Genetic Variation and Atherosclerosis

A family history of atherosclerosis is independently associated with an increased incidence of cardiovascular events. The genetic factors underlying the importance of inheritance in atherosclerosis are starting to be understood. Genetic variation, such as mutations or common polymorphisms has been shown to be involved in modulation of a range of risk factors, such as plasma lipoprotein levels, inflammation and vascular calcification. This review presents examples of present studies of the role of genetic polymorphism in atherosclerosis

Estudo molecular de dislipidemias familiares

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012A hipercolesterolemia familiar (FH) é uma dislipidemia de transmissão autossómica dominante que se caracteriza por níveis elevados de colesterol no plasma e aparecimento prematuro de aterosclerose e de doença cardiovascular. Mutações nos genes LDLR, APOB e PCSK9 são causa de FH. A dislipidemia familiar combinada (FCHL) é uma hiperlipidemia poligénica também relacionada com a ocorrência de DCV prematura. O seu fenótipo tem sido associado a alterações nos genes LPL, APOC2, APOC3, APOA4 e APOA5. O objectivo deste trabalho consistiu na caracterização clínica e molecular de indivíduos com suspeita clínica de dislipidemia familiar, nomeadamente FH. Foi também objectivo do estudo a avaliação dos critérios clínicos da FH. A amostra de estudo consistiu em 40 casos-índex (crianças e adultos de ambos os sexos) referenciados ao INSA por suspeita clínica de FH. A caracterização bioquímica permitiu avaliar o fenótipo do doente e inferir sobre a aplicação de critérios de diagnóstico clínico de FH. A caracterização molecular incluiu a pesquisa de alterações nos genes LDLR, APOB e PCSK9, recorrendo às metodologias de dHPLC, sequenciação automática e MLPA. Em 5 doentes que apresentavam também TG elevados foi efectuada a pesquisa de alterações nos genes LPL, APOC2, APOC3, APOA4 e APOA5, por sequenciação automática dos mesmos. O estudo molecular dos 40 casos-índex estudados identificou 15 (37,5%) indivíduos com FH. Em 31-39% dos 609 casos-índex portugueses já estudados no EPGH, o diagnóstico clínico resultante da aplicação dos 2 critérios clínicos de FH aplicados não coincide com os resultados do estudo molecular. Relativamente à FCHL, o estudo molecular somente identificou alterações que estão descritas como polimorfismos. A identificação precoce de casos-índex com dislipidemia familiar e dos seus familiares em risco pode aumentar a qualidade e a esperança de vida destes indivíduos, por aplicação de uma orientação terapêutica adequada, que possa possibilitar a redução da incidência de doenças cardiovasculares prematuras.Familial hypercholesterolaemia (FH) is an autosomal dominant disorder that is characterized by increased levels of cholesterol and premature arteriosclerosis and cardiovascular disease. Mutations in LDLR, APOB and PCSK9 genes are responsible for FH. Familial combined hyperlipidaemia (FCHL) is a polygenic disorder also related with the presence of premature cardiovascular disease. The phenotype of FCHL has been associated with defects in the LPL, APOC2, APOC3, APOA4 and APOA5 genes. The aim of this work was the clinical and molecular characterization of patients with a clinical diagnosis of familial dyslipidaemia, particularly FH. Another purpose of this study was the evaluation of the clinical criteria for FH. The sample was composed of 40 index patients (adults and children of both sexes), referred to INSA with clinical diagnosis of FH. The biochemical characterization allowed the evaluation of the phenotype of the patient and made possible the inference of the application of clinical diagnostic criteria of FH. The molecular characterization included the search for defects in LDLR, APOB and PCSK9 genes, using the techniques of dHPLC, automated sequencing and MLPA. In 5 of these patients that also presented high levels of TG, the search for defects in LPL, APOC2, APOC3 APOA4 and APOA5 genes was done by automated sequencing. The molecular study of the 40 índex patients had identified 15 (37,5%) patients with an alteration in one of the three genes associated with FH. In 31-39% of the total 609 Portuguese índex patients that had already been studied by PFHS, the clinical diagnosis obtained from the application of the clinical criteria didn´t match with the results from the molecular study. The molecular study of genes associated with FCHL had just identified genetic defects that are referred as polymorphisms. An early identification of index patients with familial dyslipidaemia and of relatives at risk can increase the quality of life and the life expectancy, by the application of an adequate therapy that can help to reduce the incidence of premature cardiovascular diseases

Comparative analysis of familial hypercholestrerolaemia in different populations

Thesis (Ph.D.) -- University of Stellenbosch, 1999.ENGLISH SUMMARY: Familial hypercholesterolaemia (FH) and familial defective apolipoprotein B-IOO (FDB) are relatively common disorders of lipid and lipoprotein metabolism caused by mutations in the low density lipoprotein receptor (LDLR) and apolipoprotein B (apo B) genes, respectively. DNA analyses at these loci were performed in 132 molecularlyuncharacterised South African, 11 Costa Rican and 13 New Zealand subjects with clinical features of heterozygous FH. Mutation R3500Q causing FDB was identified in a relatively large proportion (~30%) of the New Zealand patients. LDLR gene defects were identified in 4 Costa Rican and 6 New Zealand FH patients. Sixty-five different LDLR gene mutations were identified in South African hypercholesterolaemics, revealing ten founder-type mutations. Haplotype analysis at the LDLR and apo B loci excluded the likelihood that mutations in these two genes underlie the FH phenotype in one of the New Zealand families. The apparently autosomal dominant hypercholesterolaemia (ADH) in this family could also not be linked to a newly identified gene locus, designated FH3. Analysis of the New Zealand study cohort, although small, demonstrated both mutational and locus heterogeneity in ADH. Analysis was also extended to include subjects from the various ethnic groups within South Africa. The high prevalence of FH in Afrikaners of European descent is in striking contrast to the reported virtual absence of this lipid disorder in the Black South African population. In addition to three previously-described Afrikaner founder mutations (D154N, D206E and V408M), four minor founder mutations, D200G, S285L, C356Y and G361V, were identified in 12 Afrikaner families. Surprisingly, a 6-bp deletion in exon 2 of the LDLR gene was detected at a relatively high frequency (28%) in Black FH patients. This finding, as well as clinical correlations performed in the patients, suggests that the expression of FH mutations in the Black population may be altered due to interaction with other genetic and/or environmental factors, therefore leading to underdiagnosis of the disease. Common LDLR gene mutations have also been described in South African Indians (P664L) and Jews (del 197), most likely as a consequence of multiple introductions of defective genes into these relatively isolated communities. Caucasoid admixture was recognised as a major factor contributing to the FH phenotype in the indigenous South African population of mixed ancestry from the Western Cape, where six founder-type mutations account for the disease in 22% of cases. The high prevalence of specific LDLR gene mutations in different population groups facilitates an improved diagnostic service for FH in South Africa.AFRIKAANSE OPSOMMING: Familiele hipercholesterolemie (FH) en familiele defektiewe apolipoprotelen B-I00 (FDB) is relatief algemene afwykings in lipied en lipoprotelen metabolisme wat onderskeidelik veroorsaak word deur mutasies in die lae digtheids lipoprotelen reseptor (LDLR) en apolipoproteleri B-I00 (apo B) gene. Molekulere DNS analise van hierdie lokusse is uitgevoer in 132 Suid Afrikaanse, 11 Costa Rikaanse en 13 New Zealandse pasiente waar die geen mutasies onderliggend, aan die kliniese beeld van heterosigotiese FH onbekend was. Mutasie R3500Q wat FDB veroorsaak was in 'n relatief groot aantal van die New Zealandse pasiente (~30%) teenwoordig. LDLR geen defekte is in 4 Costa Rikaanse en 6 New Zealandse FH pasiente geldentifiseer. Vyf en sestig verskillende LDLR geen mutasies is aangetoon in die Suid Afrikaanse populasie waarvan tien stigtergeen mutasies is. Haplotipe analise van die LDLR en apo B lokusse het die moontlikheid uitgesluit dat mutasies in hierdie twee gene verantwoordelik is vir die FH fenotipe in een van die New Zealandse families. Die waarskynlik outosomaal dominante hipercholesterolemie (ODH) in hierdie familie kon ook nie toegeskryf word aan 'n nuwe geidentifiseerde geen lokus genaamd FH3 nie. Analise van die New Zealandse studie paneel het dus beide mutasie en lokus heterogeniteit in ODH gedemonstreer. Analise was uitgebrei deur die toevoeging van individue van verskeie etniese groepe van Suid-Afrika. Die hoe voorkoms van FH in Afrikaners van Europese afkoms is , in opvallende kontras met die voorheen vermeende feitlike afwesigheid van hierdie lipied afwyking in die Swart Suid-Afrikaanse populasie. Afgesien van drie bekende Afrikaner stigter mutasies (D154N, D206E en V408M), is nog vier relatief algemene mutasies, D200G, S285L, C356Y en G361V, ge'identifiseer in 12 Afrikaner families. 'n Onverwagse bevinding was die opsporing van 'n 6-bp delesie in ekson 2 van die LDLR geen teen 'n relatief hoe frekwensie (28%) in Swart FH pasiente. Hierdie bevinding, sowel as kliniese korrelasies wat in hierdie groep pasiente uitgevoer is, impliseer dat FH moontlik ondergediagnoseer word in die Swart populasie weens interaksie van defektiewe LDLR gene met ander genetiese en/of omgewingsfaktore. Algemene LDLR geen mutasies is ook beskryf in Suid Afrikaanse Indiers (P664 L) en J ode (del 197), heel waarskynlik as 'n gevolg van veelvuldige oordrag van defektiewe gene in hierdie relatief geisoleerde gemeenskappe. Kaukasier vermenging is herken as 'n belangrike faktor onderliggend aan die FH fenotipe in die inheemse W es-Kaapse kleurling populasie van Suid-Afrika, waar ses stigter-tipe mutasies verantwoordelik is vir die siekte in 22% van' gevalle. Die hoe voorkoms van spesifieke LDLR geen mutasies in verskillende populasie groepe maak populasie-gerigte DNA dililgnose van FH moontlik in Suid Afrika

Estudo bioquímico e molecular de famílias com hipercolesterolemia familiar

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012Introdução: A hipercolesterolemia familiar (FH) é uma patologia genética, caracterizada clinicamente por um aumento dos níveis de colesterol-LDL (c-LDL) no plasma, sendo maioritariamente causada por mutações no gene do receptor das LDL (gene LDLR). Mutações missense nos genes APOB ou PCSK9 podem causar fenótipos semelhantes. A FH sozinha ou em conjugação com outros factores de risco cardiovasculares pode promover o desenvolvimento de doenças cardiovasculares (DCVs) prematuras. De acordo com a frequência da doença (1:500 indivíduos) estima-se que em Portugal existam cerca de 20.000 doentes com FH, porém esta encontra-se sub-diagnosticada. Até à data, apenas foram diagnosticados aproximadamente 550 doentes. O método de cascade screening (CS) permite a rápida identificação de indivíduos com FH numa família e foi descrito como custo-efectivo na identificação de novos doentes com FH. Objectivos: O principal objectivo deste trabalho é, através do CS, aumentar o número de casos identificados com FH. Pretendeu-se ainda realizar a caracterização bioquímica de casos índex e respectivos familiares com FH, a fim de tentar correlacionar o genótipo e o fenótipo destes e compreender de que forma a FH e outros factores de risco cardiovascular influenciam o desenvolvimento de DCVs nesta população. Métodos: Para alcançar os objectivos propostos, estudaram-se 45 famílias, perfazendo um total de 194 indivíduos, 45 casos índex (cujo estudo molecular já havia sito realizado) e 149 familiares. As amostras de soro de casos índex e familiares foram analisados por electroforese de lipoproteínas para avaliação da presença de sdLDL e por métodos enzimáticos e colorimétricos automatizados para determinar a concentração de sdLDL e outras apolipoproteínas no soro. Após extracção de DNA dos familiares realizou-se a amplificação e sequenciação dos exões em que se detectou uma mutação no respectivo caso índex (gene LDLR, APOB). Foi ainda determinado o genótipo APOE de todos os indivíduos estudados. Os resultados da sequenciação foram analisados através do software staden package. Por fim realizou-se uma análise estatística de forma a interpretar os resultados obtidos. Resultados: A partir do estudo molecular aos 149 familiares, foram diagnosticados com FH 83 indivíduos (20 crianças e 63 adultos). Determinou-se que 71,79% das crianças e 76,62% dos adultos apresentam um genótipo APOE E3/E3, não se obtendo diferenças estatisticamente significativas entre os diferentes alelos e o perfil bioquímico. Relativamente à estratificação das sdLDL, 65,52% das crianças e 72,88% dos adultos apresentam, maioritariamente, subfracções maiores e menos densas das LDL. Observou-se ainda que os níveis de Lipoproteína(a) são tendencialmente superiores em indivíduos com DCV. Da amostra total, 47,93% dos indivíduos com FH não estão ainda medicados. Porém, mesmo os indivíduos medicados apresentam um perfil lipídico de risco para o desenvolvimento de uma DCV prematura. Conclusão: O CS é um método cujo custo-benefício é extremamente eficaz. Sendo, no entanto, necessário, aumentar a adesão dos familiares. O CS permite identificar precocemente indivíduos com FH, que apresentam elevado risco cardiovascular, necessitando de intervenções farmacológica e de controlo de outros factores de risco adequadas. Os resultados obtidos demonstram que mais de 50% dos casos não tem o seu risco cardiovascular controlado, sendo essencial uma melhor compreensão sobre a dinâmica existente entre as DCVs e a FH.Familial Hypercholesterolemia (FH) is a genetic condition, clinically characterized by increased levels of LDL-cholesterol circulating in plasma and is mainly caused by mutations in receptor LDL (LDLR gene), but missense mutations in APOB and PCSK9 genes may also cause similar phenotypes. FH combined with the presence of other cardiovascular risk factors, may promote the development of premature cardiovascular diseases (CVD). According to the frequency of FH (1:500 individuals) estimated in Portugal, there are about 20.000 cases of FH, but is under-diagnosed. To date, only approximately 550 FH patients were identified. The Cascade Screening (CS) method allows the rapid identification of FH individuals with a family and was described as a cost-effective method to identify new FH patients. Objectives: The main goal of this work is, through CS, increase the number of individuals diagnosed with FH. It is also proposed to perform a biochemical characterization of index cases with FH and their relatives to try to correlate genotype and phenotype of these subjects and understand how the FH and other cardiovascular risk factors may influence the development of CVD in our population. Methods: To achieve these objectives we studied 45 families in a total of 194 individuals: 45 index cases (whose molecular study had already been done) and 149 relatives. Serum samples from index cases and relatives were analyzed for lipoproteins by electrophoresis and stratification of sdLDL. Through an automated enzymatic and colorimetric methods were quantified sdLDL and others apolipoproteins in serum samples, respectively. After de DNA extraction of relatives, the amplification and sequencing of the exon in which a mutation was detected in the respective index case (LDLR or APOB gene) was performed. It was further determined the APOE genotype for all the subjects in study. The sequencing results were analyzed using staden package software. At last, was performed a statistical analysis in order to interpret the results. Results: From the molecular study of 149 relatives, 83 were diagnosed with FH (20 children and 63 adults). It was determined that 71,79% of the children and 76,62% of the adults have E3/E3 APOE genotype. Through the sdLDL stratification, it was observed that 65.52% of children and 72.88% of adults have, mostly sub fractions larger and less dense LDL. It was also observed that the levels of Lipoprotein(a) tends to be higher in subjects with CVD. Of the total sample, 47,93% of individuals with FH are not yet treated. However, even individuals taking medication have a lipid risk for the development of a premature CVD. Conclusion: CS is a extremely cost-effective method. However, is necessary the increase of relatives adherence. The CS allows the early identification of individuals with FH who have high cardiovascular risk, requiring pharmacologic intervention and control of other cardiovascular risk factors. The results show that over than 50% of cases do not have their cardiovascular risk controlled and so is essential a better understanding of the dynamics between the CVD and FH

Software and database for the analysis of mutations in the human LDL receptor gene.

The low-density lipoprotein receptor (LDLr) plays a pivotal role in cholesterol homeostasis. Mutations in the LDLr gene (LDLR), which is located on chromosome 19, cause familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by severe hypercholesterolemia associated with premature coronary atherosclerosis. To date almost 300 mutations have been identified in the LDLR gene. To facilitate the mutational analysis of the LDLR gene, and promote the analysis of the relationship between genotype and phenotype, a software package along with a computerized database (currently listing 210 entries) have been created