Functional Analysis of a Novel Single Nucleotide Polymorphism in Human SLC7A1 Gene

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Abstracts

ABSTRACTS

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

Heart, Lung and Circulation 2007;16:S1–S201

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