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LOCAL POLYMORPHISMS INFLUENCE STK39 GENE EXPRESSION BUT ARE NOT ASSOCIATED WITH BLOOD PRESSURE
Abstracts / Atherosclerosis 207 (2009) e1–e13
LOCAL POLYMORPHISMS INFLUENCE STK39 GENE EXPRESSION BUT ARE NOT ASSOCIATED WITH BLOOD PRESSURE C. Kay ∗ , M.S. Keavney
Cunnington, P.
Avery, M.
Santibanez-Koref, B.
Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, NE1 3BZ, UK Background: Blood pressure (BP) has significant heritability, but the genes responsible remain largely unknown. Single nucleotide polymorphisms (SNPs) in the STK39 gene were recently associated with hypertension by genome-wide association in an Amish population; in vitro data from transient transfection experiments using reporter constructs suggested that altered STK39 expression might mediate the effect. However, other large studies have not implicated STK39 in hypertension. We determined whether reported SNPs influenced STK39 expression in vivo, or were associated with BP in a large British Caucasian cohort. Methods: 1372 members of 247 Caucasian families ascertained through a hypertensive proband were genotyped for reported risk variants (rs6749447, rs3754777, rs35929607) using Sequenom technology. The Merlin programme was used for family-based association testing (corrected for covariants). Cis-acting influences on expression were assessed in vivo using allelic expression ratios in cDNA from peripheral blood cells in 35 individuals heterozygous for a transcribed SNP in STK39 (rs1061471) and quantified by mass spectrometry (Sequenom). Results: Despite documented heritability of BP in this population, and proven power to detect small genetic effects in previous studies, no significant association was seen between SNPs and systolic or diastolic BP in clinic (n = 1138) or ambulatory (n = 903) measurements (all P > 0.05). The tested SNPs were all associated with allelic expression differences in peripheral blood cells (P < 0.05), with the strongest association for the intronic SNP rs6749447 (P < 0.0002). Conclusions: The tested SNPs correlate with in vivo STK39 allelic expression, supporting previous in vitro data, but are not associated with blood pressure in our British Caucasian cohort. doi:10.1016/j.atherosclerosis.2009.09.052 −203A/C POLYMORPHISM OF CHOLESTEROL 7ALPHAHYDROXYLASE (CYP7A1) GENE AND DIURNAL VARIATION IN CYP7A1 ACTIVITY J. Kovar 1,2 , M. Zimolova 1,2 , M. Lenicek 3 , L. Vitek 3 , M. Jirsa 1 , R. Poledne 1,2 1
Institute for Clinical and Experimental Medicine, Prague, Czech Republic 2 Centre of Cardiovascular Research, Prague, Czech Republic 3 1st Faculty of Medicine, Charles University, Department of Clinical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic The −203A/C polymorphism of cholesterol 7alpha-hydroxylase (CYP7A1) gene, the key regulatory enzyme in bile salt synthesis, affects responsiveness of serum cholesterol to dietary fat and cholesterol. However, no relationship between enzyme activity and this polymorphism has been demonstrated so far. Therefore, we studied whether this polymorphism can affect the diurnal variation of CYP7A1 activity. The diurnal variation of 7-alpha-hydroxycholest-4-en-3-one [C4], a marker of CYP7A1 activity, was measured in 16 healthy male volunteers homozygous either for −203A or for −203C variants. The study of diurnal variation was carried out on three occasions: after short term treatment with cholestyramine [Q], chenodeoxycholic acid (CDCA) and without any treatment. No differences
e9
between −203A and −203C carriers were found in diurnal variation of CYP7A1 activity in control experiment and on CDCA, however, the CYP7A1 activity throughout the day rose considerably more in −203C allele carriers after treatment with Q. The short term Q administration resulted in 9% decrease of cholesterolemia in −203C allele carriers and had no effect in −203A allele carriers. The results of this study are supported by findings from dual luciferase assay in HepG2 cells, in which −764 to +14 fragment of CYP7A1 gene promoter carrying −203C variant has five times higher promoter activity than the fragment carrying −203A variant. In conclusion, −203C allele of CYP7A1 gene can be regulated to a much higher extent than −203A allele in accordance with its anticipated role in determination of cholesterolemia hyperresponsiveness. Acknowledgement The study was supported by grant No. 1M0510 from MEYS CR. doi:10.1016/j.atherosclerosis.2009.09.053 PRO-PROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE9 (PCSK9) VARIANT DATABASE: PRELIMINARY ANALYSIS S.E. Leigh ∗ , T. Jakubcova, R.A. Whittall, S.E. Humphries Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, The Rayne Building, Royal Free & University College London Medical School, 5 University Street, London WC1E 6JJ, UK Background: Disease-causing variants in PCSK9 were first identified in 2003 in patients with autosomal dominant hypercholesterolemia. Since that time >100 additional variants have been identified in subjects exhibiting high, low and normal cholesterol levels, classified as “gain of function”, “loss of function” or “neutral” respectively. This study aims to place all published PCSK9 variants in a publicly available database and assess the potential biological significance of each variant. Methods: Following a PubMed literature search, variant data was loaded onto the Leiden Open Source Variant Database (LOVD) platform on the UCL web server. Predicted effects of amino acid substitutions were assessed using the literature and publicly available computer programs. Results: The database lists 154 entries, (100 unique variants), 66% are exonic DNA substitutions (∼1/3rd each of “gain”, “loss” and “neutral” variants). Three DNA rearrangements have been reported; two result in “loss of function” and one is “neutral”. Of the 30 non-exonic variants (5 UTR n = 5, introns n = 22 and 3 UTR n = 3) 27 appear to be “neutral” and 3 to result in “gain of function” (2 in 5 UTR, 1 in intron 11). The use of predictive computer programs has not provided clear results, although useful trends are appearing. Conclusion: PCSK9 variants are of considerable interest because they result in elevated or reduced cholesterol levels in patients and the general population. The UCL PCSK9 variant database provides a publicly available resource for researchers investigating the role of this gene in disease. Funding: This work and the variant database are supported by the British Heart Foundation. doi:10.1016/j.atherosclerosis.2009.09.054
LOCAL POLYMORPHISMS INFLUENCE STK39 GENE EXPRESSION BUT ARE NOT ASSOCIATED WITH BLOOD PRESSURE C. Kay ∗ , M.S. Keavney
Cunnington, P.
Avery, M.
Santibanez-Koref, B.
Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, NE1 3BZ, UK Background: Blood pressure (BP) has significant heritability, but the genes responsible remain largely unknown. Single nucleotide polymorphisms (SNPs) in the STK39 gene were recently associated with hypertension by genome-wide association in an Amish population; in vitro data from transient transfection experiments using reporter constructs suggested that altered STK39 expression might mediate the effect. However, other large studies have not implicated STK39 in hypertension. We determined whether reported SNPs influenced STK39 expression in vivo, or were associated with BP in a large British Caucasian cohort. Methods: 1372 members of 247 Caucasian families ascertained through a hypertensive proband were genotyped for reported risk variants (rs6749447, rs3754777, rs35929607) using Sequenom technology. The Merlin programme was used for family-based association testing (corrected for covariants). Cis-acting influences on expression were assessed in vivo using allelic expression ratios in cDNA from peripheral blood cells in 35 individuals heterozygous for a transcribed SNP in STK39 (rs1061471) and quantified by mass spectrometry (Sequenom). Results: Despite documented heritability of BP in this population, and proven power to detect small genetic effects in previous studies, no significant association was seen between SNPs and systolic or diastolic BP in clinic (n = 1138) or ambulatory (n = 903) measurements (all P > 0.05). The tested SNPs were all associated with allelic expression differences in peripheral blood cells (P < 0.05), with the strongest association for the intronic SNP rs6749447 (P < 0.0002). Conclusions: The tested SNPs correlate with in vivo STK39 allelic expression, supporting previous in vitro data, but are not associated with blood pressure in our British Caucasian cohort. doi:10.1016/j.atherosclerosis.2009.09.052 −203A/C POLYMORPHISM OF CHOLESTEROL 7ALPHAHYDROXYLASE (CYP7A1) GENE AND DIURNAL VARIATION IN CYP7A1 ACTIVITY J. Kovar 1,2 , M. Zimolova 1,2 , M. Lenicek 3 , L. Vitek 3 , M. Jirsa 1 , R. Poledne 1,2 1
Institute for Clinical and Experimental Medicine, Prague, Czech Republic 2 Centre of Cardiovascular Research, Prague, Czech Republic 3 1st Faculty of Medicine, Charles University, Department of Clinical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic The −203A/C polymorphism of cholesterol 7alpha-hydroxylase (CYP7A1) gene, the key regulatory enzyme in bile salt synthesis, affects responsiveness of serum cholesterol to dietary fat and cholesterol. However, no relationship between enzyme activity and this polymorphism has been demonstrated so far. Therefore, we studied whether this polymorphism can affect the diurnal variation of CYP7A1 activity. The diurnal variation of 7-alpha-hydroxycholest-4-en-3-one [C4], a marker of CYP7A1 activity, was measured in 16 healthy male volunteers homozygous either for −203A or for −203C variants. The study of diurnal variation was carried out on three occasions: after short term treatment with cholestyramine [Q], chenodeoxycholic acid (CDCA) and without any treatment. No differences
e9
between −203A and −203C carriers were found in diurnal variation of CYP7A1 activity in control experiment and on CDCA, however, the CYP7A1 activity throughout the day rose considerably more in −203C allele carriers after treatment with Q. The short term Q administration resulted in 9% decrease of cholesterolemia in −203C allele carriers and had no effect in −203A allele carriers. The results of this study are supported by findings from dual luciferase assay in HepG2 cells, in which −764 to +14 fragment of CYP7A1 gene promoter carrying −203C variant has five times higher promoter activity than the fragment carrying −203A variant. In conclusion, −203C allele of CYP7A1 gene can be regulated to a much higher extent than −203A allele in accordance with its anticipated role in determination of cholesterolemia hyperresponsiveness. Acknowledgement The study was supported by grant No. 1M0510 from MEYS CR. doi:10.1016/j.atherosclerosis.2009.09.053 PRO-PROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE9 (PCSK9) VARIANT DATABASE: PRELIMINARY ANALYSIS S.E. Leigh ∗ , T. Jakubcova, R.A. Whittall, S.E. Humphries Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, The Rayne Building, Royal Free & University College London Medical School, 5 University Street, London WC1E 6JJ, UK Background: Disease-causing variants in PCSK9 were first identified in 2003 in patients with autosomal dominant hypercholesterolemia. Since that time >100 additional variants have been identified in subjects exhibiting high, low and normal cholesterol levels, classified as “gain of function”, “loss of function” or “neutral” respectively. This study aims to place all published PCSK9 variants in a publicly available database and assess the potential biological significance of each variant. Methods: Following a PubMed literature search, variant data was loaded onto the Leiden Open Source Variant Database (LOVD) platform on the UCL web server. Predicted effects of amino acid substitutions were assessed using the literature and publicly available computer programs. Results: The database lists 154 entries, (100 unique variants), 66% are exonic DNA substitutions (∼1/3rd each of “gain”, “loss” and “neutral” variants). Three DNA rearrangements have been reported; two result in “loss of function” and one is “neutral”. Of the 30 non-exonic variants (5 UTR n = 5, introns n = 22 and 3 UTR n = 3) 27 appear to be “neutral” and 3 to result in “gain of function” (2 in 5 UTR, 1 in intron 11). The use of predictive computer programs has not provided clear results, although useful trends are appearing. Conclusion: PCSK9 variants are of considerable interest because they result in elevated or reduced cholesterol levels in patients and the general population. The UCL PCSK9 variant database provides a publicly available resource for researchers investigating the role of this gene in disease. Funding: This work and the variant database are supported by the British Heart Foundation. doi:10.1016/j.atherosclerosis.2009.09.054