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

REGULATION OF LOW DENSITY LIPOPROTEIN RECEPTOR SPLICING EFFICIENCY

Low density lipoprotein receptor (LDLR) is an apolipoprotein E (apoE) receptor and may play a role in Alzheimer’s disease (AD) development. A single nucleotide polymorphism (SNP), rs688, that has been identified to modulate the splicing efficiency of LDLR exon 12 and is associated with higher cholesterol and AD in some case-control populations. The exon 12 deleted mRNA is predicted to produce a soluble form of LDLR that fails to mediate apoE uptake. To gain additional insights, in this study, I seek to understand the regulation of LDLR splicing efficiency. To identify functional cis-elements within LDLR exon 12, I mutated several conserved putative exonic splicing enhancers (ESEs) to neutralize their affinity to serine/arginine-rich (SR) proteins. Transfection of wild type (WT) or mutant LDLR minigenes in HepG2 cells was performed, and splicing efficiency evaluated by quantitative RT-PCR. The results showed that two functional ESEs within exon 12, near rs688, are critical to LDLR splicing. To identify splicing factors that modulate exon 12 splicing, I co-transfected an LDLR minigene and vectors encoding different SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). After quantifying the splicing efficiency, I found that SRp20 and SRp38 increased exon 11- skipping. Moreover, ectopic expression of SRp38-2 and hnRNP G increased exon 11&12-skipping. Interestingly, the actions of hnRNP G did not require its RNA recognition motif (RRM). To further investigate the role of theses splicing factors on LDLR splicing, I quantified the expression level of these splicing factors as well as LDLR splicing efficiency in human brain and liver. I found that SRp38 mRNA expression is associated with LDLR splicing efficiency. In conclusion, this study discovered that rs688 is located close to the two functional ESEs within LDLR exon 12, and revealed a role of SRp38 in LDLR splicing efficiency

Molecular analysis of the LDLR gene in coronary artery disease patients from the Indian population

BACKGROUND: Cardiovascular disease is a leading cause of mortality in Indian population. Mutations in LDLR, APOB and PCSK9 genes may lead to Familial Hypercholesterolemia, an autosomal dominant disorder which in turn leads to cardiovascular diseases. The primary objective of this study is to analyze these genes in CAD patients of Indian population. METHODS: A total of 30 patients were selected out of 300 CAD patients based on UK-Simon Broome criteria from South India. The gDNA was isolated by organic extraction method and the exons and exon-intron boundaries of LDLR gene, APOB (exon 26) and PCSK9 (exon 7) were screened by PCR-high resolution melt analysis. The amplicons showing shift in melting pattern were sequenced to find out the variation. RESULTS: This study reports three novel variations, an intronic deletion c.694+8_694+18del in intron 4, a synonymous variation c.966 C>T [p. (N322=)] in exon 7 and a deletion insertion c.1399_1340delinsTA [p. (T467Y)] in exon 10, two recurrent variations c.862G>A [p. (E288K)] in exon 6 and a splice site variation c.1845+2T>C in exon-intron junction of exon 12 in LDLR gene and PCSK9 gene had c.1180+17C>T change in intron 7. However there are no pathogenic variations in APOB and PCSK9 genes in Indian population. In silico analysis predicted all the variations as pathogenic except the synonymous variation. CONCLUSION: This report adds five new variations to the spectrum of LDLR variations in Indian population. This study also suggests that UK Simon Broom criteria can be followed to categorize FH patients in Indian population

Predominance of a 6 bp deletion in exon 2 of the LDL receptor gene in Africans with familial hypercholesterolaemia

The original publication is available at http://jmg.bmj.com/In South Africa, the high prevalence of familial hypercholesterolaemia (FH) among Afrikaners, Jews, and Indians as a result of founder genes is in striking contrast to its reported virtual absence in the black population in general. In this study, the molecular basis of primary hypercholesterolaemia was studied in 16 Africans diagnosed with FH. DNA analysis using three screening methods resulted in the identification of seven different mutations in the coding region of the low density lipoprotein (LDLR) gene in 10 of the patients analysed. These included a 6 bp deletion (GCGATG) accounting for 28% of defective alleles, and six point mutations (D151H, R232W, R385Q, E387K, P678L, and R793Q) detected in single families. The Sotho patient with missense mutation R232W was also heterozygous for a de novo splicing defect 313+1G→A. Several silent mutations/polymorphisms were detected in the LDLR and apolipoprotein B genes, including a base change (g→t) at nucleotide position −175 in the FP2 LDLR regulatory element. This promoter variant was detected at a significantly higher (p<0.05) frequency in FH patients compared to controls and occurred in cis with mutation E387K in one family. Analysis of four intragenicLDLR gene polymorphisms showed that the same chromosomal background was identified at this locus in the four FH patients with the 6 bp deletion. Detection of the 6 bp deletion in Xhosa, Pedi, and Tswana FH patients suggests that it is an ancient mutation predating tribal separation approximately 3000 years ago.Harry and Doris Crossley FoundationSouth African Medical Research CouncilUniversity of StellenboschBritish Heart Foundation (grant no PG/96013)Publisher's versio

The genetics of familial hypercholesterolaemia and establishing familial hypercholeserolaemia genetic testing as a clinical diagnostic service

Familial hypercholesterolaemia (FH) is a monogenically inherited disorder of lipoprotein metabolism caused by a mutation in the low density lipoprotein receptor gene (LDLR). However in a few individuals, the defect lies in the gene for apolipoprotein B (APOB), the ligand for the LDL-receptor, and this is called familial defective apolipoprotein B-100 (FDB), while in others the receptor function is apparently normal and the defect must lie elsewhere. Mutation studies are the most practical way in which one can identify the cause of heterozygous hypercholesterolaemia. A genetic diagnostic service for FH has been established and the various aspects of setting up are described, with unusual cases being reported. The mutations identified are described and mutation detection rates were calculated for groups of paediatric and adult probands from the UK. The feasibility of altemative mutation screening methods and the specificity and sensitivity of reducing the number of tests has been assessed from the results obtained over the last four years. A quantitative fluorescent multiplex PCR screen was adapted to analyse LDLR rearrangements which would improve the genetic diagnosis of FH individuals. One assay based on exons 1, 8, 10, 12 and 16 were optimised and tested on known major rearrangements. A group of FH probands from the USA were then analysed with this multiplex assay. The inter and intra-assay variation were very wide, so a second method was designed to overcome these problems, universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR). The multiplex set developed analysed exons 3, 5, 8, 14, and 17 of LDLR, and the method could also be used to detect major rearrangements in other genes. The method was evaluated by conducting a trial on 15 reported deletions and duplications. Two groups of FH patients from the UK were screened with this UPQFM-PCR assay. The influence of LDLR & APOB mutations on the cholesterol-lowering response of the HMG-CoA reductase inhibitor simvastatin was investigated in patients with heterozygous FH. Data suggest that there may be a difference in cholesterol-lowering between 'severe' and 'mild' LDLR mutations. Future developments and transferring the findings into a clinical genetic service are discussed

GenMAPP 2: New features and resources for pathway analysis

BACKGROUND: Microarray technologies have evolved rapidly, enabling biologists to quantify genome-wide levels of gene expression, alternative splicing, and sequence variations for a variety of species. Analyzing and displaying these data present a significant challenge. Pathway-based approaches for analyzing microarray data have proven useful for presenting data and for generating testable hypotheses. RESULTS: To address the growing needs of the microarray community we have released version 2 of Gene Map Annotator and Pathway Profiler (GenMAPP), a new GenMAPP database schema, and integrated resources for pathway analysis. We have redesigned the GenMAPP database to support multiple gene annotations and species as well as custom species database creation for a potentially unlimited number of species. We have expanded our pathway resources by utilizing homology information to translate pathway content between species and extending existing pathways with data derived from conserved protein interactions and coexpression. We have implemented a new mode of data visualization to support analysis of complex data, including time-course, single nucleotide polymorphism (SNP), and splicing. GenMAPP version 2 also offers innovative ways to display and share data by incorporating HTML export of analyses for entire sets of pathways as organized web pages. CONCLUSION: GenMAPP version 2 provides a means to rapidly interrogate complex experimental data for pathway-level changes in a diverse range of organisms

\u3cem\u3ePPAP2B\u3c/em\u3e EXPRESSION LIMITS LESION FORMATION IN MURINE MODELS OF ATHEROSCLEROSIS

Coronary artery disease (CAD) is the leading cause of death in both men and women worldwide and is defined as a narrowing of the coronary arteries due to accumulation of atherosclerotic plaques. Genome-wide association studies have identified risk loci within the gene PPAP2B that confers increased risk of developing CAD. Evidence suggests these aforementioned SNPs are regulating PPAP2B expression in a cis-manner through the interruption of transcription factor binding sites. PPAP2B encodes the lipid phosphate phosphatase 3 enzyme that plays a key role in degrading bioactive lysophosphatidic acid (LPA). LPA has a plethora of effects on vascular tissue and is implicated in increasing inflammation and exacerbating the development of atherosclerotic lesions in mice. Interestingly, PPAP2B expression is increased in murine models of atherosclerosis and both global and smooth muscle cell-specific deletion increases the development of lesions compared to control mice. LPP3-deficient mice with increased atherosclerosis show significant increases in LPA accumulation in their proximal aorta as well as increased expression of inflammatory markers and positive staining for leukocyte marker CD68. Globally deficient mice also show substantial increases in ICAM-1 staining in their aortic root lesions relative to controls. Preliminary evidence also suggests that total LPA content, and specifically unsaturated LPA species, increase in the atherogenic LDL-C fractions of plasma in hyperlipidemic mice prone to developing atherosclerosis. Taken together, these data suggest that as CAD develops, LPA accumulates in atherosclerotic plaques, and the intrinsic mechanism of defense is to upregulate LPP3 through transcription factor- mediated effects on PPAP2B; however, individuals harboring the previously mentioned risk alleles are unable to increase PPAP2B expression and thus experience unchecked inflammation and exacerbated development of atherosclerosis

Alteraciones en genes del metabolismo lipídico y enfermedad cardiovascular

Las enfermedades cardiovasculares (ECV) ocupan el primer lugar entre las causas de morbilidad y mortalidad a nivel mundial. La observaci&oacute;n bien documentada de la predisposici&oacute;n familiar a padecer ECV, junto al avance vertiginoso en t&eacute;cnicas de an&aacute;lisis de ADN y la disponibilidad de secuencias del genoma humano, han orientado la investigaci&oacute;n de alteraciones g&eacute;n&eacute;ticas relacionadas con el desarrollo de ECV. Debido a que la ECV est&aacute; directamente relacionada con alteraciones en los niveles plasm&aacute;ticos de l&iacute;pidos, el principal esfuerzo en investigaci&oacute;n gen&eacute;tica de ECV est&aacute; dirigido a la identificaci&oacute;n de mutaciones o polimorfismos en genes involucrados en la s&iacute;ntesis, transporte y metabolismo de lipoprote&iacute;nas. Las alteraciones en algunos genes candidatos, tales como LDLR, APOB-100, APOE, LPA, LPL, HL, CETP, APOA1, APOA2 y LCAT han sido bien caracterizadas, demostr&aacute;ndose su asociaci&oacute;n a riesgo ECV en diversas poblaciones. Los estudios gen&eacute;ticos no solo permiten definir la contribuci&oacute;n de alteraciones de genes en el desarrollo del fenotipo de ECV, sino tambi&eacute;n mejorar la comprensi&oacute;n de su fisiopatolog&iacute;a y definir nuevas alternativas de tratamiento de la enfermedad cardiovascular a trav&eacute;s de la identificaci&oacute;n de nuevas mol&eacute;culas y posibles blancos terap&eacute;uticos.The cardiovascular diseases (CVD) is one of the main leading causes of death worldwide. The observation documented of familial predisposition to suffer CVD, combined with vertiginous advance in DNA analysis techniques and the availability of human genome sequences, has led to the investigation of genetic alterations related to development of ECV. Because the ECV directly is related to alterations in the plasma lipid levels, the main effort in genetic investigation of ECV is directed to the identification of mutations or polymorphisms in genes involved in lipoprotein synthesis, transport and metabolism. The alterations in some genes candidates, such as LDLR, APOB-100, APOE, LPA, LPL, HL, CETP, APOA1, APOA2 and LCAT have been well characterized, demonstrating its association to risk ECV in different populations. The genetic studies not only allow to define the contribution of genes alterations in the development of the ECV phenotype, but also to improve the understanding of their physiopatology and to define new alternatives of treatment of the cardiovascular disease through the identification of new molecules and possible therapeutic targets