Identification and Molecular Characterization of New PCSK9 Missense Mutations Associated with Familial Hypecholesterolemia (FH)

Genetics – Posters 212 Perindopril Reduces Large Artery Stiffness and Aortic Root Diameter in Patients with Marfan Syndrome A. Ahimastos 1,∗ , A. Aggarwal 2 , K. D’Orsa 1 , M. 1 2 1 Formosa , R. Savarirayan , A. Dart , B. Kingwell 1 1 Baker

Heart Research Institute, Melbourne, Victoria, Australia; 2 Royal Melbourne Hospital, Melbourne, Victoria, Australia Background: Aortic stiffness is elevated in Marfan syndrome (MFS) contributing to aortic dilatation and rupture, the major cause of premature death in this population. Given the known beneficial effects of angiotensin converting enzyme inhibitors (ACEIs) on arterial stiffness, we hypothesised that perindopril therapy would reduce aortic stiffness and attenuate aortic dilatation in MFS patients. Methods: Seventeen MFS patients (aged 33 ± 6 (mean ± S.D.)) on standard ␤-blocker therapy were randomised to also receive either perindopril (10 mg od, n = 10) or placebo (n = 7) for 24 weeks in a randomised, double blind study. Indices of arterial stiffness were assessed globally via systemic arterial compliance (SAC) and augmentation index (AIx), and regionally via carotidfemoral (PWVc) and femoral-dorsalis pedis (PWVp) pulse wave velocity. Aortic root diameter was assessed via a transthoracic echocardiogram. Data are expressed as percent or absolute change from baseline for the placebo and perindopril groups. Results: Perindopril reduced arterial stiffness as indicated by increased SAC (perindopril 62 ± 11% versus placebo −4.30 ± 1%, p < 0.0001), reduced AIx (perindopril −23.50 ± 3% versus placebo 3 ± 1%, p < 0.0001), reduced PWVc (perindopril −21 ± 2% versus placebo 5 ± 2%, p < 0.0001) and PWVp (perindopril −20 ± 2% versus placebo 2 ± 1%, p < 0.0001). In addition, perindopril significantly reduced aortic root diameter (perindopril −0.28 ± 0.04 cm versus placebo 0.11 ± 0.03 cm, p < 0.0001). While perindopril marginally reduced mean blood pressure (perindopril −3.5 ± 0.5 mmHg versus placebo 1.1 ± 0.7 mmHg, p < 0.001), importantly, the observed changes in both stiffness (p = 0.01–0.03) and aortic diameter (p < 0.01) remained significant when mean blood pressure was included as a covariate. Conclusion: ACEIs reduce aortic stiffness and aortic root diameter in MFS patients and may potentially protect against aortic rupture. doi:10.1016/j.hlc.2007.06.217

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213 Functional Estrogen Receptor Alpha Promoter Polymorphism is Associated with Improved LDL Cholesterol G.A. Figtree 1,3,∗ , S.M. Channon 3 , H. Watkins 3

Grieve 2 , P.

Gleeson 3 , K.M.

1 Department of Cardiology, Royal North Shore Hospital, Sydney, Australia; 2 NHMRC Clinical Trials Centre, Sydney, Australia; 3 Department of Cardiology, University of Oxford, UK

Estrogen receptor alpha (ER␣) mediates beneficial actions on cholesterol metabolism. Genetic variations in ER␣ may therefore influence serum cholesterol levels. We have recently identified a novel polymorphism in the estrogen receptor negative transcriptional element (ERNE T > C) that abolishes the negative regulatory effect of the element in hepatic cell lines. We have now examined for the association of ERNE T > C with serum cholesterol levels in 248 randomly chosen subjects (131 females, 117 males) aged 20–28 years. The effect of the ERNE (T > C) locus on LDL cholesterol was modelled using ANOVA controlling for variations in gender, age and the interaction of gender and ER␣ genotype. Using this model the association of the C allele toward improved LDL cholesterol was significant (p = 0.018; n = 223). There was a highly significant interaction of sex and ERNE genotye in the prediction of LDL cholesterol, consistent with a possible modification of gender effect on LDL by ER␣ genotype (p = 0.008). A similar association was observed between ERNE genotype and ApoB (p = 0.03), with a highly significant interaction term for sex and ERNE genotype (p = 0.01). The association of LDL with ER␣ genotype appeared greatest in the female subgroup in which individuals possessing the variant C allele had a substantially reduced LDL cholesterol compared with TT individuals. The association of the variant C allele with reduced LDL cholesterol shown in this study may be explained by its reversal of negative transcriptional regulation by the element in which it lies. doi:10.1016/j.hlc.2007.06.218 214 Identification and Molecular Characterization of New PCSK9 Missense Mutations Associated with Familial Hypecholesterolemia (FH) Vivienne M. Homer 1,∗ , Francesca Charlton 2 , Andrew D. Laurie 1 , David A. Marais 3 , Nicola J. Hurndell 1 , David R. Sullivan 4 , Philip J. Barter 2 , Kerry-Ann Rye 2 , Peter M. George 1 , Gilles C. Lambert 2,5 1 Canterbury Health Laboratories, Christchurch, New Zealand; 2 The Heart Research Institute, Sydney, Australia; 3 The Univer-

sity of Cape Town, Cape Town, South Africa; 4 The Royal Prince Alfred Hospital, Sydney, Australia; 5 Universit´e de Nantes Inserm U539 CHU Hotel-Dieu, Nantes, France Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an inhibitor of the LDL-receptor. In humans, PCSK9 “gain of function” mutations are associated with FH,

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whereas mutations inactivating PCSK9 are associated with reduced plasma LDL and cardiovascular events. Characterization of the naturally occurring mutations reported to date has provided some insights into PCSK9’s mechanism of action but it has not been possible to distinguish the phenotypic effect of some “gain of function” from some “loss of function” mutations based on their autocatalytic cleavage and secretion pattern. In the present study, we analysed the PCSK9 exons and intronic junctions of FH patients previously found to be negative for LDL-receptor or apolipoprotein B100 mutations. The previously reported S127R French mutation was found in a South-African family, whereas new heterozygous missense mutations D129G and A168E were found in two New Zealand families. PCSK9 overexpression studies in HuH7 hepatoma cells shows that both S127R and D129G PCSK9 mutants have 75% reduced autocatalytic activity compared to wild type, whereas the A168E mutant is processed normally. The S127R and D129G mutants were not secreted in the culture media, and cellular LDL binding was decreased by 25–30% in cells overexpressing these mutants. Our study indicates that (1) the region within the prodomain of PCSK9 encompassing the S127 and D129 residues is critical for PCSK9 autocatalytic activity and secretion, and that (2) two non-secreted naturally occurring mutants of PCSK9 inhibit LDL-receptor expression and activity, suggesting that PCSK9 mediated inhibition of the LDL-receptor in the liver occurs intracellularly. doi:10.1016/j.hlc.2007.06.219 215 Misdiagnosis of Long QT Syndrome as Epilepsy J.M. MacCormick 1,2,∗ , H. McAlister 2 , J. Crawford 2 , J. French 2 , I. Crozier 2 , A. Shelling 2 , C. Nel 2 , M.I. Rees 2 , J.R. Skinner 1,2 , on behalf of the Cardiac Inherited Disease Group 1 Paediatric

and Congenital Cardiac Services, Starship Children’s Hospital, Auckland, New Zealand; 2 Cardiac Inherited Diseases Group (CIDG), New Zealand Background: Following two recent sudden unexpected deaths of local children diagnosed with seizure disorders, post-mortem genetic analysis revealed long QT syndrome (LQTS). We aimed to establish the frequency of misdiagnosis and delayed recognition of LQTS in living genotype-positive probands. Methods: Probands were identified through the New Zealand CIDG Registry and medical records were reviewed retrospectively. Results: Thirty-one living genotype-positive LQTS probands were identified. Genetic mutations were consistent with LQT1 in 18 (58%), LQT2 in 10 (32%) and LQT3 in 3 (10%). Median age at diagnosis was 21 years (range: 0–54). In 13 cases (39%) there had been presentation with loss of consciousness to a hospital or specialist outpatient clinic prior to diagnosis of LQTS. In this group the median period from initial presentation until diagnosis was 2.4

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years (range 2 months–23 years). Ten of the 13 had at least one electrocardiogram recorded prior to diagnosis. Retrospective review revealed previously unrecognised prolonged QTc intervals in eight cases (QTc range 470–650 ms). The QTc had not been recorded in four cases and was recorded incorrectly in four. Ten of 31 probands (32%) underwent electroencephalograms for suspected primary seizures; epilepsy was diagnosed in 5 (16%); and anticonvulsants prescribed for 4 (13%). Conclusions: Although electrocardiograms are being integrated into the routine work-up for syncope, interpretation errors are frequent. Misdiagnosis of LQTS as a seizure disorder is common. We postulate that our figures may underestimate the rate of misdiagnosis, as this study does not include those presenting with sudden death and those yet to be diagnosed. doi:10.1016/j.hlc.2007.06.220 216 Functional Analysis of a Novel Single Nucleotide Polymorphism in Human SLC7A1 Gene Zhiyong Yang ∗ , David M. Kaye Wynn Department of Metabolic Cardiology, Baker Heart Research Institute, Melbourne, Australia Background: Previously we have reported that a novel single nucleotide polymorphism in the 3 untranslated region (UTR) of the member 1 gene of human solute carrier family 7 (SLC7A1) may account for the apparent link between altered endothelial function, L-arginine and nitric oxide metabolism, and predisposition to essential hypertension (Circulation, in press). The polymorphism and its surrounding sequences are 5 -GGGGCG(G/T)GGC-3 . It has been well documented that SP1 protein recognises GC/GT boxes and interacts with DNA through three C2 H2 -type zinc fingers located at the C-terminal domain, where the consensus SP1 binding site is 5 (G/T)GGGCGG(G/A)(G/A)(C/T)-3 . We set to investigate if SP1 might play the role in differential binding to the polymorphism site. Methods: We used HeLa nuclear extracts and recombinant human SP1 protein as binding proteins, SP1 consensus oligo, 38-bp fragments containing different alleles of the polymorphism as probes in the gel shift assay. Results: The allele C fragment had similar binding patterns with SP1 consensus oligo. However, although both allele C and T probes could bind to HeLa nuclear extracts, only allele C probe bound to recombinant human SP1 protein. The binding of allele C-SP1 protein could be blocked by the prior incubation of cold allele C probe or the consensus SP1 oligo, but not cold allele T probe or unrelated consensus AP2 oligo. Conclusion: These findings provide further evidence that 3 UTR polymorphism plays important role in gene expression and regulation, probably via binding or attenuated binding to SP1 protein or SP1-associated proteins. doi:10.1016/j.hlc.2007.06.221