Origem ancestral da doença de Machado Joseph : abordagem molecular

A doença de Machado-Joseph ou ataxia espinocerebelar do tipo 3 (MJD/SCA3) é uma doença neurodegenerativa de herança autossômica dominante, causada por expansões de repetições nucleotídicas CAG no gene ATXN3. A MJD/SCA3 é responsável por 69% dos casos de SCAs no Brasil e 84% no estado do Rio Grande do Sul (RS). Estudos prévios indicaram a presença de dois eventos mutacionais responsáveis pela origem ancestral do alelo mutante, um ligado ao haplótipo ACA e outro ao haplótipo GGC. O objetivo deste estudo foi compreender a presença e origem da MJD/SCA3 no Brasil, especialmente no estado do RS, onde há um alto número de pacientes diagnosticados. As análises foram realizadas em 262 indivíduos com diagnóstico molecular [220 do RS e 42 de outros estados do Brasil (1-MG, 9-PA, 1-PB, 13-RJ, 1-RN e 17-SP)] e em 50 indivíduos normais. Foram incluídos no trabalho a análise de 3 SNP intragênicos: (G669A, G987C e A1118C) e 4 STR extragênicos [(TAT)n, (CA)n, (AC)n e (GT)n]. A linhagem mais frequente nos alelos expandidos dos pacientes foi ACA (92% de todos os casos e 97% dos casos do RS), mas as linhagens GGC e AGA também foram encontradas. Nos controles, a linhagem GGC foi encontrada em 80% dos alelos normais, diferenciando-os dos alelos expandidos (p<0,05, teste x²). A análise dos STR mostrou que 85% dos pacientes da linhagem ACA compartilham um mesmo haplótipo (11-21-14-15), cujos pacientes eram provenientes dos estados do PA, PB, RJ, RS e SP. A linhagem GGC teve 54% dos pacientes compartilhando um haplótipo comum no grupo (16-22-18-19), provenientes dos estados de MG, PA, RJ e SP. A presença da MJD/SCA3 no Brasil é associada historicamente com a colonização portuguesa, principalmente com a colonização açoriana. A análise realizada nesse trabalho, com o uso dos marcadores moleculares, confirma a presença do mesmo haplótipo encontrado em pacientes portugueses, reforçando a ligação da presença da MJD/SCA3 com a colonização portuguesa. A alta frequência de um único haplótipo no RS confirma a hipótese de um efeito fundador no estado.The Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is a neurodegenerative disorder with autosomal dominant inheritance, caused by CAG repeats expansions in the ATXN3 gene. MJD/SCA3 is responsible for 69% of cases of SCAs in Brazil and 84% of cases from Rio Grande do Sul (RS) state. Previous studies indicated that two mutational events are responsible for ancestral origin of the mutant allele, one linked to ACA haplotype and other to GGC haplotype. The aim of this study was to understand the presence and origin of MJD/SCA3 in Brazil, especially in the state of Rio Grande do Sul. Analyses were performed in 262 patients with molecular diagnosis [220 from RS and 42 from other states in Brazil (1-MG, 9-PA, 1-PB, RJ-13, 1-RN, and 17-SP)] and 50 normal individuals. The study included the analysis of three intragenic SNPs (G669A, G987C and A1118C) and four STR outside the gene [(TAT)n, (CA)n (CA)n and (GT)n]. The most common lineage in patients’ mutant alleles was ACA (92% of all cases and 97% of RS cases), but lineages GGC and AGA were also found. In controls, the GGC lineage was found in 80% of normal alleles, being different from expanded alleles (p <0.05, X² test). STR analysis showed that 85% of patients from ACA lineage share the same haplotype (11-21-14-15), and those patients were from the following states: PA, PB, RJ, RS and SP. GGC lineage had 54% of patients sharing a comun haplotype (16-22-18-19) and patients were from MG, PA, RJ and SP states. The presence of MJD/SCA3 in Brazil is historically associated to the Portuguese colonization, especially from Azorean background. The analysis performed in this work using molecular markers confirms the presence of the same haplotype found in Portuguese patients, confirming the link to the Portuguese colonization. The high frequency of a single haplotype in RS confirms the hypothesis of a founder effect in the state

Biomarcadores na Doença de Machado-Joseph

A doença de Machado-Joseph (MJD), também conhecida como ataxia espinocerebelar do tipo 3 (SCA3), é uma doença neurodegenerativa de início tardio. SCA3/MJD é a mais comum dentre as ataxias espinocerebelares no mundo, tendo uma alta prevalência no sul do Brasil (seis casos em cada 100.000 indivíduos). SCA3/MJD é caracterizada por degeneração do cerebelo e o principal sintoma é a ataxia; no entanto, os sintomas são variados. A doença inicia normalmente entre os 30-40 anos de idade, podendo se manifestar em qualquer idade até na infância e apresenta uma progressão lenta. A doença é causada por uma mutação no gene ATXN3, onde uma expansão de repetições CAG (acima de 52 repetições) leva o indivíduo a manifestar a doença em algum momento da vida, sendo que o número de repetições CAG está associado à idade de início e gravidade da doença. No entanto, o fato das repetições CAG não explicarem completamente essas características e a progressão da doença ser lenta, se faz necessário o estudo de biomarcadores para identificação do início da doença e sua progressão. Nesta tese, serão abordados possíveis biomarcadores de estado e progressão na SCA3/MJD. Através da revisão de estudos com biomarcadores em SCA3/MJD, identificou-se que a proteína NSE, a glutationa peroxidase, o reflexo vestíbulo-ocular e vídeo-oculografia são bons candidatos a biomarcadores de estado da doença e as escalas clínicas ainda são os melhores biomarcadores de progressão. Analisando a composição corporal, foi identificado que o IMC está associado a repetições CAG expandidas. No entanto, não achamos associação com a doença, provavelmente devido à seleção dos nossos casos. Além disso, os indivíduos com SCA3/MJD apresentaram maior sensibilidade periférica à insulina quando comparados aos controles. Essa sensibilidade não está associada ao índice de massa corporal (IMC) e se correlaciona com a idade e gravidade da doença. O estudo de proteínas apresentou alteração em algumas neurotrofinas, onde NGF mostrou níveis mais baixos em pacientes com SCA3/MJD do que em controles, além de diminuir os níveis ao longo de 48 semanas. Uma correlação positiva foi observada entre NGF e Neurological Examination Score for Spinocerebellar Ataxia (NESSCA), além de uma correlação inversa entre BDNF e Scale for the assessment and rating of ataxia (SARA). Além disso, confirmamos níveis mais elevados de NSE. O CTNF diminui com a idade em controles, mas correlação semelhante em SCA3/MJD não atingiu significância estatistica. A análise de mRNA mostrou que pacientes com SCA3/MJD apresentam expressão reduzida de HDAC6 e que o uso de lítio aumenta a expressão dos genes HDAC6 e de GSK3β. O presente trabalho inclui o estudo de um amplo espectro de possíveis biomarcadores e é um dos poucos estudos que inclui análises prospectivas. Candidatos a biomarcadores de estado e progressão foram apresentados nesse estudo, contribuindo para a compreensão da fisiopatologia da doença e para possível uso em futuros ensaios clínico.Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a late-onset neurodegenerative disease. SCA3/MJD is the most common spinocerebellar ataxias worldwide, being highly prevalent in South Brazil, where it reaches six cases per 100,000 individuals. SCA3/MJD is characterized by cerebellum degeneration and ataxia is the main symptom of the disease. However, symptoms are diverse and progression is slow. The onset of disease usually begins at 30-40 years old, but onset can be seen at any age, even during childhood. SCA3/MJD is caused by a mutation at the ATXN3 gene, where an expansion of CAG repeats (above 52 repeats) leads to disease onset at some point in life. CAG repeat length is inversely correlated with age of onset and severity. The fact that CAG length does not explain all disease characteristics and the disease presents a slow disease progression; biomarkers studies are required for identification of disease onset and progression. In this thesis, it will be shown some candidates to state and progression biomarkers in SCA3/MJD. Through review of studies on biomarkers, it was identified NSE protein, glutathione peroxidase, vestibulo-ocular reflex and video-oculography as good candidates to biomarkers of disease state and clinical scales are the best progression biomarkers to date. Analyzing body composition, it was identified an association between BMI and CAG expanded repeats. However, no association was found with the disease, probably due to the selection of our cases. In addition, individuals with SCA3/MJD had higher peripheral sensitivity to insulin when compared to controls. This sensitivity was not associated with body mass index (BMI), and correlated with age and disease severity. Alterations in neurotrophins were demonstrated, where lower levels of NGF were shown in SCA3/MJD patients than in controls, with further reduction during 48 weeks long period. A positive correlation was seen between NGF and Neurological Examination Score for Spinocerebellar Ataxia (NESSCA), and an inverse correlation was seen between BDNF and Scale for the assessment and rating of ataxia (SARA) in SCA3/MJD at baseline. In addition, higher NSE levels were confirmed. CTNF was shown to decrease with age in controls, and this correlation was not observed in SCA3/MJD. mRNA analyses showed that X SCA3/MJD patients have reduced expression of HDAC6, and that treatment with lithium increases HDAC6 and GSK3β genes expression. We have studied a broad spectrum of possible biomarkers, and this is one of few studies including prospective analyses. Candidates to state and progression biomarkers were presented in this study, contributing to the understanding of disease physiopathology, and for a possible application in future clinical trials

State biomarkers for Machado Joseph disease : validation, feasibility and responsiveness to change

Machado-Joseph disease (SCA3/MJD) is the most common spinocerebellar ataxia worldwide, and particularly so in Southern Brazil. Due to an expanded polyglutamine at ataxin-3, SCA3/MJD presents a relentless course with no current disease modifying treatment. Clinical scales used to measure SCA3/MJD progression present moderate effect sizes, a major drawback for their use as main outcomes in clinical trials, given the rarity and slow progression of the disease. This limitation might be overcome by finding good surrogate markers. We present here a review of studies on peripheral and neurophysiological markers in SCA3/MJD that can be candidates for state biomarkers. Data on markers already studied were summarized, giving emphasis on validation against clinical scale, and responsiveness to change. While some biological fluid compounds and neurophysiological parameters showed poor responsiveness, others seemed to be good candidates. Some potential candidates that are waiting for responsiveness studies were serum levels of neuron specific enolase, vestibulo-ocular reflex and video-oculography. Candidates evaluated by RNA and microRNA expression levels need further studies to improve their measurements. Data on peripheral levels of Beclin-1 and DNAJB1 are promising but still incipient. We conclude that several potential candidates should follow onto validating studies for surrogate state biomarkers of SCA3/MJD

FOXO1 controls protein synthesis and transcript abundance of mutant polyglutamine proteins, preventing protein aggregation

FOXO1, a transcription factor downstream of the insulin/insulin like growth factor axis, has been linked to protein degradation. Elevated expression of FOXO orthologs can also prevent the aggregation of cytosine adenine guanine (CAG)-repeat disease causing polyglutamine (polyQ) proteins but whether FOXO1 targets mutant proteins for degradation is unclear. Here, we show that increased expression of FOXO1 prevents toxic polyQ aggregation in human cells while reducing FOXO1 levels has the opposite effect and accelerates it. Although FOXO1 indeed stimulates autophagy, its effect on polyQ aggregation is independent of autophagy, ubiquitin–proteasome system (UPS) mediated protein degradation and is not due to a change in mutant polyQ protein turnover. Instead, FOXO1 specifically downregulates protein synthesis rates from expanded pathogenic CAG repeat transcripts. FOXO1 orchestrates a change in the composition of proteins that occupy mutant expanded CAG transcripts, including the recruitment of IGF2BP3. This mRNA binding protein enables a FOXO1 driven decrease in pathogenic expanded CAG transcript- and protein levels, thereby reducing the initiation of amyloidogenesis. Our data thus demonstrate that FOXO1 not only preserves protein homeostasis at multiple levels, but also reduces the accumulation of aberrant RNA species that may co-contribute to the toxicity in CAG-repeat diseases