A ataxia espinocerebelar tipo 2 (SCA2) é causada por uma expansão em uma sequência repetitiva de códons CAG (CAGexp) no exon 1 do gene ATXN2. O trato expandido de poliglutaminas está associado a um ganho de função tóxica, expressa predominantemente em neurônios. A SCA2 é uma condição neurodegenerativa autossômica dominante, com início, em média, entre os 30 e os 40 anos, manifesta-se por distúrbios neurológicos progressivos que levam os afetados à cadeira de rodas e à sobrevida reduzida. Embora propensa a forças seletivas negativas como a antecipação, a freqüência da SCA2 parece ser estável nas populações onde foi observada, sugerindo a existência de mecanismos seletivos favoráveis simultâneos. Para averiguar essa hipótese, o presente estudo estimou o fitness, a segregação e o papel da antecipação em uma coorte de famílias com diagnóstico molecular de SCA2. Dados de famílias com diagnóstico molecular realizado no Serviço de Genética Médica do Hospital de Clínicas e armazenados em banco de dados de acesso restrito aos investigadores serviram de base para identificar a população em estudo. Entre 2016 e 2017, sujeitos adultos e pertencentes a essas famílias foram convidados a participar desse estudo. Após apresentarem seu consentimento informado, esses sujeitos forneceram dados sobre todos os seus parentes ligados ao ramo familiar onde a SCA2 segregou, permitindo a construção de heredogramas completos. Os dados de ao menos dois informantes por família deveriam ser convergentes para serem estudados. As variáveis em estudo foram obtidas de todos os sujeitos: data de nascimento, sexo, relações de parentesco, ordem de nascimento, status sintomático e número de filhos. A data defalecimento e a idade de início do primeiro sintoma foram anotadas, quando adequado. O fitness genético (ou o sucesso reprodutivo) dos portadores de SCA2 foi considerado igual a w= W afetados /W não afetados, onde W corresponde à mediana do número de filhos do grupo. Como o genótipo não estava disponível para a maioria dos membros das famílias SCA2, o fenótipo foi usado para classificar os sujeitos como afetados ou não afetados. Para reduzir os efeitos de potenciais vieses de memória e de aferição, dois critérios de inclusão foram utilizados: (1) ser mais velho do que um desvio-padrão acima da média da idade de início dos sintomas do grupo total; e (2) existir a informação completa da história reprodutiva tanto o sujeito como do seu genitor transmissor. A análise de segregação comparou o número de irmãos afetados com o dos não afetados; como critério de inclusão, novamente todos os irmãos estudados deveriam ser mais velhos do que um desvio padrão da média de idade de início da coorte completa. Vinte e uma das trinta e uma famílias com diagnóstico de SCA2 foram recuperadas: os sujeitos de uma família negaram-se a participar do estudo, enquanto as demais dez famílias foram perdidas do follow-up. Os heredogramas estudados incluíram 1.017 pessoas, 164 das quais sintomáticas – e 84 delas ainda vivas. Como a idade de início média foi de 36.6 (14.9) anos, sujeitos afetados e não afetados com mais de 52 anos de idade foram utilizados para estimar padrões de fitness e segregação. Sessenta e sete sujeitos afetados e 97 sujeitos não afetados foram incluídos na análise do fitness e tiveram a mediana de 3 e 2 filhos respectivamente, o que resultou em um fitness genético igual a 1,5 (p <0,025) . Finalmente, o fitness variou de acordo com a idade de início dos sujeitos, tornando-se menor do que o fitness dos sujeitos não afetados quando a idade de início dos sintomas era menor ou igual a 20 anos de idade. 9 Entre os sujeitos incluídos na análise de segregação, havia 137 não-afetados (59,6%) e 93 afetados (40,4%) e essa proporção foi significativamente diferente da esperada em uma segregação randômica de alelos (p = 0,04). A idade de início foi inversamente proporcional ao tamanho do CAGexp em 49 indivíduos onde a informação molecular estava disponível (rho= -0.708, p< 0.0001, Spearman); cada repetição CAG adicional associou-se a uma redução de 1,57 anos na idade de início (p<0.003). A diferença média entre a idade de início dos sujeitos e a de seus genitores afetados foi de -10.77 anos, ou seja, a idade no início das gerações apontou para a antecipação como um fenômeno bastante frequente. Portanto, nossos resultados apontaram que três forças seletivas podem afetar a recorrência da SCA2. A SCA2 teve um fitness genético aumentado nos portadores com idades de início medianas ou tardias. Em contraste, nós observamos uma distorção na segregação ou favorecendo os gametas com alelos normais – em mais de 95% das vezes, portadores de alelos com 22 repetições (CAG)22 – ou desfavorecendo os gametas com alelos expandidos. Nosso resultado, inclusive, sugere que a alta frequência do alelo (CAG)22 nos cromossomos humanos seja explicada por essa vantagem seletiva. Por fim, nossos dados sugerem que a antecipação é a ocorrência mais frequente nas transmissões entre afetados na comparação entre as idades de início nas gerações subsequentes. Essa última observação propõe que a transmissão do CAGexp instável tende a expandi-lo ainda mais; isso precisa ser confirmado pela observação de irmandades completamente genotipadas, em futuros estudos. Se ela se confirmar, a tendência à antecipação deverá se associar a um fitness reduzido nesses afetados. Em conclusão, deduzimos que embora o fitness dos afetados com idades de início médias seja maior do que a dos não afetados, a frequência da SCA2 não está aumentando na população, pois outras forças seletivas agem 10 em sentido contrário (seleção negativa): a segregação distorcida e que favorece a transmissão do alelo normal e a tendência à antecipação. / Spinocerebellar ataxia type 2 (SCA2) is caused by an expansion in a repetitive sequence of CAG codons (CAGexp) in exon 1 of the ATXN2 gene. The expanded polyglutamine tract is associated with a gain in toxic function, expressed predominantly in neurons. SCA2 is an autosomal dominant neurodegenerative condition, with onset between 30 and 40 years of age, manifestations due to progressive neurological disorders that lead to those affected in the wheelchair and reduced survival. Despite being a community of companies, as an anticipation, a frequency of SCA2, which is a place for the communities where it was observed, suggesting the existence of simultaneous favorable selective mechanisms. To ascertain this hypothesis, the present study, fitness estimate, a segregation and role of anticipation in a cohort of families with molecular diagnosis of SCA2. Data from families with molecular diagnoses performed at the Medical Genetics Service of the Hospital de Clínicas and stored in databases of restricted access to the researchers served as a basis to identify the population under study. Between 2016 and 2017, adult subjects belonging to these families were invited to participate in this study. After presenting their informed consent, these subjects provided data on all their relatives related to the family branch where the SCA2 segregated, allowing the construction of complete heredograms. Data from at least two informants per family should be convergent to be studied. The study variables were obtained from all subjects: date of birth, gender, relationship, birth order, symptomatic status and number of children. The date of death and the age of onset of the first symptom were noted where appropriate. The genetic fitness (or reproductive success) of patients with SCA2 was considered equal to w = W affected / W unaffected, where W was the median number of children in the group. 12 As the genotype was not available for most members of the SCA2 families, the phenotype was used to classify the subjects as affected or unaffected. To reduce the effects of potential memory and bias biases, two inclusion criteria were used: (1) to be older than a standard deviation above the mean age of symptom onset of the total group; and (2) there is complete information on the reproductive history of both the subject and the transmitting parent. The segregation analysis compared the number of siblings affected with that of the unaffected; as inclusion criterion, again all siblings studied should be older than a standard deviation from the mean age of complete cohort start age. Twenty-one of the 31 families diagnosed with SCA2 were recovered: subjects from one family refused to participate in the study, while the remaining 9 families were lost from follow-up. The heredograms studied included 1,017 people, 164 of whom were symptomatic - and 84 of them were still alive. As the mean age at onset was 36.6 (14.9) years, affected and unaffected subjects over 52 years of age were used to estimate fitness and segregation patterns. Sixty-seven affected subjects and 97 unaffected subjects were included in the fitness analysis and had a median of 3 and 2 children respectively, which resulted in a genetic fitness equal to 1.5 (p <0.025). The vast majority of affected children were born before the onset of symptoms of these parents. Finally, the fitness varied according to the age of onset of subjects, becoming smaller than the fitness of the unaffected subjects when the age of onset of symptoms was less than or equal to 20 years of age. Among the subjects included in the segregation analysis, there were 137 unaffected (59.6%) and 93 affected (40.4%), and this proportion was significantly different from that expected in a random segregation of alleles (p = 0.04 ). The age of onset was inversely proportional to the size of CAGexp in 49 individuals where molecular information was available (rho = -0.708, p <0.0001, 13 Spearman); each additional CAG repeat was associated with a reduction of 1.57 years at the onset age (p <0.003). The mean difference between the age of onset of the subjects and that of their affected parents was -10.77 years: that is, the age at the beginning of the generations pointed to the anticipation as a frequent phenomenon. Therefore, our results indicated that three selective forces may affect the recurrence of SCA2. SCA2 had an increased genetic fitness in patients with median or late onset ages. In contrast, we observed a distortion in segregation or favoring gametes with normal alleles - more than 95% of the time, with alleles with 22 replicates (CAG) 22 - or disfavoring the gametes with expanded alleles. Our result even suggests that high frequency of the allele (CAG) 22 on human chromosomes is explained by this selective advantage. Finally, our data suggest that anticipation is the most frequent occurrence in transmissions between affected, in the comparison between the ages of onset in subsequent generations. This last observation proposes that the transmission of unstable CAGexp tends to expand it further; this needs to be confirmed by the observation of completely genotyped brotherhoods in future studies. If it is confirmed, the tendency to anticipate should be associated with a reduced fitness in this affected. In conclusion, although the fitness of the affected individuals with the mean onset ages is increased, the frequency of ACS2 is not increasing in the population because other selective forces act in the opposite direction (negative selection): distorted segregation and favors the transmission of the allele normal and the tendency to anticipate.

Pbp1 is a component of glucose deprivation induced stress granules and is involved in P-body-dependent granule assembly. We have recently shown that Pbp1 plays an important role in the interplay between three sensory protein kinases in yeast: AMP-regulated kinase (Snf1 in yeast), PAS kinase 1 (Psk1 in yeast), and the target of rapamycin complex 1 (TORC1), to regulate glucose allocation during nutrient depletion. This signaling cascade occurs through the SNF1-dependent phosphorylation and activation of Psk1, which phosphorylates and activates poly(A)- binding protein binding protein 1 (Pbp1), which then inhibits TORC1 through sequestration at stress granules. In this study we further characterized the regulation of Pbp1 by PAS kinase through the characterization of the role of the Psk1 homolog (Psk2) in Pbp1 regulation, and the identification of functional Pbp1 binding partners. Human ataxin-2 (ATXN2) is the homolog of yeast Pbp1 and has been shown to play an important role in the development of several ataxias. In this study we have also provided the evidence that human ataxin-2 can complement Pbp1 in yeast, and that human PAS kinase can phosphorylate human ataxin-2. Further characterizing this interplay between PAS kinase and Pbp1/ATXN2 aid in understanding pathways required for proper glucose allocation during nutrient depletion, including reducing cell growth and proliferation when energy is low. In addition, it yields valuable insights into the role of ataxin-2 in the development of devastating ataxias.

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI&lt;23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V. Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose. Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries. / On the aetiology of ALS: A comprehensive genetic study

Nutrient sensing kinases lie at the heart of cellular health and homeostasis, allowing cells to quickly adapt to changing environments. Target of Rapamycin (TOR) and PAS kinase (PASK, or PASKIN) are two such nutrient kinases, conserved from yeast to man. In yeast, these kinases each have paralogs. The two TOR paralogs in yeast mimic the mammalian TORC1 and TORC2 complexes, except both Tor1 and Tor2 may contribute to TORC1 or TORC2 function. The two PAS kinase paralogs are paired with the TOR paralogs, meaning that both Psk1 and Psk2 regulate TORC1, while Psk2 suppresses a temperature-sensitive allele of Tor2. Herein we review the evolutionary models for these paralogs, their function in yeast and mammalian cells, as well as the overlapping function of PAS kinase and TOR. We also use Rice University’s Direct Coupling Analysis algorithms to analyze co-evolutionary relationships and identify potential interaction sites between PAS kinase and several of its substrates.