com a presença de bócio (p=0,03), enquanto A946T foi associado com níveis de anti-TPO mais baixos (p=0,047). Os níveis de anti-GAD, IA2 e Resumo IAA não foram associados aos polimorfismos. CONCLUSÃO: Encontramos associações genéticas diferentes entre os pacientes com DAIT, sugerindo que as crianças possuem provavelmente padrões genéticos distintos, apesar do menor tempo de exposição a fatores ambientais / Autoimmune thyroid diseases (AITD) represented by Graves\' disease (GD) and Hashimoto\'s thyroiditis (HT), have multifactorial causes, including genetic and environmental factors. Several genes are involved, including CTLA4, PTPN22 and more recently IFIH1, especially when associated with type 1 diabetes (T1D). OBJECTIVES: To determine the allelic and genotypic frequencies of the polymorphisms: -318C/T, A49G and CT60 of CTLA4, R620W of PTPN22 and A946T of IFIH1 in pediatric patients with GD, HT and T1D associated with HT and in a control population and determine association with clinical and laboratory features. MATERIAL AND METHODS: We studied 142 patients under 21 years at diagnosis. Clinical and laboratory data were obtained from medical records. Genotyping was performed by real time PCR for all polymorphisms. Clinical and laboratory data were analyzed, such as gender, age of onset, goiter, anti-GAD, IA2 and IAA levels and TRAb and anti-TPO levels. RESULTS: Sixty-two patients were diagnosed with GD, with mean age at diagnosis (MAD) of 10.8 ± 4.4 years, 43 females; HT was present in 44 patients, 37 girls, MAD 10.3 (± 2.9 years) and type 1 diabetes associated with HT was present in 36 patients, 21 girls, MAD 6.2 (± 4.0 years) at diagnosis of T1D and 11.6 (± 4.6 years) at diagnosis of HT. Control group consisted of 81 subjects without diabetes, normal thyroid function and absence of antithyroid antibodies. All polymorphisms were in Hardy-Weinberg equilibrium. The polymorphism -318C/T was not associated with any of the groups. The risk allele G of A49G polymorphism was more frequent in patients with HT (p=0.047) and the pathogenic genotype (AG and GG) was more frequent in patients with GD (p=0.049). The risk allele G of CT60 polymorphism was more frequent only in patients with GD (p=0.035). The risk allele T of R620W polymorphism was more frequent in patients with T1D associated with HT (p=0.043). The risk allele T of A946T polymorphism was more frequent in patients with T1D associated with HT (p=0.009), as well as the pathogenic genotype (CT and TT) compared to control group (p=0.007). When all AITD is grouped, we observed association with A49G (p=0.024) and R620W (p=0.047). Only when patients with HT are grouped, we found differences in A49G (p=0.018) and A946T (p=0.041). CT60 polymorphism was associated with a shorter duration of drug therapy on GD group (p=0.004), but no association with TRAb levels or presence of goiter were found. In T1D with HT, the risk allele of A49G was more often found in males (p=0.04); R620W was associated with presence of goiter (p=0.03), while A946T was associated with anti-TPO levels (p=0.047). The anti-GAD, IAA and IA2 levels were not associated with any polymorphisms. CONCLUSION: We found different genetic associations among patients with AITD, suggesting that children are likely to have distinct genetic background, despite shorter exposure to environmental factors

Alzheimer's disease (AD) is the most common form of dementia, currently affecting around 17--25 million people worldwide. The typical neuropathological hallmarks of AD are amyloid beta (Abeta) deposition, presence of neurofibrillary tangles and neuronal cell death. Evidence from ongoing studies on the pathogenesis of AD, suggests that several different mechanisms are involved in neurons loss and thus decline of cognitive function. These include the metabolism of amyloid peptide, inflammation, cholesterol metabolism, and hormonal factors. / I have focused on the role of inflammation in the progression of AD. The inflammation hypothesis is based on findings of (1) elevated levels of inflammatory cytokines, such as IL-1, IL-6, TNFalpha, (2) the reduced levels of anti-inflammatory cytokines, like IL-10 in CSF and the blood of AD patients, and (3) activated microglia in the histological section of the patient's brain. On the other hand, the effects of the ApoE gene and differential age of onset between the two sexes suggested a modulation role for cholesterol and sex hormone like estrogen, which may influence the inflammatory response in the brain, so as to modulate the risk of AD. / In this project, the genetic risk factors predisposing to AD were investigated by genetic association studies of candidate genes. Candidate genes were shortlisted by two approaches. (I) Linkage-based candidate genes: Candidate genes were identified from reported loci with linkage to AD genome scan studies. Previous linkage studies of AD families revealed linked loci at 1p36, 1q23, 3p14, 4q32, 6p21, 6q27, 9q22, 10q24, 13q32, 15q26, 19q13 and 21q22. Several candidate genes from these loci including TNFalpha-related genes, TLR2, IGF-1, IFNalpha and MTHFR were selected for this project. (II) Hypothesis-based candidate genes: Candidate genes were selected according to their possible involvement in the inflammation hypothesis of AD. Under the hypothesis-based candidate gene approach, genes that might contribute to the inflammatory response of amyloid deposition were identified. These genes were validated by their expression level in the central nervous system. A further priorization step was carried out to select those genes showing a higher degree of inter-individual variation. Therefore, these genes were more likely to have a genetic/inherited variation at the population level. In other words, they are more likely to be the predisposition genes than genes without inter-individual variation (house-keeping genes are examples of genes showing little inter-individual variation). In this project, genes involved in the inflammatory pathway in the brain, such as IL-10 and HLA-A, and also genes that interact with the inflammatory pathway such as cholesterol related enzymes and estrogen receptors were investigated under the hypothesis-based approach. / This project is based on a case-control genetic association study which comprised of NINCDS-ADRDA diagnosed Chinese patients with AD (n=259) and age-matched non-demented subjects (n=248). Three genes PTGS2 (encoding for COX-2), MxA and ESR1 were selected for an intensive study by investigating their linkage disequilibrium pattern and using tagSNP strategy. TagSNPs selected for each gene were genotyped to investigate their association with the risk of AD. / This study showed that MTHFR, IL-10, HLA-A, CYP46A1, PTGS2 (COX-2) and ESR1 were associated with the risk of AD, and MxA, identified for the first time, was associated with the age of onset of AD. In conclusion, the results of my study further suggested the roles of inflammation in the pathogenesis of AD. / Ma Suk Ling. / "June 2006." / Advisers: Linda C. W. Lam; Nelson L. S. Tang. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1417. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 169-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In conclusion, gene expression profiling in blood may have potential to be an adjuvant marker for early detection of AD. Expression marker panel is more informative than single gene expression signature. Further validation in prospective studies will substantiate its clinical application and explore its potential to differentiate AD from other dementias and to predict the progression from MCI to AD. (Abstract shortened by UMI.) / In the study, the profile of 12 target gene expression levels in peripheral blood cells were determined in 96 AD, 145 MCI and 167 normal controls (NC) by quantitative real-time RT-PCR. The genes were identified with (i) high expression in blood and brain; (ii) differential expression between AD and control; (iii) AD related candidate genes. Then, a list of genes were selected including CTSB, CTSD, DDT, ITPKB, NDUFA6, NRD1, PIN1, SNX2, TSC1, UQCRC1, CNR2, GSTM3. Seven genes were found to be differentially expressed between AD and NC group, with upregulation of CTSB, CTSD, DDT, TSC1 and UQCRC1, and downregulation of ITPKB and PIN1 in AD patients. Expression levels of two genes were increased in the MCI compared with NC group, including CTSB and CTSD. In addition, an upregulation of CTSD, UQCRC1, NRD1 and downregulation of ITPKB were observed in AD subjects in comparison to the MCI group (Mann-Whitney U test, p<0.05). After adjusting for confounding factors of age, gender, education level, ApoE4 status and the total CIRS score, expression level of any single gene was not associated with the classfication of AD or MCI (Logistic regression, p>0.05). A five gene biomarker panel, including DDT, ITPKB, PIN1, TSC1 and UQCRC1 was identified with logistic regression analysis. The function of Logit(P)= ln(P/(1-P))= b0+b1RatioDDT+ b2RatioITPKB + b3Ratio PIN1 +b4 RatioTSC1+b5Ratio UQCRC1 were defined as the probability of a subject to be diagnosed as "AD" or "MCI' by using 5-gene biomarker panel. ROC analysis showed that the AUC for the 5-gene biomarkers panel in differentiating between AD and NC, between MCI and NC, between AD and MCI were 0.79 (95% CI, 0.72-0.86; p<0.001), 0.61 (95% CI, 0.53-0.69; p=0.007) and 0.68 (95% CI, 0.60-0.76; p<0.001) respectively. The 5-gene combination was found to discriminate AD subjects from normal controls with good sensitivity and specificity of 70.7% and 86.7% respectively at an optimal cut-off point of 0.486. Low sensitivity (42.4%) and acceptable specificity (76.2%) were observed at a cut-off threshold of 0.505 when differentiating MCI from NC subjects. Between AD and MCI subjects, gene combination showed a sensitivity of 61% and specificity of 73.7% at a cut-off value of 0.496. / Several genes including CTSD, DDT, NDUFA6, TSC1 and UQCRC1 were found in association with the cognitive and psychiatric symptoms, indicating the role of genetic factors in moderating the presence of cognitive and NP profiles in demented individuals. / The aim of the present study is to evaluate the gene expression profiling of peripheral leukocytes in Chinese subjects with Alzheimer's disease (AD) and explored its potential of clinical application. Behavioral phenotypes of cognitive performance and neuropsychiatric assessment were also investigated in association with gene expression in AD. Persons with mild cognitive impairment (MCI), as an at-risk state between normal aging and clinical dementia, was also assessed in consideration that the information may provide a better understanding of the mechanisms involved in clinical progression of AD. / The genes identified in this study were involved in processes implicated in neurodegneration, including protein isomerization (PIN1), calcium disequilibrium and mitochondria insufficiency (ITPKB and UQCRC1), increased inflammatory response (DDT), apoptosis (CTSB and CTSD) and neurogeneration (NRD1 and TSC1). / Fu, Yan. / Adviser: Chiu Wa Lam. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 132-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum com uma prevalência aproximada de 3% em pacientes com mais de 64 anos. A doença é esporádica, mas o parkinsonismo primário (PP) familiar, decorrente de defeitos genéticos específicos, tem sido encontrado em cerca de 10% dos casos diagnosticados como DP. Os objetivos deste trabalho são analisar o DNA de pacientes com PP acompanhados no ambulatório do Grupo de Estudo de Distúrbios do Movimento da Clínica Neurológica do Hospital das Clínicas da FMUSP que apresentam início precoce (< 40 anos) ou história familiar positiva com o intuito de rastrear mutações responsáveis pela doença e descrever as características clínicas desse grupo de pacientes e dos familiares acometidos. Entre Janeiro de 2004 a Janeiro de 2006 foram selecionados 53 probandos com PP, sendo que 29 eram esporádicos, 16 com história familiar sugestiva de herança autossômica dominante (AD) e 8 com história familiar sugestiva de herança de autossômica recessiva (AR). No total, 100 amostras de DNA foram coletadas, 70 de pacientes ou familiares com PP, 1 com parkinsonismo secundário ao uso de neuroléptico e o restante de familiares sem PP. Dos casos afetados, 45 eram do sexo masculino e 25 feminino, a idade média de início dos sintomas foi de 38,3 anos (10-72) e a média de idade no momento da investigação foi de 49,8 anos (22-72). Todos apresentaram instalação assimétrica do quadro, curso lento e progressivo e boa resposta ao tratamento com levodopa ou agonista dopaminérgico. Pacientes com padrão de herança AD foram testados para a mutação Gli2019Ser que é o defeito mais comum do gene LRRK2 (PARK8) sendo encontradas duas famílias afetadas. A análise mutacional dos genes PARK6 e PARK7 está em andamento. Todos os casos esporádicos e com padrão de transmissão AR foram testados para mutações do gene PARK2 e foram encontradas as seguintes mutações homozigóticas em 4 famílias: 255delA, deleção de exon 3-4, deleção do exon 2-3 e uma nova mutação IVS1+1G/T. Num paciente com parkinsonismo juvenil (idade de início dos sintomas <21 anos) foi encontrada uma nova mutação homozigótica no gene ATP13A2 (PARK9) no exon 15 que determina a substituição Gli504Arg na proteína codificada. Em grande parte dos casos estudados os achados genéticos e clínicos são similares aos descritos na literatura. Entretanto, encontramos novas mutações do gene PARK2 e PARK9 e no paciente com a mutação ATP13A2 os achados clínicos diferem em alguns aspectos da descrição clássica. / Parkinson disease (PD) is the second most common neurodegenerative disorder affecting approximately 3% of the population over age 64. Most cases of PD manifest in sporadic form, but familial primary parkinsonism (PP) due to specific genetical abnormalities has been found in about 10% of cases diagnosed as PD. The aims of this study were to analyze the DNA of PP patients seen at the Group for the Study of Movement Disorders of the Neurology Department of Hospital das Clinicas of the University of São Paulo who presented early onset of the disease (< 40 years of age) or positive family history, with the purpose of screening possible candidate mutations for the disease, and to describe the clinical features of this group of patients and affected members of their families. Between January 2004 and January 2006, 53 probands were selected of whom, 29 were sporadic cases, 16 had probable autosomical dominant (AD) pattern of inheritance, and 8 autosomical recessive (AR). In total 100 samples of DNA were collected, 70 from PP patients or affected relatives, one case with neuroleptic-induced parkinsonism, and the rest from not affected members. Forty five affected individuals were men and 25 women, the median age of the symptoms onset was 38.3 years (10-72), and the median age at the moment of the examination was 49.8 years (22-72). All patients had asymmetric installation of the disease, slow progression of the PP, and good response to levodopa or dopaminergic agonist therapy. Patients with AD inheritance were screened for Gly2019Ser mutation, which is the most common defect in PD due to LRRK2 gene, and two families carried this mutation. The screening of PARK6 and PARK7 is ongoing. All sporadic and AR inheritance cases were tested for mutation of (PARK2) and the following mutation were found in 4 families in homozygous state: 255delA, exon 3-4 deletion, exon 2-3 deletion, and a novel mutation IVS1+1G/T. In a juvenile parkinsonism proband (age of onset < 21 years) a novel missense homozygous mutation in ATP13A2 (PARK9) gene was found in exon 15 which resulted the Gly504Arg change in the encoded protein. In general the genetical and clinical findings of this series of patients are similar to those reported in the literature, although novel mutation in PARK2 and PARK9 were obtained. Some clinical features of the patient with ATP13A2 mutation differed from the classical descriptions.

Background: Parkinson’s disease (PD) is a complex disease with a multi-factorial aetiology, comprising both genetic and environmental risk factors. The disease pathology is progressive and neurodegenerative where dopaminergic nerve cell death occurs predominantly in the substantia nigra pars compacta (SNpc) with the subsequent loss of the dopamine neurotransmitter in the basal ganglia. The most significant risk factors for PD include an advancing age and a family history of the disease, while environmental and lifestyle risk factors such as pesticide exposure and smoking are widely accepted as risk altering exposures. Currently up to 10% of PD is attributed to Mendelian inherited PD at one of 13 PARK loci in 9 genes. The pursuit of common susceptibility alleles for idiopathic PD has proven challenging with only a few loci reproducibility associated with an altered risk. The aim of this thesis is to study, using a candidate gene case-control design, the potential role of genetic variants in PD. The APOE candidate gene was hypothesized to modify the risk of PD as it is a proven modifier of Alzheimer’s disease (AD). The common pathological finding in PD of elevated levels of iron within the SNpc is proposed to increase the oxidative state of the nerve cells and predispose the dopaminergic neurons to apoptosis. Therefore, susceptibility alleles within the candidate genes that regulate iron metabolism and homeostasis are hypothesized to alter iron metabolism and predispose to iron-induced neurodegeneration in PD. Missense variants and common “tagging” SNPs with the HFE, Transferrin and Transferrin Receptor genes are investigated extensively in this thesis. Finally, autosomal recessively inherited PD can result from mutations in the parkin gene at the PARK2 locus. The final hypothesis explored in this thesis suggests that non-deleterious missense variants in the parkin gene modify the risk for developing sporadic PD. Further genetic variation in the parkin gene such as exon rearrangements is a frequently reported mutation where heterozygosity for these rearrangements may increase the risk of PD. Heterozygous deletions or duplications of exons in the parkin gene provide technical challenges for their detection. In this thesis a novel assay for the detection of these mutations is investigated. Methods: Genotyping was performed using PCR-RFLP for genetic variants in the APOE (E2 and E4 alleles), HFE (C282Y, H63D and S65C), Transferrin receptor (TfR; S142G), Transferrin (Tfn; P570S and G258S), IREB2 genes (L159V) and the parkin gene (S167N, R366W and V380L) in a cohort of 425 PD cases and 387 controls recruited from throughout Queensland, Australia. A tagged SNP high-throughput genotyping approach was then employed to try to replicate single SNP associations in 6 iron-related genes using a cohort of 1034 PD cases and 774 controls. These genetic variants were analysed for direct association with PD risk, age of onset effects as well as potential gene x gene (GxG) and gene x environment (GxE) interactions. Additionally, a quantitative PCR assay was developed to detect heterozygous deletions and duplications within the parkin gene and utilised to screen 43 YOPD cases for these mutations. Results: The initial study of the HFE C282Y variant revealed a significant protective association with PD in the two independent cohorts studied. Further study did not reveal significant associations with PD for the other HFE variants or missense variants within the Tfn and TfR genes. When analysed for GxE interactions, the C282Y, P589S and G277S variants showed evidence for an increased risk of PD in synergy with pesticide and herbicide exposure. Carriers of the risk variant and with toxin exposure were at two-fold increased risk of PD, although the number of individuals in this category was small. A further investigation of the role of common genetic polymorphisms in iron genes revealed only one of the 20 SNPs genotyped using high-throughput multiplex methods, remained significantly associated with PD after correction for age and sex. The rs198855 SNP is downstream of the HFE gene and further implicates a role for HFE in PD. The APOE E4 allele demonstrated modifying effects for the age of PD onset, restricted to the female cases. Analysis of the parkin missense variants also demonstrated a modifying effect on the age of PD onset in carriers of the S167N variant, with putative interactions between the APOE E4 allele, a family history of PD and toxin exposure that further reduced the age of onset. Twenty individuals of the 43 YOPD cases screened demonstrated heterozygous parkin exon rearrangements using the novel qPCR method. Conclusions: Non-synonymous variants within iron-related genes or the parkin gene putatively interact with herbicide and pesticide exposure to increase the risk of PD or modify the phenotype, highlighting the need for future studies to address the multi-factorial aetiology of PD in their study design and analysis. This thesis provides evidence for the association between genetic variation within the HFE locus and PD and for the APOE E4 allele as a modifier of PD.

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum com uma prevalência aproximada de 3% em pacientes com mais de 64 anos. A doença é esporádica, mas o parkinsonismo primário (PP) familiar, decorrente de defeitos genéticos específicos, tem sido encontrado em cerca de 10% dos casos diagnosticados como DP. Os objetivos deste trabalho são analisar o DNA de pacientes com PP acompanhados no ambulatório do Grupo de Estudo de Distúrbios do Movimento da Clínica Neurológica do Hospital das Clínicas da FMUSP que apresentam início precoce (< 40 anos) ou história familiar positiva com o intuito de rastrear mutações responsáveis pela doença e descrever as características clínicas desse grupo de pacientes e dos familiares acometidos. Entre Janeiro de 2004 a Janeiro de 2006 foram selecionados 53 probandos com PP, sendo que 29 eram esporádicos, 16 com história familiar sugestiva de herança autossômica dominante (AD) e 8 com história familiar sugestiva de herança de autossômica recessiva (AR). No total, 100 amostras de DNA foram coletadas, 70 de pacientes ou familiares com PP, 1 com parkinsonismo secundário ao uso de neuroléptico e o restante de familiares sem PP. Dos casos afetados, 45 eram do sexo masculino e 25 feminino, a idade média de início dos sintomas foi de 38,3 anos (10-72) e a média de idade no momento da investigação foi de 49,8 anos (22-72). Todos apresentaram instalação assimétrica do quadro, curso lento e progressivo e boa resposta ao tratamento com levodopa ou agonista dopaminérgico. Pacientes com padrão de herança AD foram testados para a mutação Gli2019Ser que é o defeito mais comum do gene LRRK2 (PARK8) sendo encontradas duas famílias afetadas. A análise mutacional dos genes PARK6 e PARK7 está em andamento. Todos os casos esporádicos e com padrão de transmissão AR foram testados para mutações do gene PARK2 e foram encontradas as seguintes mutações homozigóticas em 4 famílias: 255delA, deleção de exon 3-4, deleção do exon 2-3 e uma nova mutação IVS1+1G/T. Num paciente com parkinsonismo juvenil (idade de início dos sintomas <21 anos) foi encontrada uma nova mutação homozigótica no gene ATP13A2 (PARK9) no exon 15 que determina a substituição Gli504Arg na proteína codificada. Em grande parte dos casos estudados os achados genéticos e clínicos são similares aos descritos na literatura. Entretanto, encontramos novas mutações do gene PARK2 e PARK9 e no paciente com a mutação ATP13A2 os achados clínicos diferem em alguns aspectos da descrição clássica. / Parkinson disease (PD) is the second most common neurodegenerative disorder affecting approximately 3% of the population over age 64. Most cases of PD manifest in sporadic form, but familial primary parkinsonism (PP) due to specific genetical abnormalities has been found in about 10% of cases diagnosed as PD. The aims of this study were to analyze the DNA of PP patients seen at the Group for the Study of Movement Disorders of the Neurology Department of Hospital das Clinicas of the University of São Paulo who presented early onset of the disease (< 40 years of age) or positive family history, with the purpose of screening possible candidate mutations for the disease, and to describe the clinical features of this group of patients and affected members of their families. Between January 2004 and January 2006, 53 probands were selected of whom, 29 were sporadic cases, 16 had probable autosomical dominant (AD) pattern of inheritance, and 8 autosomical recessive (AR). In total 100 samples of DNA were collected, 70 from PP patients or affected relatives, one case with neuroleptic-induced parkinsonism, and the rest from not affected members. Forty five affected individuals were men and 25 women, the median age of the symptoms onset was 38.3 years (10-72), and the median age at the moment of the examination was 49.8 years (22-72). All patients had asymmetric installation of the disease, slow progression of the PP, and good response to levodopa or dopaminergic agonist therapy. Patients with AD inheritance were screened for Gly2019Ser mutation, which is the most common defect in PD due to LRRK2 gene, and two families carried this mutation. The screening of PARK6 and PARK7 is ongoing. All sporadic and AR inheritance cases were tested for mutation of (PARK2) and the following mutation were found in 4 families in homozygous state: 255delA, exon 3-4 deletion, exon 2-3 deletion, and a novel mutation IVS1+1G/T. In a juvenile parkinsonism proband (age of onset < 21 years) a novel missense homozygous mutation in ATP13A2 (PARK9) gene was found in exon 15 which resulted the Gly504Arg change in the encoded protein. In general the genetical and clinical findings of this series of patients are similar to those reported in the literature, although novel mutation in PARK2 and PARK9 were obtained. Some clinical features of the patient with ATP13A2 mutation differed from the classical descriptions.

Inflammatory bowel disease (IBD), which includes the subtypes Crohn's disease (CD) and ulcerative colitis (UC), is a common disease particularly in the Western world. IBD is characterised by inflammation of the small intestine and/or colon. The two subtypes affect different gut locations but both show an increased intestinal permeability or the 'leaky gut syndrome'. This led to the hypothesis that tight junction (TJ) proteins expressed in the epithelium may affect the intestinal permeability as a cause or effect of IBD.Initially, variants in the CARD15, IL23R and ATG16L1 genes, previously associated with an increased risk of IBD, were genotyped in a cohort of 500 IBD (295 CD and 205 UC) patients and 877 matched controls. These variants were significantly associated in our cohort. A random effects meta-analysis was undertaken on all previously reported CD associations with the variant rs2241880 from ATG16L1 (n=25, p=0.0017, OR: 1.36 95% CI 1.12-1.66) and with rs11209026 from IL23R (n=26, p=0.0006, OR: 0.37 95% CI 0.21-0.67), showing pooled odds ratios consistent with those reported in our cohort. Individuals carrying >1 CARD15 mutant variant were found to have a 2.5 fold increased risk of CD (p=0.0001). Candidate TJ proteins were chosen on the basis of previous reported associations and through the investigation of the claudin proteins which are abundant at TJs. Twenty one candidate genes were selected and 79 variants successfully genotyped in up to 1063 IBD (502 CD and 478 UC) and 870 control patients. Significant associations were detected with variants in the CLDN1, CLDN5 and CDH1 genes with CD; CLDN5, CLDN8 and CDH1 variants were associated to IBD; and the rs7791132 variant (between CLDN4 and ELN) and a CDH1 variant were associated to UC. The CLDN1 rs6809685 variant trended towards association in a Toronto ascertained IBD replication cohort (genotypic p=0.04, allelic p=0.06) suggesting this may be a novel IBD susceptibility variant. Small intestinal biopsies from CD patients with known rs6809685 genotypes showed a dose dependent reduced immunohistochemical staining of claudin 1 with carriage of the mutant G allele. Claudin 1 helps seal TJs and reduced levels may increase risk of CD.Peroxisome proliferator activator receptors (PPARs) can directly affect TJ proteins and could therefore affect intestinal permeability. Twelve PPARγ variants were genotyped in up to 1050 IBD (502 CD and 467 UC) and 725 control patients. Significant genotypic associations were found with the rs2067819 variant in CD (p=0.05) and IBD (p=0.02), and also the rs13099634 variant in UC (P=0.02). There was a strong gender difference particularly for rs2067819 and rs4135247, where allelic associations were highly significant and increased risk of IBD in men (p=0.01 and p=0.007 respectively). However no significant associations were found in the female cohort. Troglitazone a PPARα agonist increased Caco2 cell transepithelial electrical resistance (TEER), a marker of TJ integrity, and increased expression of claudins -3 and -4. In contrast, the PPARα antagonist GW6471 reduced the TEER without causing cell death and PPARγ ligands did not affect TEER measurements. In summary, using a robust cohort of cases and controls the data indicates that variants in genes encoding TJ proteins may affect susceptibility to IBD and that PPARs can regulate these proteins altering intestinal permeability.

Nicotinic acid (NA) has been clinically used for over 50 years to regulate lipid plasma levels. It is the only drug in current clinical use that significantly raises HDL cholesterol and reduces inflammatory markers. However, mechanistic understanding into its wide range of actions remains unclear. The recent identification of the Gi-coupled protein receptor HCAR2, for which NA is a potent agonist, provides intriguing insight due to its anti-lipolytic action and restricted, yet specific, expression in adipose tissue and immune cells. The HCAR2 gene is 96% homologous to HCAR3, but the HCAR3 receptor shares neither the specificity for NA, nor the range of functional effects. Moreover, the close homology makes it difficult to separate the genetic variability and regulation of the two genes. To this end, I resequenced HCAR2 and HCAR3 in a selected population to characterize the variability of the two genes and to inform the subsequent design of specific genotyping assays. The Oxford Biobank, which is a random population-based collection of 30-50 year old men and women in Oxfordshire with a wide range of collected phenotypes, was used to explore genetic associations. A preliminary trend with HDL and rs7314976 in HCAR2 motivated the further search associations. However after increasing the sample size, the HDL association did not reach significance. When looking at inflammatory phenotypes, a 20% lower level of systemic hsCRP was found in males with a promoter region variant in HCAR3 (N=1808, p=0.007 for rs55718746). Replication of this finding in two relevant cohorts (NPHS-II, N=2185 and Whitehall, N=4228) resulted in conflicting findings. After optimising the specific detection of both HCAR2 and HCAR3 transcripts, I characterized gene expression in human AT biopsies. This revealed an 18% increase in HCAR2 expression in the female abdominal depot (N=106, p<0.0001) and a reduction in abdominal HCAR2 in both males (β=-0.37, p<0.001, N=107) and females (β=-0.251, p=0.005, N=106) with increasing adiposity. The rs55718746 variant in HCAR3 was also seen to influence expression of both HCAR2 (N=182, p=0.018 in the abdominal depot) and HCAR3 (N=198, p=0.005) but surprisingly in opposite directions, establishing it as the first cis-eQTL for this genomic region. Finally, I used human adipocyte in vitro culture systems to setup a pilot to study the anti-inflammatory effects of NA. The gene expression of HCAR2 and HCAR3 increased significantly with adipocyte differentiation in vitro. NA led to a drop in IL-6 transcript abundance in two out of three of the in vitro differentiated human adipocytes. In conclusion, genetic variability in HCAR2 and HCAR3 shows weak associations with cardiovascular disease risk phenotypes relating to their respective pathways. The relevance of HCAR2 and HCAR3 gene expression and the role of the receptor in the control of inflammation will require further studies.

Type 2 diabetes (T2D) has a genetic component which is only partially understood. The majority of genetic variance in disease susceptibility is unaccounted for, whilst the precise transcripts and molecular mechanisms through which most risk variants exert their effect is unclear. A complete understanding of T2D susceptibility mechanisms could have benefits in risk prediction, and in drug discovery through the identification of novel therapeutic targets. Work presented in this thesis aims to define relevant transcripts and disease mechanisms at known susceptibility loci, and to identify disease association with classes of genetic variation other than common single nucleotide polymorphisms (SNPs). KCNQ1 contains intronic variants associated with T2D susceptibility and β-cell dysfunction, but only maternally-inherited alleles confer increased disease risk. It maps within an imprinted domain with an established role in congenital and islet-specific growth phenotypes. Using human adult islet and foetal pancreas samples, I refined the transcripts and developmental stage at which T2D susceptibility must be conferred by demonstrating developmentally plastic monoallelic and biallelic expression. I identified a potential risk mechanism through the effect of T2D risk alleles upon DNA methylation. The disease-associated regions identified through genome-wide association (GWA) studies often contain multiple transcripts. I performed mRNA expression profiling of genes within loci associated with raised proinsulin/insulin ratios in human islets and metabolically relevant tissues. Some genes (notably CT62) were not expressed and therefore excluded from consideration for a risk effect, whilst others (for example C2CD4A) were highlighted as good regional candidates due to specific expression in relevant tissues. GWA studies for T2D risk have focused predominantly upon common single nucleotide polymorphisms. As part of a consortium conducing GWA analysis for copy number variation (CNV) and T2D risk, I optimised and compared alternative methods of CNV genotyping, before using this information to validate two signals of disease association. I genotyped three rare single nucleotide variants emerging from an association study with T2D risk based on imputed data, providing an indication of imputation accuracy and more powerful disease association analysis. These data underscore the challenge of translating association signals to causal mechanisms, and of identifying alternative forms of genomic variation which contribute to T2D risk. My work highlights candidates for functional analysis around proinsulin-associated loci, and makes significant progress towards uncovering risk mechanisms at the KCNQ1 locus.

The rising prevalence of type 2 diabetes (T2D) is a global problem, and suggests that we need better therapeutic strategies against this disease. The glycolytic enzyme glucokinase (GCK) catalyses the phosphorylation of glucose, and is a well-established T2D drug target. Rare GCK mutations cause monogenic beta-cell dysfunction, whilst common genetic variants within GCK are associated with fasting plasma glucose (FPG) levels and T2D risk. Since GCK is expressed in both the pancreas and liver, pharmacological GCK activation provides the promise of a two-pronged attack on hyperglycaemia. In vivo, GCK activity is modulated by the hepatic inhibitor glucokinase regulatory protein (GKRP, gene GCKR). GKRP negatively regulates GCK activity competitively with respect to glucose, and is controlled by fructose 6- and fructose 1-phosphate (F6P and F1P), which compete with each other for binding and enhance or diminish GCK inhibition respectively. GKRP also sequesters GCK in the nucleus and paradoxically stabilises the enzyme. As GCK and its regulatory protein are fundamental to glucose homeostasis, we aimed to investigate the role of genetic variation in both GCK and GCKR to further our understanding of these important T2D drug targets in a system that would be relevant to man. I demonstrated that two novel GCK mutations (T103S and V389L) identified in patients with hyperinsulinaemic hypoglycaemia were kinetically activating and through structural modelling identified a novel regulatory site for GCK activation by small molecular activators. Genome-wide association studies (GWAS) identified GCKR as a regulator of FPG and triglyceride levels, and showed a role for GKRP in T2D risk. Unlike most GWAS hits, this signal included a non-synonymous variant within GCKR (P446L), thus facilitating functional studies. P446L-GKRP was characterised kinetically and at the cellular sequestration-level. This variant showed diminished F6P-mediated modulation, which was proposed to reduce hepatic GCK inhibition, increase glycolytic flux (decreasing FPG), and feed metabolites into liver pathways (elevating triglycerides). As GCKR was not expressed at functional levels within human islets, this phenotype was thought to be driven by the liver. Preliminary analysis at the cellular level was inconclusive, with optimisation required to study human P446L-GKRP in this cellular system. Finally, I showed that mutations within GCKR are not a common cause of “GCK-Like” phenotypes in man, despite the regulatory protein directly modulating GCK activity. These data provide further insight as to the pathogenic consequences of perturbing GCK activity. This must be considered if this enzyme is to be the subject of therapeutic intervention in T2D.