Oxidative Modifications of Apolipoprotein(a): Implications for Proinflammatory and Prothrombotic Roles of Lipoprotein(a) in the Vasculature

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) have been identified as a causal risk factor for calcific aortic valve disease (CAVD) and coronary heart disease (CHD). Relationships have recently been identified for genetic factors, such as single nucleotide polymorphisms (SNPs) in the LPA gene, specifically r10455872 and rs3798220, that have been correlated with increased Lp(a) plasma levels and risk of cardiovascular disease (CVD). Apo(a) bears striking homology with the zymogen plasminogen and possesses several similar structural features. A key feature shared between these proteins is the presence of multiple repeats of a kringle domain, which possesses the ability to bind to exposed lysine residues with high affinity. Apo(a) contains several copies of a plasminogen like KIV domain, one of which, KIV10, has been implicated in many proinflammatory processes in vitro. It has been hypothesized that the proinflammatory potential of Lp(a)/apo(a) is derived from the ability to be covalently modified by an oxidized phosphatidylcholine (oxPC) moiety. The work in this dissertation assesses the mechanism by which the oxPC moiety on apo(a) stimulates interleukin-8 (IL-8) production in macrophages. Targeted mutagenesis was used to determine a role for the KIV10 strong lysine binding site (sLBS) in the covalent addition of the oxPC moiety on apo(a) and identified the site of covalent oxPC modification at the amino acid level. Furthermore, characterization of the I4399M variant of apo(a), resulting from the rs3798220 SNP, from a perspective of its distinct structural and functional properties, revealed roles for the polymorphism on the structure and permeability of purified fibrin and plasma clots. The enhanced prothrombotic potential of this variant may be a result of an oxidized methionine residue, as identified by mass spectrometry. The identification of distinct functional properties associated with the oxidative modification of Lp(a)/ apo(a) offers insights into its proatherosclerotic and prothrombotic potentials

Molecular Genetic Analysis of the PLP1 Gene in 38 Families with PLP1-related disorders: Identification and Functional Characterization of 11 Novel PLP1 Mutations

<p>Abstract</p> <p>Background</p> <p>The breadth of the clinical spectrum underlying Pelizaeus-Merzbacher disease and spastic paraplegia type 2 is due to the extensive allelic heterogeneity in the X-linked <it>PLP1 </it>gene encoding myelin proteolipid protein (PLP). <it>PLP1 </it>mutations range from gene duplications of variable size found in 60-70% of patients to intragenic lesions present in 15-20% of patients.</p> <p>Methods</p> <p>Forty-eight male patients from 38 unrelated families with a PLP1-related disorder were studied. All DNA samples were screened for <it>PLP1 </it>gene duplications using real-time PCR. <it>PLP1 </it>gene sequencing analysis was performed on patients negative for the duplication. The mutational status of all 14 potential carrier mothers of the familial <it>PLP1 </it>gene mutation was determined as well as 15/24 potential carrier mothers of the <it>PLP1 </it>duplication.</p> <p>Results and Conclusions</p> <p><it>PLP1 </it>gene duplications were identified in 24 of the unrelated patients whereas a variety of intragenic <it>PLP1 </it>mutations were found in the remaining 14 patients. Of the 14 different intragenic lesions, 11 were novel; these included one nonsense and 7 missense mutations, a 657-bp deletion, a microdeletion and a microduplication. The functional significance of the novel <it>PLP1 </it>missense mutations, all occurring at evolutionarily conserved residues, was analysed by the <it>MutPred </it>tool whereas their potential effect on splicing was ascertained using the <it>Skippy </it>algorithm and a neural network. Although <it>MutPred </it>predicted that all 7 novel missense mutations would be likely to be deleterious, <it>in silico </it>analysis indicated that four of them (p.Leu146Val, p.Leu159Pro, p.Thr230Ile, p.Ala247Asp) might cause exon skipping by altering exonic splicing elements. These predictions were then investigated <it>in vitro </it>for both p.Leu146Val and p.Thr230Ile by means of RNA or minigene studies and were subsequently confirmed in the case of p.Leu146Val. Peripheral neuropathy was noted in four patients harbouring intragenic mutations that altered RNA processing, but was absent from all <it>PLP1</it>-duplication patients. Unprecedentedly, family studies revealed the <it>de novo </it>occurrence of the <it>PLP1 </it>duplication at a frequency of 20%.</p

Exploring the determinants of metal sensing in Salmonella typhimurium using FrmR, a non-metal sensing RcnR/CsoR family member

Salmonella FrmR, a member of the RcnR/CsoR family of metalloregulators, has been characterised during the course of this work and was coincidentally confirmed to bind specifically to the frmRA operon, which encodes a putative Zn(II)-requiring class III alcohol dehydrogenase. FrmR shares a high degree of similarity with Ni(II)/Co(II)-sensing RcnR, in particular conserving two residues of a so-called WXYZ motif required to detect metals. Metal-binding properties of FrmR were therefore extensively investigated in vitro and its ability, or otherwise, to respond to metals explored in vivo. FrmR binds Zn(II), Cu(I), Co(II) and Ni(II), adopting different geometries, and always involving a mercapto group from the only cysteine residue (Cys35). Moreover, KZn(II)FrmR is only slightly below the range of affinity found for other zinc sensors. Since FrmR fails to sense metals in cells, where only formaldehyde is detected, questions about which parameters are required in metal regulation in Salmonella and, in general, in bacteria were investigated. A single-point mutation (Glu64 His) allows FrmR to sense cellular zinc and cobalt. FrmR and E64HFrmR have been consequently used as a case of study to test hypotheses about the mechanisms determining which metals are detected by a given sensor in cells. In addition, the ability of FrmR to detect cellular formaldehyde has been investigated, and a reaction mechanism tested by site-directed mutagenesis in vitro. Salmonella Ni(II)/Co(II)-sensing RcnR has been characterised, and employed to test the specificity of formaldehyde responsiveness of FrmR. By a single point-mutation (Ser2 Pro), the Ni(II)/Co(II)-sensing RcnR has been successfully switched to a formaldehyde sensor in vitro, further endorsing the proposed mechanism. Investigation of FrmR structure has been pursued by producing apo- and Zn(II)-bounded FrmR and E64HFrmR crystals, which were then analysed at the Diamond Light Source. The best dataset has been processed to obtain a 3D-structure

Identification and Characterization of 15 Novel GALC Gene Mutations Causing Krabbe Disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region. © 2010 Wiley-Liss, Inc

Apolipoprotein(a) Secretion is Modulated by Sortilin, Proprotein Convertase Subtilisin/Kexin Type 9, and Microsomal Triglyceride Transfer Protein

Elevated plasma lipoprotein(a) (Lp(a)) levels are a causal risk factor for cardiovascular disease (CVD), but development of specific Lp(a) lowering therapeutics has been hindered by insufficient understanding of Lp(a) biology. For example, the location of the noncovalent interaction that precedes the extracellular disulfide linkage between apolipoprotein(a) (apo(a)) and apolipoprotein B-100 (apoB-100) in Lp(a) biosynthesis is unclear. In this study we modulated known intracellular regulators of apoB-100 production and then assessed apo(a) secretion from human HepG2 cells expressing 17-kringle (17K) apo(a) isoform variants using pulse-chase analysis. Treating 17K-expressing HepG2 cells withproprotein convertase subtilisin-kexin type 9(PCSK9) significantly increased apo(a) secretion. Treating the same cell line with Lomitapide, a microsomal triglyceride transfer protein (MTP) inhibitor, significantly decreased apo(a) secretion. Overexpression of human sortilin variants (F404Y and K302E) significantly increased apo(a) secretion relative to wild-type. Our findings suggest a role for sortilin, PCSK9, and MTP in modulating Lp(a) levels through effects on apo(a) secretion, possibly through impacting the intracellular bioavailability of apoB-100

Struktur-Funktionsbeziehungen bei Apolipoproteinen : genetische Varianten am Apolipoprotein B-Lokus als Ursache atherogener Hypercholesterinämien

Das Apolipoprotein (Apo) B­100 ist der wesentliche Proteinbestandteil der Low Density Li­ poproteine (LDL). Es spielt als Ligand bei der rezeptorvermittelten Aufnahme der LDL aus dem zirkulierenden Blut in die Leber und andere periphere Gewebe eine wichtige Rolle. Erhöhte Plasmakonzentrationen der LDL gelten als unabhängiger Risikofaktor in der Genese der Koronaren Herzkrankheit (KHK). Eine gestörte Interaktion des LDL­Rezeptors mit seinem Liganden Apo B­100 vermindert die Endozytose der im Blut befindlichen LDL und kann damit eine primäre Hyperlipoproteinämie (HLP) des Typ IIa verursachen. Die Rezeptorbindungsregion des Apo B­100, dessen Primärstruktur 4536 Aminosäuren um­ fasst, ist im carboxyterminalen Bereich des Moleküls lokalisiert. Im Gegensatz zu einer Viel­ zahl bekannter LDL­Rezeptormutationen wurden lediglich drei Mutationen am Apo B­100 bekannt, die als Auslöser der Typ IIa HLP in Frage kommen. Diese werden in der Literatur als Familiär Defektes Apo B­100 (FDB) beschrieben. Bei allen drei FDB­Varianten (FDB 3500Q , FDB 3500W , FDB 3531C ) liegen Punktmutationen innerhalb des Exons 26 des Apo B­100 Gens vor. In der hier vorliegenden Arbeit wurde versucht, weitere, bisher unbekannte Punktmutationen am Apo B­100, die als Ursache bindungsdefekter LDL in Frage kommen, zu finden und zu charakterisieren. Die zunächst durchgeführte Sequenzierung der genomischen DNA einer Pa­ tientin mit bindungsdefekten LDL, die negativ für FDB war, zeigte keine Abweichungen von der Wildtyp­DNA­Sequenz im Bereich der Rezeptorbindungsregion. Es wurde daraufhin eine genetische Screening­Strategie für bindungsdefekte Apo B­100 entwickelt, die für die Analyse größerer Probenmengen geeignet erschien. Hierzu wurden zunächst parallel zwei Verfahren, der single­strand conformation polymorphism (SSCP) und die Temperatur Gradienten Gel E­ lektrophorese (TGGE) getestet. Letztere erwies sich als geeignete Methode. Ein Kollektiv von 297 Typ IIa HLP­Patienten, deren LDL­Cholesterin > 1,55 g/L und Trigly­ zerid­Konzentrationen <2,0 g/l waren, wurde zusammengestellt. Die Blutproben stammten von nicht miteinander verwandten, im Rhein/Main­Gebiet ansässigen Personen kaukasischer Abstammung. Ein DNA Bereich von 2,8 Kilobasenpaaren korrespondierend zu den Aminosäureresten 3131­ 3837 und 4269­4498 des Apo B­100 wurde in einzelnen Abschnitten amplifiziert und mit He­ teroduplex­TGGE analysiert. Es wurden neun Substitutionen identifiziert. Diese waren: FDB 3500Q , FDB 3500W , L3350L, Q3405E, R3611Q, I4287V, N4311S, A4454T, und T4457M. Bei 21 Personen (7,1%) wurde FDB 3500Q nachgewiesen. Dies entspricht nach Extrapolation auf die Gesamtbevölkerung einer Mutationshäufigkeit von 1,4% in unserer Region. Es konnte festgestellt werden, dass bei allen Merkmalsträgern die R3500Q Mutation mit einem einheitli­ chen Haplotypen kosegregiert, so dass eine stammesgeschichtliche Verwandtschaft der Muta­ tionsträger belegt ist. FDB 3500W wurde bei zwei HLP­Patienten diagnostiziert. Dies war insofern überraschend, da diese Mutation zuvor lediglich bei einer Person kaukasischer Abstammung in Großbritannien gefunden worden war. Beide R3500W Mutationen waren mit unterschiedlichen, bisher unbe­ kannten Haplotypen assoziiert. LDL der beiden heterozygoten Merkmalsträger für FDB 3500W zeigten in einem an normalen humanen Fibroblasten durchgeführten Bindungsassay eine deut­ lich verminderte Rezeptorbindungsaffinität, jedoch lag diese etwas höher als die der LDL ei­ ner FDB 3500Q heterozygoten Person. Es ist daher anzunehmen, dass der Austausch eines Argi­ nins durch Tryptophan an Position 3500 einen geringeren Bindungsverlust der LDL bewirkt als der Austausch von Arginin zu Glutamin. Zwei der in der TGGE identifizierten Punktmutationen stellten bekannte Apo B­100 Poly­ morphismen (MspI 3611 und N4311S) dar, eine weitere stille Mutation an Kodon L3350L wur­ de bei drei Personen festgestellt. Daneben fand sich ein Merkmalsträger mit einer Punktmuta­ tion an Kodon 4457 (T4457M). Zwei zuvor beschriebene Substitutionen, Q3405E und A4454T, traten mit einer Prävalenz von 1,3%, bzw. 6,4% im untersuchten Kollektiv auf. LDL eines heterozygoten Merkmalträgers für Q3405E hatten normale Bindungsaffinität an LDL­ Rezeptoren, zeigten jedoch eine signifikant erniedrigte Internalisation und Degradation im in vitro­Bindungstest. Schliesslich wurde eine bisher unbekannte Mutation des Apo B­100 am Aminosäurerest I4287V, die mit einer Allelfrequenz von 1% im untersuchten Kollektiv auf­ trat, funktionell überprüft. Dabei zeigte sich sowohl im Fibroblasten Bindungsassay als auch in einem zweiten in vitro­Testverfahren, dem U­937 Zellen Wachstumsassay keine Assoziati­ on der I4287V Mutation mit bindungsdefekten LDL in der Familie einer heterozygoten Merkmalsträgerin. Eine funktionelle Relevanz dieses Aminosäureaustausches ist daher un­ wahrscheinlich. An einem Tryptophanrest an Position 4369 des Apo B­100, der bei der Formation der dreidi­ mensionalen Struktur des Proteins mit Arginin 3500 interagiert, wurden Nukleotidveränderun­ gen zunächst durch gezielte Mutagenese des Kodon 4369 erzeugt, um sicherzustellen, dass diese Mutationen durch TGGE nachweisbar sind. Nachfolgend wurde bei den Typ IIa HLP­ Patienten nach Mutationen an dieser Position gesucht. Es konnte festgestellt werden, dass im untersuchten Kollektiv keine Punktmutationen am Aminosäurerest 4369 existierten. Die Vermutung, dass weitere klinisch relevante Apo B­100 Mutationen mit einem dem FDB konformen Merkmalsbild bei Typ IIa HLP­Patienten vorhanden sind, konnte nicht bestätigt werden. Andererseits wurde eine überraschend hohe Prävalenz (1:72) des FDB 3500Q im Rhein/Main Gebiet festgestellt. Aufgrund der gemeinsamen Abstammung aller Merkmalsträ­ ger und der hohen Mutationsfrequenz in dieser Region ist es denkbar, dass sich die vermutlich vor ca. 6000 Jahren datierte Founder­Mutation in diesem Teil Deutschlands ereignete. Da es offensichtlich auch, zwar seltener auftretende, Fälle des FDB 3500W in der hier untersuch­ ten Region gibt, sind Analyse­Methoden, wie etwa die TGGE, die sich als ein hochsensibles Nachweisverfahren multipler Punktmutationen erwies, bei der differentialdiagnostischen Ab­ klärung von Fettstoffwechselstörungen solchen Methoden vorzuziehen, die nur auf die Substi­ tution FDB 3500Q testen

Genetic functional studies of low density lipoprotein- cholesterol (LDL-C) associated variants and the genetic spectrum of familial hypercholesterolemia in different ethnic groups

Since the end of the 20th century, cardiovascular disease has been the major cause of death worldwide. Hypercholesterolemia is major risk factor for CVD. Family-based linkage analyses in patients with familial hypercholesterolemia (FH) have revealed the major loci that are involved in cholesterol hemostasis. These include low density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type-9 protein (PCSK9), and apolipoprotein B (APOB). However, these discovered genes only explain disease etiology in a proportion of patients with the clinical phenotype, and roughly 60% of patients who are diagnosed with FH are negative for any FH-causative mutation. This suggests that (1) there are undetected new mutations in known genes, (2) that disease-causing mutations might occur in unidentified gene(s), (3) disease may occur as a result of an accumulation of common small-effect LDL-C raising variants. My project has three main aims. First, the identification of mutations in patients clinically diagnosed with FH in two ethnic groups: UK (n=69) and Iran (n=16). Several novel mutations were identified in LDLR [UK: p.(Cys392Tyr), p.(Tyr553Ser) and p.(Tyr553*); Iran: p.(Leu479Gln) and p.(Glu668*)] and PCSK9 [p.(Arg357Cys)] using next generation sequencing technology. The second aim was to investigate the functional role explaining the Genome-Wide Association (GWAS) LDLR “hit” Single Nucleotide Polymorphism (SNP) rs6511720. This SNP and one other in strong linkage disequilibrium (LD) (rs57217136) were both found to act as a cis-regulatory element, where the sequence around the rare alleles of the SNPs is a target for proteins that enhance gene transcription. The third aim was studying the Annexin A2 (ANXA2) gene. AnxA2 recently was found to be involved in the LDL-R pathway through PCSK9. The study aimed to identify and determine the functional role of common SNPs that are associated with LDL-C. I showed that the ANXA2 common SNP rs17845226 (V98L) minor allele, was associated with significantly higher levels of LDL-C and a higher risk of CHD in a large prospective study of healthy UK men (NPHSII). The SNP shows strong LD with SNPs in the intragenic region, and I showed that the minor alleles of rs17191344 and rs11633032 are targets for proteins that repress gene transcription and the subsequent lower levels of AnxA2 protein means that there will be higher levels of PSCK9-mediated degradation of the LDL-R and this will lead to an increase in LDL-C levels. Finally, I identified that rs116928563 in the 3'UTR of ANXA2 was a potential site for a micro-RNA (miRNA-155*) binding, however, the study failed to result in any definite conclusions about the influence of miRNA-155 in ANXA2 expression and LDL-C levels. However, it gives insight into how a SNP also may affect gene expression at the post-transcription level by creating or destroying micro-RNA binding sites

Hypertension, hypercholesterolemia, hyperaldosteronism: a genetic perspective for personalized therapy.

Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, hyperaldosteronism, or other causes of secondary hypertension are not present. Essential hypertension accounts for 80-90% of all cases of hypertension; it is a heterogeneous disorder, with different patients having different causal factors that may lead to high BP. Life-style, diet, race, physical activity, smoke, cultural level, environmental factors, age, sex and genetic characteristics play a key role in the increasing risk. Conversely to the essential hypertension, secondary hypertension is often associated with the presence of other pathological conditions such as dyslipidaemia, hypercholesterolemia, diabetes mellitus, obesity and primary aldosteronism. Amongst them, primary aldosteronism represents one of the most common cause of secondary hypertension, with a prevalence of 5-15% depending on the severity of blood pressure. Besides high blood pressure values, a principal feature of primary aldosteronism is the hypersecretion of mineralcorticoid hormone, aldosterone, in a manner that is fairly autonomous of the renin-angiotensin system. Primary aldosteronism is a heterogeneous pathology that may be divided essentially in two groups, idiopathic and familial form. Despite all this knowledge, there are so many hypertensive cases that cannot be explained. These individuals apparently seem to be healthy, but they have a great risk to develop CVD. The lack of known risk factors makes difficult their classification in a scale of risk. Over the last three decades a good help has been given by the pharmacogenetics/pharmacogenomics, a new area of the traditional pharmacology that try to explain and find correlations between genetic variation, (rare variations, SNPs, mutations), and the risk to develop a particular disease

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