- Email: [email protected]
A genetic variant of the human aldosterone synthase gene causes salt-dependent hypertension in transgenic mice
Journal of the American Society of Hypertension 9(4S) (2015) e73–e74
GENETICS/GENE THERAPY/PROTEOMICS P-121 Circadian blood pressure monitoring in human and normotensive/ spontaneously hypertensive rats after embryo transfer into different wombs and cross-suckling Mary S. Lee,1 John S. Lee,2 Jong Y. Lee,3 Silvia H. Azar,3 Franz Halberg.3 1 Northwestern University Feinberg School of Medicine &UMN School of Medicine, Chicago /Minneapolis, MN, United States; 2Princeton University & UMN School of Medicine, Princeton /Minneapolis, MN, United States; 3 UMN School of Medicine, Minneapolis, MN, United States Daily (circadian) rhythm characteristics are considered essential parameters for recognizing and treating increased risks in blood pressure (BP). Ambulatory BP in 20 adolescents and adults were monitored automatically around the clock at 30-minute to hourly intervals and analyzed by the least square rhythmometry method. The results were compared with telemetered circadian BP in rats after one-cell homozygous embryo-transfer into spontaneously hypertensive (SHR, pup:shr) or normotensive (WKY, pup:wky) rats’ oviducts (embryos: s,w; oviduct-uterus: S,W) and cross-suckled at birth (nurses S,W). All subjects showed significant circadian fluctuations. The circadian response peaked in the late afternoon hours in most human subjects and early morning hours in rats. Circadian double amplitudes (2A) in the human subjects varied from 8 to 26 mm Hg with higher 2A in elder adults, and 3-8 mm Hg in rats with significantly higher fluctuations in SHR strain groups (7.50.7 for sSS, 8.30.6 for sSW vs. 4.70.3 mm Hg for wWW). In individualized rats’ peak hour comparisons, most groups showed clustered peaks around the light-on hours, while sWS and wSS groups showed individual rats’ peak hour variations (19:18 through 08:06 and 02:58 through 09:10 hours, respectively). The hypertensiveprone shr strain showed significantly lowered BPs in a normotensive WKY uterine environment and/or by WKY nursing mothers, indicating that environment can strongly modify genetic influence, yet the lowered shr MESORs by the WKY environments remained above the MESORs encountered in wky donors. Chronomes broader than circadian should be considered in interpreting BP responses as a gauge of vascular disease status. Keywords: Circadian blood pressure monitoring, embryo transfer; crosssuckling; spontaneously hypertensive and normotensive rat; vascular disease risk.
P-122 A genetic variant of the human aldosterone synthase gene causes salt-dependent hypertension in transgenic mice Brahmaraju Mopidevi, Nitin Puri, Ashok Kumar. The University of Toledo, Toledo, OH, United States Aldosterone, synthesized by the enzyme CYP11B2, induces positive sodium-balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the hCYP11B2 gene to human hypertension. We have identified three SNPs, in linkage disequilibrium, in the hCYP11Be gene: T/A at -663, T/C at -470 and C/T at -344. Variants ACT occur together and form the haplotype I (Hap I) while variants TTC constitute haplotype II (Hap II). We hypothesize that these SNPs, when present together, will lead to haplotype-dependent transcription of the hCYP11B2 gene, differentially increase aldosterone and affect blood pressure. To this end, novel
transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype II or I variant are used in the study. ChIP assay, using anti-RNA pol II antibody, shows increased Pol II binding to the chromatin from Hap I TG mice in adrenal (2.8 fold higher, p<0.05) and renal tissues (1.3 fold higher, p<0.05) as compared to chromatin extracts from Hap II-TG mice. Immunoblot analysis shows upregulation of the hCYP11B2 in adrenal (2.7 fold higher, p<0.05) and renal tissues (1.35 fold higher, p<0.05) of Hap I vs. Hap II-TG mice; no significant difference was observed in mCYP11B2 between the two haplotypes. Complementary ELISA shows higher circulating levels (p<0.05) of aldosterone in Hap I mice (150448.7 pg/mL) as compared to both, Hap II (778142.8 pg/mL) and C57 mice (74028.9 pg/mL). Importantly, we observed increased baseline blood pressure in Hap I TG mice (Hap I1172.5 vs. Hap II- 1091.9 mm Hg, p<0.05), an effect accentuated by high-salt diet (Hap I- 1352.6 vs. Hap II- 1222.2 mm Hg, p<0.05). Elevated aldosterone was accompanied by up-regulation (p<0.05) of proinflammatory markers in renal tissues from Hap I-TG mice (IL1b, MCP1, ICAM). Thus, this study identifies -344T as a reporter polymorphism for Hap I of the hCYP11B2 gene. SNPs in Hap I promote increased transcription and expression of the gene, in multiple tissues, with resultant elevation of plasma aldosterone levels. Pathophysiological impact of this haplotype-dependent transcriptional regulation of the hCYP11B2 is highlighted by increased inflammation and blood pressure in TG mice with the Hap I of this transgene. Keywords: ldosterone; polymorphism; hypertension
P-123 Modelling the association between variants in the igf-axis and systolic blood pressure as a gene-network Priyakumari G. Parmar. Auckland University of Technology, Auckland, New Zealand It is well accepted that lifestyle and environmental factors play an important role toward causation of elevated blood pressure (BP) levels. High BP is a major modifiable risk factor for cardiovascular diseases (CVD). The prevalence of high BP (or essential/primary hypertension) is recognised to be due to epistasis and the interplay of gene-gene and gene-environment interactions which is correlated with genomic complexity. However, in the current large scale genome-wide framework it is practically impossible to model millions of Single Nucleotide Polymorphisms (SNPs) generated by genome-wide association studies along with repeatedly collected phenotypic measures, due to space and heavy computational constraints. Here I have analysed Systolic Blood Pressure (SBP) with the Insulin-like Growth Factor (IGF) network (which has previously been shown to be associated with blood pressure and cardiovascular health) using a twostage mixed effects gene-set analyses as an example of a proposed method to meet the need to develop sophisticated approaches that model the multilevel variation simultaneously and incorporate gene or pathway-level data into the model whilst not being technologically exhausting. The gene interaction network summarised SNP data within the nine IGF-axis genes (two ligands; IGF1 and IGF2, two receptors; IGF1R and IGF2R, five binding proteins; IGFBP1-5) and their association with the longitudinal SBP outcome from a total of 2279 individuals from The Western Australian Pregnancy (Raine) Cohort Study with one or more measures of SBP recorded between the ages of 5 and 17 years, adjusting for BMI, age as a discrete variable (<10 years, 11-14 years, 15+ years) and sex, with age and sex interacting with all gene-gene interactions.
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved.
GENETICS/GENE THERAPY/PROTEOMICS P-121 Circadian blood pressure monitoring in human and normotensive/ spontaneously hypertensive rats after embryo transfer into different wombs and cross-suckling Mary S. Lee,1 John S. Lee,2 Jong Y. Lee,3 Silvia H. Azar,3 Franz Halberg.3 1 Northwestern University Feinberg School of Medicine &UMN School of Medicine, Chicago /Minneapolis, MN, United States; 2Princeton University & UMN School of Medicine, Princeton /Minneapolis, MN, United States; 3 UMN School of Medicine, Minneapolis, MN, United States Daily (circadian) rhythm characteristics are considered essential parameters for recognizing and treating increased risks in blood pressure (BP). Ambulatory BP in 20 adolescents and adults were monitored automatically around the clock at 30-minute to hourly intervals and analyzed by the least square rhythmometry method. The results were compared with telemetered circadian BP in rats after one-cell homozygous embryo-transfer into spontaneously hypertensive (SHR, pup:shr) or normotensive (WKY, pup:wky) rats’ oviducts (embryos: s,w; oviduct-uterus: S,W) and cross-suckled at birth (nurses S,W). All subjects showed significant circadian fluctuations. The circadian response peaked in the late afternoon hours in most human subjects and early morning hours in rats. Circadian double amplitudes (2A) in the human subjects varied from 8 to 26 mm Hg with higher 2A in elder adults, and 3-8 mm Hg in rats with significantly higher fluctuations in SHR strain groups (7.50.7 for sSS, 8.30.6 for sSW vs. 4.70.3 mm Hg for wWW). In individualized rats’ peak hour comparisons, most groups showed clustered peaks around the light-on hours, while sWS and wSS groups showed individual rats’ peak hour variations (19:18 through 08:06 and 02:58 through 09:10 hours, respectively). The hypertensiveprone shr strain showed significantly lowered BPs in a normotensive WKY uterine environment and/or by WKY nursing mothers, indicating that environment can strongly modify genetic influence, yet the lowered shr MESORs by the WKY environments remained above the MESORs encountered in wky donors. Chronomes broader than circadian should be considered in interpreting BP responses as a gauge of vascular disease status. Keywords: Circadian blood pressure monitoring, embryo transfer; crosssuckling; spontaneously hypertensive and normotensive rat; vascular disease risk.
P-122 A genetic variant of the human aldosterone synthase gene causes salt-dependent hypertension in transgenic mice Brahmaraju Mopidevi, Nitin Puri, Ashok Kumar. The University of Toledo, Toledo, OH, United States Aldosterone, synthesized by the enzyme CYP11B2, induces positive sodium-balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the hCYP11B2 gene to human hypertension. We have identified three SNPs, in linkage disequilibrium, in the hCYP11Be gene: T/A at -663, T/C at -470 and C/T at -344. Variants ACT occur together and form the haplotype I (Hap I) while variants TTC constitute haplotype II (Hap II). We hypothesize that these SNPs, when present together, will lead to haplotype-dependent transcription of the hCYP11B2 gene, differentially increase aldosterone and affect blood pressure. To this end, novel
transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype II or I variant are used in the study. ChIP assay, using anti-RNA pol II antibody, shows increased Pol II binding to the chromatin from Hap I TG mice in adrenal (2.8 fold higher, p<0.05) and renal tissues (1.3 fold higher, p<0.05) as compared to chromatin extracts from Hap II-TG mice. Immunoblot analysis shows upregulation of the hCYP11B2 in adrenal (2.7 fold higher, p<0.05) and renal tissues (1.35 fold higher, p<0.05) of Hap I vs. Hap II-TG mice; no significant difference was observed in mCYP11B2 between the two haplotypes. Complementary ELISA shows higher circulating levels (p<0.05) of aldosterone in Hap I mice (150448.7 pg/mL) as compared to both, Hap II (778142.8 pg/mL) and C57 mice (74028.9 pg/mL). Importantly, we observed increased baseline blood pressure in Hap I TG mice (Hap I1172.5 vs. Hap II- 1091.9 mm Hg, p<0.05), an effect accentuated by high-salt diet (Hap I- 1352.6 vs. Hap II- 1222.2 mm Hg, p<0.05). Elevated aldosterone was accompanied by up-regulation (p<0.05) of proinflammatory markers in renal tissues from Hap I-TG mice (IL1b, MCP1, ICAM). Thus, this study identifies -344T as a reporter polymorphism for Hap I of the hCYP11B2 gene. SNPs in Hap I promote increased transcription and expression of the gene, in multiple tissues, with resultant elevation of plasma aldosterone levels. Pathophysiological impact of this haplotype-dependent transcriptional regulation of the hCYP11B2 is highlighted by increased inflammation and blood pressure in TG mice with the Hap I of this transgene. Keywords: ldosterone; polymorphism; hypertension
P-123 Modelling the association between variants in the igf-axis and systolic blood pressure as a gene-network Priyakumari G. Parmar. Auckland University of Technology, Auckland, New Zealand It is well accepted that lifestyle and environmental factors play an important role toward causation of elevated blood pressure (BP) levels. High BP is a major modifiable risk factor for cardiovascular diseases (CVD). The prevalence of high BP (or essential/primary hypertension) is recognised to be due to epistasis and the interplay of gene-gene and gene-environment interactions which is correlated with genomic complexity. However, in the current large scale genome-wide framework it is practically impossible to model millions of Single Nucleotide Polymorphisms (SNPs) generated by genome-wide association studies along with repeatedly collected phenotypic measures, due to space and heavy computational constraints. Here I have analysed Systolic Blood Pressure (SBP) with the Insulin-like Growth Factor (IGF) network (which has previously been shown to be associated with blood pressure and cardiovascular health) using a twostage mixed effects gene-set analyses as an example of a proposed method to meet the need to develop sophisticated approaches that model the multilevel variation simultaneously and incorporate gene or pathway-level data into the model whilst not being technologically exhausting. The gene interaction network summarised SNP data within the nine IGF-axis genes (two ligands; IGF1 and IGF2, two receptors; IGF1R and IGF2R, five binding proteins; IGFBP1-5) and their association with the longitudinal SBP outcome from a total of 2279 individuals from The Western Australian Pregnancy (Raine) Cohort Study with one or more measures of SBP recorded between the ages of 5 and 17 years, adjusting for BMI, age as a discrete variable (<10 years, 11-14 years, 15+ years) and sex, with age and sex interacting with all gene-gene interactions.
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved.