Stroke genetics and genomics

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2011This project presents a comprehensive approach to the identification of new genes that influence the risk for developing stroke. Stroke is the leading cause of death in Portugal and the third leading cause of death in the developed world. It is even more disabling than lethal, and the persistent neurological impairment and physical disability caused by stroke have a very high socioeconomic cost. Moreover, the number of affected individuals is expected to increase with the current aging of the population. Stroke is a “brain attack” cutting off vital blood and oxygen to the brain cells and it is a complex disease resulting from environmental and genetic factors. Major known risk factors include family history, age, hypertension, hypercholesterolemia, diabetes, cardiovascular disease, smoking and alcohol consumption. The common forms of stroke can be classified in two major clinical types: ischemic stroke (IS; most frequent) or hemorrhagic (10-20% of cases) stroke. Identification of genes increasing susceptibility to stroke could have far-reaching public health impact. The genetic component of this disease has been demonstrated in twin, family and animal model studies, and mutations have been found in several genes in rare classical Mendelian forms of stroke. However, very few susceptibility genes for the common forms of stroke have been identified and association studies have mostly reported conflicting results. In this project, to accomplish our goals in the study of IS, we first performed some candidate gene association analysis. Concomitantly, we applied the genomic convergence (GC) approach combining whole genome linkage screens, expression analysis, and case-control association studies. This unified, comprehensive, and multidisciplinary approach has not yet been implemented in other studies of stroke but the availability of new genetic, molecular and statistical tools, as well as their success in the study of other complex diseases, made such an approach both timely and essential. Since phosphodiesterase 4D (PDE4D) and arachidonate 5-lipoxygenase-activating protein (ALOX5AP) genes have been in recent years controversially implicated in the risk of IS, we assessed their association with IS in a Portuguese cohort. PDE4D degrades second messenger cyclic adenosine monophosphate, a key signal transduction molecule in different cell types, including inflammatory, vascular endothelial and smooth muscle cells. ALOX5AP is involved in the initial steps of leukotriene synthesis and is secreted by various types of inflammatory cells clustering at the injured sites in blood vessels. We genotyped 67 single nucleotide polymorphisms (SNPs) in the 5’end of PDE4D and 24 SNPs in ALOX5AP and both 10kb flanking regions on 565 Caucasian Portuguese patients and 518 unrelated controls. These SNPs are either tagging SNPs from HapMap or SNPs previously found associated. We tested their allelic, genotype and haplotype associations with IS risk, using standard qui-square tests (χ2) and multivariate logistic regression to adjust the analyses of association with risk for confounding factors, namely hypertension, diabetes and ever smoking. None of the previously associated SNPs were found associated with IS risk in our cohort, and considering the number of tests performed, we found no major involvement of other variants in these genes in stroke susceptibility in the Portuguese population. Only the SNP rs7442640 in PDE4D shows an association (p-value = 0.006) with IS risk when genotypic tests were adjusted for covariates, and SNP rs4491352 downstream of the ALOX5AP shows a modest evidence of association with IS risk (0.017< p-value < 0.025) for allelic and unadjusted genotypic tests. Performing a meta-analysis including all recently published studies and our Portugese and Spanish samples for SNP41, SNP45, SNP56, SNP87, and SNP89 (found associated in the original report) in PDE4D, no significant association results with IS risk were found. However, we found that SNP rs10507391 (or SG13S114) in ALOX5AP, which is part of the HapA haplotype, was associated with IS risk as described in the original study both in the Iberian population and in the meta-analysis performed. These results suggest that PDE4D may not constitute a major risk factor for IS in the Portuguese or Spanish populations, contrasting with ALOX5AP which may confer an increased risk of IS in the Iberian and other populations. We also assessed the association of the kalirin gene (KALRN) with IS in our Portuguese cohort since several recent studies have implicated its variants with susceptibility to cardiovascular and metabolic phenotypes, but no studies have yet been performed in stroke. Cerebrovascular and cardiovascular diseases are both complex disorders resulting from the interplay of genetic and environmental factors, and may share several susceptibility genes. KALRN is involved, among others, in the inhibition of inducible nitric oxide synthase, in the regulation of ischemic signal transduction, and in neuronal morphogenesis, plasticity and stability. Our goal was to determine whether SNPs in the KALRN region on 3q13, which includes the ropporin gene (ROPN1), predispose to IS in our cohort of Portuguese patients and controls. We genotyped 34 tagging SNPs in the KALRN and ROPN1 chromosomal region on 565 IS patients and 517 unrelated controls from our Portuguese case-control sample, and performed genotype imputation for 405 markers on chromosome 3. We tested the singlemarker and haplotype association of these SNPs with IS as explained above. One SNP in the ROPN1- KALRN intergenic region (rs4499545) and two SNPs in KALRN (rs17286604 and rs11712619) showed significant (0.003 < p-value < 0.049) allelic and genotypic (unadjusted and adjusted for hypertension, diabetes and ever smoking) association with IS risk. Thirty-two imputed SNPs also showed an association at p-value < 0.05, and actual genotyping of three of these polymorphisms (rs7620580, rs6438833 and rs11712039) validated their association. Furthermore, rs11712039 was associated with IS (0.001 < p-value < 0.01) in the genome-wide association study (GWAS) published by Ikram and co-authors (2009). These studies suggest that variants in the KALRN constitute risk factors for IS, and that KALRN may be a common genetic risk factor for vascular diseases. Additionally, we tested the association with the IS risk of the complement inhibitor factor H gene (CFH), as well as of several candidate genes related to neuroprotection: erythropoietin (EPO), heme-oxigenase 2 (HO2), and kallikrein 1 (KLK1) genes. CFH has been suggested to play an important role in the complement inhibition in atherosclerotic lesions and has been consistently associated with an increased risk for age-related macular degeneration (AMD) and myocardial infarction (MI) which share several risk factors with stroke. On the other hand, EPO, HO2 and KLK1 are neuroprotectors, for instance, in brain hypoxia and ischemia (EPO) and against induced stroke (KLK1). The polymorphism in CFH (rs1061170) previously associated with AMD and MI seems to be modestly associated (388 Portuguese patients and 461 controls; 0.030 < p-value < 0.035) with the IS risk in allelic and unadjusted genotypic tests, but no haplotype tagging SNP in this gene was clearly associated with IS in the GWAS performed by Ikram et al. (2009). Although we only study one polymorphism in the gene, these results did not justify a more in depth analysis. We genotyped 3, 3 and 5 tagging SNPs in the coding and 10 kb flanking regions of the EPO, HO2 and KLK1, respectively, on 565 IS patients and 518 controls from our Portuguese sample. No single-marker and haplotype associations were found for the studied SNPs in these neuroprotector genes, suggesting that they do not constitute genetic risk factors of IS. To identify novel susceptibility genes for IS, we applied the proposed GC approach. We performed gene expression analysis in peripheral blood mononuclear cells of 20 IS cases and 20 ageand sex-matched controls using Affymetrix GeneChip Human U133 Plus 2.0 arrays, which represent 47,000 human transcripts and variants. We identified several affected biological pathways in stroke patients, such as the cell adhesion molecules pathway. 16 out of the differentially expressed genes among cases and controls (1.2 fold-change cut-off and uncorrected p-value < 0.05) map to linkage peaks reported in published human whole-genome linkage studies. All tagging SNPs from these prioritized genes and from their 10 kb flanking regions (a total of 191 SNPs) were genotyped in 565 IS cases and 520 controls from our Portuguese biobank. Single-marker and haplotype association tests were performed. Association results suggest that variants in 6 (HEMGN, GFI1B, TMTC4, TTC7B, SDC4 and TUBB1) out of the 16 prioritized genes may constitute risk factors for IS in the Portuguese population. Several of the associated SNPs in these genes are also part of associated haplotypes. SNPs like the intronic rs9582406 and rs946845 polymorphisms in TMTC4, and the intronic rs2284278 in SDC4, were associated (0.015 < p-value < 0.050) with IS risk in all tests performed. On the other hand, the intronic SNP rs1535321 in TTC7B showed an association (p-value = 0.009) in allelic and unadjusted genotypic tests, even though no association in the adjusted test for hypertension, diabetes and ever smoking was verified. To follow-up these results, SNPs with single low-stringency significance association (p-value < 0.05) in at least one of the tests performed, and some SNPs that define associated haplotypes, were then genotyped in a Spanish dataset. A total of 570 Caucasian IS cases and 390 controls were included, and allelic, genotype and haplotype association tests with IS risk were conducted using also χ2 tests and multivariate logistic regression to adjust the analyses for hypertension, diabetes, dyslipidemic status and cigarette smoking. The same analyses were performed for the atherothrombotic, cardioembolic and lacunar forms of stroke. We found some significant associations with IS risk in TMTC4, TTC7B and SDC4, however, the only replicated SNP was rs9582406 in TMTC4 which was associated with IS risk in unadjusted tests (p-value = 0.019) in the Spanish casecontrol dataset. This SNP is also associated with the risk of atherothrombotic and lacunar forms of stroke for allelic and genotypic tests (0.011 < p-value < 0.049). For TTC7B and SDC4, the single SNPs and haplotypes associated in the Spanish sample were not the same as in the Portuguese cohort. The SNP rs6073708 in SDC4 was associated in the Spanish dataset for all tests performed (0.027 < p-value < 0.029). However, none of the studied SNPs were clearly replicated in the recent well-powered GWAS reported by Ikram and colleagues (2009). The overall results suggest that HEMGN and GFI1B (that were not replicated in the Spanish dataset) may constitute risk factors for IS in the Portuguese population, being important the enlargement of the Portuguese sample to validate the positive results. Similarly, TUBB1, which could not be genotyped in the Spanish cohort for technical problems, may constitute a risk factor for IS in the Portuguese population but should be further studied in other datasets to validate and understand its role in IS. TMTC4 may constitute a risk factor for IS in the Iberian population, and TTC7B and SDC4, with the observed heterogeneity of their significant association results among cohorts, may be novel risk factors for IS, being likely that their true susceptibility variants have not been studied yet. TTC7B was one of the top hits for major cardiovascular diseases in the Framingham Heart Study 100K project GWAS (Larson et al. 2007). For this gene, several SNPs and haplotypes in the intron 5 – intron 6 region associated in the Portuguese and Spanish datasets individually and combined, were modestly associated in the GWAS reported by Ikram and colleagues (2009). Multiple independent lines of evidence therefore support the role of TTC7B in stroke susceptibility, but further work is warranted to pinpoint the exact risk variant and to elucidate its pathogenic potential. In this project, given the very large number of SNPs tested, none of the significant findings would survive to multiple testing correction. However, it is generally accepted that replication in multiple independent datasets remains the gold-standard of association studies, even for modest associations. If our findings could be confirmed in other independent datasets and a most complete study of other possible genetic variants in the loci of interest could be performed, we think that functional studies of the genes and of their causative variants will allow a significant improvement of our knowledge on stroke disease. Deep sequencing may have to be used to precisely identify the true susceptibility genetic variants, or rare variants that the association studies have no power to detect. We suggest that identifying the genetic determinants of stroke using different strategies and populations and analysing them in an integrate view as performed in this project, is a most complete form to study the stroke in order to improve our knowledge of the disease.Este projecto apresenta uma abordagem multifactorial para a identificação de novos genes que influenciem o risco de sofrer acidentes vasculares cerebrais (AVCs). Os AVCs são a principal causa de morte em Portugal e a terceira maior causa de morte no conjunto dos países desenvolvidos. São ainda mais incapacitantes do que letais e os distúrbios neurológicos persistentes e a incapacidade física que provocam têm um custo socioeconómico muito elevado. Além disso, o número de pessoas afectadas deverá aumentar com o actual envelhecimento da população. Os AVCs são um “ataque cerebral” que interrompe o fluxo de sangue e oxigénio para as células de determinadas regiões do cérebro e são uma doença complexa resultante de factores genéticos e ambientais. Os principais factores de risco conhecidos incluem a idade, hipertensão, hipercolesterolemia, diabetes, doenças cardiovasculares, consumo de tabaco e de álcool e história familiar. As formas mais comuns dos AVCs podem ser classificadas em dois grandes tipos clínicos: AVCs isquémicos (AVCI; mais frequentes), ou hemorrágicos (10-20% dos casos). A identificação de novos genes que aumentem a susceptibilidade para se sofrer AVCs pode ter um forte impacto na saúde pública. A componente genética da doença tem sido demonstrada em estudos feitos em gémeos, famílias e modelos animais, e foram encontradas mutações em diversos genes em formas mendelianas raras de AVCs. No entanto, poucos genes de susceptibilidade para as formas comuns de AVCs foram até hoje identificados e os estudos de associação realizados apresentam geralmente resultados contraditórios. Neste projecto, para atingir os objectivos propostos no estudo dos AVCIs, começaram por realizar-se estudos de associação em alguns genes candidatos. Em paralelo, foi aplicada a abordagem de convergência genómica (CG) que combina estudos de ligação em todo o genoma, análises de expressão génica e estudos de associação em casos-controlos. Esta abordagem unificada, abrangente e multidisciplinar foi pela primeira vez implementada no estudo de AVCs. A disponibilidade de novas ferramentas genéticas, moleculares e estatísticas, bem como o seu sucesso no estudo de outras doenças complexas, tornam esta nova abordagem oportuna e essencial. Uma vez que os genes que codificam a fosfodiesterase 4D (PDE4D) e a proteína activadora de araquidonato 5-lipoxigenase (ALOX5AP) têm sido nos últimos anos controversamente implicados com o risco de sofrer AVCIs, avaliou-se neste projecto a sua associação com os AVCIs na nossa amostra portuguesa. A proteína PDE4D degrada o segundo mensageiro adenosina monofosfato cíclica, uma molécula de transdução de sinal chave em diferentes tipos de células inflamatórias, vasculares endoteliais e musculares lisas. A proteína ALOX5AP está envolvida nos passos iniciais da síntese de leucotrienos e é secretada por vários tipos de células inflamatórias aglomeradas em locais lesados de vasos sanguíneos. Foram genotipados 67 polimorfismos de um só nucleótido (SNPs) na extremidade 5' do gene PDE4D e 24 SNPs localizados no gene ALOX5AP e ambas regiões flanqueadoras de 10kb, em 565 pacientes caucasianos portugueses e 518 controlos independentes. Os SNPs seleccionados são tagging SNPs do projecto HapMap ou SNPs que foram encontrados previamente associados. Testaram-se as associações dos seus alelos, genótipos e haplótipos com o risco sofrer AVCIs, utilizando testes padrão do qui-quadrado (χ2) e regressões logísticas com múltiplas variáveis para ajustar as análises de associação com o risco para outros factores conhecidos, como a hipertensão, diabetes e consumo de tabaco. Nenhum dos SNPs previamente associados foram replicados na nossa amostra, e considerando o número de testes realizados, não foi encontrada nenhuma associação significativa de outras variantes destes genes na susceptibilidade para sofrer AVCs na população portuguesa. Apenas o SNP rs7442640 do gene PDE4D se encontra associado (p-value = 0,006) com o risco de sofrer AVCIs nos testes genotípicos ajustados para covariáveis e o SNP rs4491352 a jusante do gene ALOX5AP apresenta uma associação moderada (0,017 < p-value < 0,025) para os testes alélicos e genotípicos não ajustados. Realizando uma meta-análise incluindo todos os estudos publicados recentemente e as nossas amostras portuguesas e espanholas para os SNP41, SNP45, SNP56, SNP87 e SNP89 (que se encontram associados na publicação original) do gene PDE4D, não foram encontrados resultados de associação com o risco de sofrer AVCIs. No entanto, verificou-se que o SNP rs10507391 (ou SG13S114) do gene ALOX5AP, que faz parte do haplótipo HapA, está associado com o risco de sofrer AVCIs tal como descrito no seu estudo original, tanto na população ibérica, como na meta-análise realizada. Estes resultados sugerem que o gene PDE4D pode não ser o principal factor de risco para os AVCIs nas populações portuguesa e espanhola, contrastando com o gene ALOX5AP que pode conferir um risco aumentado para se sofrerem AVCIs na Península Ibérica e outras populações. Avaliou-se também a associação do gene kalirin (KALRN) com os AVCIs na nossa amostra portuguesa uma vez que vários estudos recentes têm implicado algumas das suas variantes com a susceptibilidade para fenótipos cardiovasculares e metabólicos, mas nenhum estudo foi ainda realizado em AVCs. As doenças cerebrovasculares e cardiovasculares são ambas complexas, resultantes da interacção de factores genéticos e ambientais, e podem partilhar diversos genes de susceptibilidade. A proteína KALRN está envolvida, entre outras funções, na inibição da enzima óxido nítrico sintase induzida, na regulação da transdução de sinais de isquémia, e na morfogénese neuronal, plasticidade e estabilidade. O nosso objectivo foi verificar se SNPs na região do gene KALRN, no cromossoma 3q13, que inclui o gene roporina (ROPN1), predispõem para os AVCIs na nossa amostra de pacientes e controlos portugueses. Foram genotipados 34 tagging SNPs na região cromossómica dos genes KALRN e ROPN1, em 565 pacientes com AVCIs e 517 controlos, e realizada a imputação de genótipos para 405 marcadores no cromossoma 3. Testaram-se as associações de cada SNP individualmente e dos haplótipos que constituem com os AVCIs. Um SNP na região intergénica ROPN1-KALRN (rs4499545) e dois SNPs no gene KALRN (rs17286604 e rs11712619) apresentam associações alélicas e genotípicas (não ajustadas e ajustadas para hipertensão, diabetes e consumo de tabaco) significativas (0,003 < p-value < 0,049) com o risco de sofrer AVCIs. Trinta e dois SNPs imputados encontram-se também associados com valores de p-value < 0,05, tendo sido validada a associação de três destes polimorfismos (rs7620580, rs6438833 e rs11712039) por genotipagem. Além disso, o SNP rs11712039 foi também associado com os AVCIs (0,001 <p-value <0,01) no estudo de associação em todo o genoma (GWAS) publicado por Ikram e seus co-autores (2009). Esses resultados sugerem que variantes no gene KALRN constituem factores de risco para os AVCIs, podendo ser factores de risco genéticos comuns das doenças vasculares. Foi também testada a associação com o risco de sofrer AVCs do factor H inibidor do complemento (CFH), bem como de vários genes candidatos relacionados com mecanismos de neuroprotecção: eritropoietina (EPO), hemoxigenase-2 (HO2) e calicreína 1 (KLK1). Tem sido sugerido que a proteína CFH desempenha um papel importante na inibição do complemento em lesões ateroscleróticas. Tem sido consistentemente associada com um risco aumentado para a degeneração macular relacionada à idade (AMD) e para os enfartes do miocárdio (MI) que compartilham vários factores de risco com os AVCs. Por outro lado, as proteínas EPO, HO2 e KLK1 são neuroprotetoras, por exemplo, em casos de hipóxia cerebral e isquémia (EPO) e contra acidente vascular cerebrais induzidos (KLK1). O polimorfismo no gene CFH (rs1061170) que foi previamente associado com a AMD e os MI parece estar modestamente associado (388 pacientes e 461 controlos portugueses; 0,030 < p-value <0,035) com o risco de sofrer AVCIs nos testes alélicos e genotípicos não ajustados, mas nenhum tagging SNP neste gene foi claramente associado com AVCIs no GWAS realizado por Ikram et al. (2009). Apesar de só ter sido estu

Variants in the inflammatory IL6 and MPO genes modulate stroke susceptibility through main effects and gene-gene interactions

A complex interplay between genetic background, clinical and life-style factors and the environment is expected to ultimately regulate the onset, acute phase and outcome of stroke. There is substantial evidence that inflammation within the Central Nervous System contributes to stroke risk, and known clinical risk factors for stroke, like atherosclerosis, diabetes, obesity, hypertension, and peripheral infection, are associated with an elevated systemic inflammatory profile. The inflammatory response is equally of major importance in recovery and healing processes after stroke. In this study we tested the genetic association of major inflammatory players IL1B (2q14), IL6 (7p21), TNF (6p21.3) and MPO (17q23.1) with stroke susceptibility and with stroke outcome at three months, in a population sample of 672 patients and 530 controls, adjusting for demographic, clinical and life-style risk factors and/or stroke severity parameters. The apparent complexity of the inflammatory mechanisms in stroke, and the multiplicity of players involved suggest a concerted process, in which implicated molecules interact to tightly regulate each other. We therefore examined both independent gene effects and the occurrence of gene-gene interactions among the tested inflammatory genes in stroke risk and stroke recovery. Two IL6 and one MPO SNP were significantly associated with stroke risk after multiple testing correction (0.022 correctedP 0.042), highlighting gene variants of low to moderate effect in stroke risk. An epistatic interaction between the IL6 and MPO genes was also identified in association with stroke susceptibility (P=0.031 after 1000 permutations). In the subset of 546 patients assessed for stroke outcome at three months using the modified Rankin Scale (mRS), we found one IL6 haplotype associated with stroke outcome (correctedP=0.024). In the present study we present supporting evidence for a role of the IL6 and MPO inflammatory genes in stroke susceptibility, and show that stroke risk is modulated by main gene effects together with clinical and life-style factors as well as by gene-gene interactions. Our findings are compatible and strengthen previous genetic and biological observations, highlighting the need of further functional studies, particularly in view of the possible utility of IL-6 as a diagnostic and/or prognostic biomarker for stroke

Genetic Variants Underlying Risk of Intracranial Aneurysms: Insights from a GWAS in Portugal

Subarachnoid hemorrhage (SAH) is a life-threatening event that most frequently leads to severe disability and death. Its most frequent cause is the rupture of a saccular intracranial aneurysm (IA), which is a blood vessel dilation caused by disease or weakening of the vessel wall. Although the genetic contribution to IA is well established, to date no single gene has been unequivocally identified as responsible for IA formation or rupture. We aimed to identify IA susceptibility genes in the Portuguese population through a pool-based multistage genome-wide association study. Replicate pools were allelotyped in triplicate in a discovery dataset (100 IA cases and 92 gender-matched controls) using the Affymetrix Human SNP Array 6.0. Top SNPs (absolute value of the relative allele score difference between cases and controls |RASdiff|≥13.0%) were selected for technical validation by individual genotyping in the discovery dataset. From the 101 SNPs successfully genotyped, 99 SNPs were nominally associated with IA. Replication of technically validated SNPs was conducted in an independent replication dataset (100 Portuguese IA cases and 407 controls). rs4667622 (between UBR3 and MYO3B), rs6599001 (between SCN11A and WDR48), rs3932338 (214 kilobases downstream of PRDM9), and rs10943471 (96 kilobases upstream of HTR1B) were associated with IA (unadjusted allelic chi-square tests) in the datasets tested (discovery: 6.84E-04≤P≤1.92E-02, replication: 2.66E-04≤P≤2.28E-02, and combined datasets: 6.05E-05≤P≤5.50E-04). Additionally, we confirmed the known association with IA of rs1333040 at the 9p21.3 genomic region, thus validating our dataset. These novel findings in the Portuguese population warrant further replication in additional independent studies, and provide additional candidates to more comprehensively understand IA etiopathogenesis.FCT grant: (CMUP-ERI/TPE/0028/2013), FCT fellowships and research contracts (SFRH/BPD/35737/2007, SFRH/BPD/70008/2010, SFRH/BD/43895/2008 and Ciência and Investigator- FCT contracts)

A systematic review and meta-analysis of enzyme replacement therapy in late-onset Pompe Disease

Pompe disease (PD) is a glycogen storage disorder caused by deficient activity of acid alpha-glucosidase (GAA). We sought to review the latest available evidence on the safety and efficacy of recombinant human GAA enzyme replacement therapy (ERT) for late-onset PD (LOPD). Methods: We systematically searched the MEDLINE (via PubMed), Embase, and Cochrane databases for prospective clinical studies evaluating ERT for LOPD on pre-specified outcomes. A meta-analysis was also performed. Results: Of 1601 articles identified, 22 were included. Studies were heterogeneous and with very low certainty of evidence for most outcomes. The following outcomes showed improvements associated with GAA ERT, over a mean follow-up of 32.5 months: distance walked in the 6-min walking test (6MWT) (mean change 35.7 m (95% confidence interval [CI] 7.78, 63.75)), physical domain of the SF-36 quality of life (QOL) questionnaire (mean change 1.96 (95% CI 0.33, 3.59)), and time on ventilation (TOV) (mean change -2.64 h (95% CI -5.28, 0.00)). There were no differences between the pre- and post-ERT period for functional vital capacity (FVC), Walton and Gardner-Medwin Scale score, upper-limb strength, or total SF-36 QOL score. Adverse events (AEs) after ERT were mild in most cases. Conclusion: Considering the limitations imposed by the rarity of PD, our data suggest that GAA ERT improves 6MWT, physical QOL, and TOV in LOPD patients. ERT was safe in the studied population. PROSPERO register: 135102

Mitochondrial haplogroup H1 is protective for ischemic stroke in Portuguese patients

<p>Abstract</p> <p>Background</p> <p>The genetic contribution to stroke is well established but it has proven difficult to identify the genes and the disease-associated alleles mediating this effect, possibly because only nuclear genes have been intensely investigated so far. Mitochondrial DNA (mtDNA) has been implicated in several disorders having stroke as one of its clinical manifestations. The aim of this case-control study was to assess the contribution of mtDNA polymorphisms and haplogroups to ischemic stroke risk.</p> <p>Methods</p> <p>We genotyped 19 mtDNA single nucleotide polymorphisms (SNPs) defining the major European haplogroups in 534 ischemic stroke patients and 499 controls collected in Portugal, and tested their allelic and haplogroup association with ischemic stroke risk.</p> <p>Results</p> <p>Haplogroup H1 was found to be significantly less frequent in stroke patients than in controls (OR = 0.61, 95% CI = 0.45–0.83, p = 0.001), when comparing each clade against all other haplogroups pooled together. Conversely, the pre-HV/HV and U mtDNA lineages emerge as potential genetic factors conferring risk for stroke (OR = 3.14, 95% CI = 1.41–7.01, p = 0.003, and OR = 2.87, 95% CI = 1.13–7.28, p = 0.021, respectively). SNPs m.3010G>A, m.7028C>T and m.11719G>A strongly influence ischemic stroke risk, their allelic state in haplogroup H1 corroborating its protective effect.</p> <p>Conclusion</p> <p>Our data suggests that mitochondrial haplogroup H1 has an impact on ischemic stroke risk in a Portuguese sample.</p

Variants of the Matrix Metalloproteinase-2 but not the Matrix Metalloproteinase-9 genes significantly influence functional outcome after stroke

<p>Abstract</p> <p>Background</p> <p>Multiple lines of evidence suggest that genetic factors contribute to stroke recovery. The matrix metalloproteinases -2 (MMP-2) and -9 (MMP-9) are modulators of extracellular matrix components, with important regulatory functions in the Central Nervous System (CNS). Shortly after stroke, MMP-2 and MMP-9 have mainly damaging effects for brain tissue. However, MMPs also have a beneficial activity in angiogenesis and neurovascular remodelling during the delayed neuroinflammatory response phase, thus possibly contributing to stroke functional recovery.</p> <p>Methods</p> <p>In the present study, the role of <it>MMP-2 </it>and <it>MMP-9 </it>genetic variants in stroke recovery was investigated in 546 stroke patients. Functional outcome was assessed three months after a stroke episode using the modified Rankin Scale (mRS), and patients were classified in two groups: good recovery (mRS ≤ 1) or poor recovery (mRS>1). Haplotype tagging single nucleotide polymorphisms (SNPs) in the <it>MMP-2 </it>(N = 21) and <it>MMP-9 </it>(N = 4) genes were genotyped and tested for association with stroke outcome, adjusting for significant non-genetic clinical variables.</p> <p>Results</p> <p>Six SNPs in the <it>MMP-2 </it>gene were significantly associated with stroke outcome (0.0018<<it>P </it>< 0.0415), two of which survived the Bonferroni correction for multiple testing. In the subset of ischemic stroke patients, association of five of these SNPs remained positive (0.0042<<it>P </it>< 0.0306). No significant associations were found for the <it>MMP-9 </it>gene.</p> <p>Conclusions</p> <p>The results presented strongly indicate that <it>MMP-2 </it>genetic variants are an important mediator of functional outcome after stroke.</p

Laser-induced breakdown spectroscopy (LIBS) for tropical soil fertility analysis

The Laser Induced Breakdown Spectroscopy (LIBS) is a promising technique for soil fertility analysis in a rapid and environmentally friendly way. This application requires the selection of an optimal modelling procedure capable of handling the high spectral resolution of LIBS. This work aimed at comparing different modelling methods of LIBS data for the determination of key fertility attributes in Brazilian tropical soils. A benchtop LIBS system was used for the analysis of 102 soil samples, prepared in the form of pressed pellets. Models for the prediction of clay, organic matter, pH, cation exchange capacity, base saturation, and the extractable nutrients P, K, Ca, and Mg were developed using univariate linear regression (ULR), multiple linear regression (MLR) and partial least squares regression (PLS). The following input data for PLS were used: (i) the full spectra from 200 to 540 nm (38,880 variables), and (ii) variables selected by the interval successive projections algorithm (iSPA). The multivariate models achieved satisfactory predictions [residual prediction deviation (RPD) > 1.40] for eight out of nine fertility attributes. However, the best performances were obtained for the PLS with the variable ranges selected by the iSPA, which achieved satisfactory predictions (RPD ≥ 1.44) for seven out of the nine soil attributes studied. The MLR method obtained lower prediction performance than the iSPA-PLS using only 21 variables. The iSPA-PLS approach allowed a reduction from 3 to 160-fold in the total of variables compared to the full LIBS spectra, making it efficient and accurate modelling method that uses reduced number of variables. Although LIBS technique proved to be efficient for predicting fertility attributes in tropical soils, further research is encouraged in order to reduce the amount of sample preparation conducted in this study