Genetics of hypertrophic cardiomyopathy in Norway.

Genetic testing for hypertrophic cardiomyopathy (HCM) became available in Norway in 2003. Here, we describe the results of this testing in probands with HCM referred until the end of 2012. The translated exons of MYBPC3, MYH7, TNNI3, TNNT2, MYL2 and MYL3 were analyzed in two groups of probands. In Group 1, comprising 696 probands above 1 year of age, a mutation was found in 203 patients (29.2%). Of those, 5.9% were carriers of two mutations. Mean age in double mutation carriers, single mutation carriers and mutation negative probands was 44 years (± 19 years), 50 years (± 5 years) and 55 years (± 6 years), respectively. In Group 2, comprising 26 infants below the age of 1, a mutation was found in 15.4%. A total of 120 different mutations were found of which 51 (42.5%) were novel.

Postmortem genetic testing of the ryanodine receptor 2 (RYR2) gene in a cohort of sudden unexplained death cases.

The aim of this investigation was to identify pathogenic variants of the ryanodine receptor 2 (RYR2) gene in a cohort of persons aged 0-40 years who died of sudden unexpected death syndrome (SUD), including a cohort of infants who died of sudden infant death syndrome (SIDS). We genetically screened 29 of the 105 exons of the RYR2 gene associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) in 74 cases of SUD without reported structural abnormalities of the heart. Cases were selected from the case database at the Institute of Forensic Medicine, and subsequent mutational screening by DNA sequencing was performed to detect variants in DNA samples extracted from blood samples of deceased persons. A total of 7 of the examined 74 cases were heterozygous for a rare sequence variant in the RYR2 gene. We identified five novel missense variants (p.Q486H, p.D1872N, p.G2367R, p.E4213D, and p.H4579Y), one synonymous variant (p.L4767L), and one previously reported missense variant (p.G4315E). Follow-up studies were possible in family members of three probands (p.Q486H, p.D1872N, and p.H4579Y), and clinical examinations were conducted in family members of two of these probands (p.Q486H and p.H4579Y). In conclusion, we identified a higher prevalence of variants in the CPVT-associated gene RYR2 than in a previously reported cohort of SIDS (9.4% vs. 1-2%). Segregation studies show that one variant (p.H4579Y) co-segregates with CPVT and is presumed to be pathogenic.

Molecular autopsy in young sudden cardiac death victims with suspected cardiomyopathy.

The aim of this investigation was to identify and characterise pathogenic mutations in a sudden cardiac death (SCD) cohort suspected of cardiomyopathy in persons aged 0-40 years. The study material for the genetic screening of cardiomyopathies consisted of 41 cases and was selected from the case database at the Institute of Forensic Medicine. Mutational screening by DNA sequencing was performed to detect mutations in DNA samples from deceased persons suspected of suffering from hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricle cardiomyopathy (ARVC). A total of 9 of the examined 41 cases had a rare sequence variant in the MYBPC3, MYH7, LMNA, PKP2 or TMEM43 genes, of which 4 cases (9.8%) were presumed to be pathogenic mutations. The presumed pathogenic mutations were distributed with one case of suspected HCM and DCM (MYH7; p.R442H), one case of suspected DCM (LMNA; p.R471H), and two cases of suspected ARVC (PKP2; p.R79X and LMNA; p.R644C). The presented data adds important information on the genetic elements of SCD in the young, and calls for expert pathological evaluation and molecular autopsy in the post-mortem examination of SCD victims with structural anomalies of the heart.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis.

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Molecular genetic analysis of long QT syndrome in Norway indicating a high prevalence of heterozygous mutation carriers.

Mutations in the KCNQ1, HERG, SCN5A, minK and MiRP1 genes cause long QT syndrome (LQTS), of which there are two forms: the Romano Ward syndrome and the Jervell and Lange-Nielsen syndrome. We have performed DNA sequencing of the LQTS-associated genes in 169 unrelated patients referred for genetic testing with respect to Romano Ward syndrome and in 13 unrelated patients referred for genetic testing with respect to Jervell and Lange-Nielsen syndrome. A total of 37 different mutations in the 5 genes, of which 20 were novel, were identified. Among patients with the most stringent clinical criteria of Romano Ward syndrome, a mutation was identified in 71%. Twelve of the 13 unrelated patients referred for genetic testing with respect to Jervell and Lange-Nielsen syndrome were provided with a molecular genetic diagnosis. Cascade genetic screening of 505 relatives of index patients with molecularly defined LQTS identified 251 mutation carriers. The observed penetrance was 41%. Although caution must be exerted, the prevalence of heterozygotes for mutations in the LQTS-associated genes in Norway could be in the range 1/100-1/300, based on the prevalence of patients with Jervell and Lange-Nielsen syndrome.

Characterization of novel mutations in the catalytic domain of the PCSK9 gene.

OBJECTIVES: To expand our understanding of the structure and function of proprotein convertase subtilisin/kexin type 9 (PCSK9) by studying how naturally occurring mutations in PCSK9 disrupt the function of PCSK9. DESIGN: Mutations in PCSK9 were identified by sequencing of DNA from subjects with hypo- or hypercholesterolemia. The effect of the identified mutations on the autocatalytic cleavage and secretion of PCSK9, as well as the effect on PCSK9-mediated degradation of the low density lipoprotein receptors, were determined in HepG2 or HEK293 cells transiently transfected with mutant PCSK9-containing plasmids. The findings were collated to the clinical characteristics of the subjects possessing these mutations, and the phenotypic effects were analysed in terms of available structural data for PCSK9. RESULTS: Five novel mutations in PCSK9 were identified. Mutation R215H was a gain-of-function mutation which causes hypercholesterolemia. Mutation G236S and N354I were loss-of-function mutations due to failure to exit the endoplasmic reticulum or failure to undergo autocatalytic cleavage, respectively. Mutations A245T and R272Q were most likely normal genetic variants. By comparing the number of patients with gain-of-function mutations in PCSK9 with the number of familial hypercholesterolemia heterozygotes among subjects with hypercholesterolemia, the prevalence of subjects with gain-of-function mutations in PCSK9 in Norway can be estimated to one in 15,000. CONCLUSION: This study has provided novel information about the structural requirements for the normal function of PCSK9. However, more studies are needed to determine the mechanisms by which gain-of-function mutations in PCSK9 cause hypercholesterolemia.

Low-density lipoprotein receptor activity in Epstein-Barr virus-transformed lymphocytes from heterozygotes for the D374Y mutation in the PCSK9 gene.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia. MATERIAL AND METHODS: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells. RESULTS: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls. CONCLUSIONS: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.

Polymorphisms in ABCG5 and ABCG8 transporters and plasma cholesterol levels.

ABCG5 and ABCG8 transporters play an important role in the absorption and excretion of sterols. Missence polymorphisms (Gln604Glu in the ABCG5 and Asp19His, Tyr54Cys, Thr400Lys, and Ala632Val in the ABCG8) in these genes have been described. In 131 males and 154 females whose dietary composition markedly changed and lipid parameters decreased over an 8-year follow-up study (total cholesterol decreased from 6.21+/-1.31 mmol/l in 1988 to 5.43+/-1.06 mmol/l in 1996), these polymorphisms were investigated using PCR. Plasma lipid levels and changes in plasma lipid levels were independent of the Gln604Glu polymorphism in ABCG5 and Asp19His and the Ala632Val polymorphisms in ABCG8. The Tyr54Cys polymorphism influenced the degree of reduction in total plasma cholesterol (delta -0.49 mmol/l in Tyr54 homozygotes vs. delta +0.12 mmol/l in Cys54 homozygotes, p<0.04) and LDL-cholesterol (delta -0.57 mmol/l in Tyr54 homozygotes vs. delta +0.04 mmol/l in Cys54 homozygotes, p<0.03) levels between 1988 and 1996 in females, but not in males. Male Thr400 homozygotes exhibited a greater decrease in total cholesterol (delta -0.90 mmol/l vs. delta -0.30 mmol/l, p<0.02) and LDL-cholesterol (delta -0.62 mmol/l vs. delta -0.19 mmol/l, p<0.04) than Lys400 carriers. No such association was observed in females. We conclude that Tyr54Cys and Thr400Lys variations in the ABCG8 gene may play a role in the genetic determination of plasma cholesterol levels and could possibly influence the gender-specific response of plasma cholesterol levels after dietary changes. These polymorphisms are of potential interest as genetic variants that may influence the lipid profile.

Mutations in ATP-cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.

Sitosterolemia is an autosomal recessive disorder caused by mutations in two adjacent genes encoding coordinately regulated ATP binding cassette (ABC) half transporters (ABCG5 and ABCG8). In this paper we describe three novel mutations causing sitosterolemia: 1) a frameshift mutation (c.336-337insA) in ABCG5 that results in premature termination of the protein at amino acid 197; 2) a missense mutation that changes a conserved residue c.1311C>G; N437K) in ABCG5 and 3) a splice site mutation in ABCG8 (IVS1-2A>G). This study expands the spectrum of the ABCG5 and ABCG8 mutations that cause sitosterolemia. Nine nonsynonymous polymorphisms are also reported: I523V, C600Y, Q604E, and M622V in ABCG5; and D19H, Y54C, T400K, A632V, and Y641F in ABCG8.

Polymorphisms in candidate genes for blood pressure regulation in young men with normal or elevated screening blood pressure.

We have previously shown correlations between cardiovascular risk factors such as blood pressure (BP), sympathetic nervous system activity, lipids and insulin resistance in young men with elevated screening BP. In the present study we aimed to: (1) compare the genotype distribution and allele frequencies of 11 polymorphisms in seven candidate genes for BP regulation in healthy 21-year-old Caucasian men, between 18 men with normal and 67 men with high screening BP, and (2) evaluate the effect of these polymorphisms in candidate genes on casual BP, BP responses to mental stress or catecholamines and metabolic parameters including insulin sensitivity. There were no differences in genotype distributions or allele frequencies between the subjects with normal and those with high screening BP. Insulin sensitivity was significantly higher in GG homozygotes in the G-261A polymorphism at the alpha 2A-adrenergic receptor (alpha(2A)AR) locus compared to GA heterozygotes (p = 0.007). Subjects who were homozygous both GG in the G-261A polymorphism at the alpha(2A)AR locus and GlyGly in the Arg16Gly polymorphism at the beta2-adrenergic (beta2AR) receptor loci had significantly higher insulin sensitivity and lower catecholamine levels during mental stress than subjects with other genotypes. Subjects who were II homozygous at the angiotensin converting enzyme (ACE) locus and AA homozygous at the angiotensin type I receptor (AT1R) locus had lower BP and a better lipid profile than the rest of the group. Thus, in this explorative study, we report an association between insulin sensitivity and a polymorphism at the alpha(2A)AR locus. We suggest the presence of gene-gene interactions in the renin-angiotensin system and the sympathetic nervous system.

Detection of mRNA for the terminal complement components C5, C6, C8 and C9 in human umbilical vein endothelial cells in vitro.

Human umbilical vein endothelial cells (HUVEC) have previously been shown to synthesize the functional terminal pathway of complement based on the detection by radioimmunoassay of the terminal complement complex (TCC) on coincubated agarose beads. In addition, C7 secretion by these cells in amounts comparable to C3, as well as C7 mRNA, has recently been demonstrated. However, it has not been possible to detect C5-6 and C8 in the fluid phase, and only trace amounts of soluble C9. Against this background we examined whether mRNA for the remaining terminal complement factors was present in HUVEC. By the use of reverse transcription (RT)-polymerase chain reaction (PCR) and Northern blot the presence of mRNA for complement factors C5, C6, C8 and C9 was demonstrated.

Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.

In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family (six mutations in ABCG8 and one in ABCG5) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.

Polymorphisms at the angiotensinogen (AGT) and angiotensin II type 1 receptor (AT1R) loci and normal blood pressure.

The M235T polymorphism at the angiotensinogen (AGT) locus and the A1166C polymorphism at the angiotensin II type 1 receptor (AT1R) locus have been reported to be associated with hypertension in several populations. We examined these polymorphisms in three samples of healthy Norwegians with respect to normal blood pressure (BP) levels. None of the genotypes defined by the polymorphisms or their combinations were associated with systolic (S) BP (SBP) or diastolic (D) BP (DBP) level. However, there was a trend in all three series that individuals carrying the C allele of the A1166C polymorphism at the AT1R locus (homozygotes as well as heterozygotes) had higher SBP, than AA homozygous individuals. The observation did not reach statistical significance in any of the series. When examining these two polymorphisms with respect to possible variability gene effects on BP in two series of monozygote (MZ) twin pairs, no such effect was detected. We could not detect any interaction between the loci studied with respect to BP level or variability. Thus, neither the AGT locus nor AT1R locus, separately analysed or together, seem to have variability gene effects or definite level gene effects on normal BP.

A DNA polymorphism at the angiotensin II type 1 receptor (AT1R) locus and myocardial infarction.

Two hundred and thirty-five survivors of myocardial infarction (MI) were compared to 384 controls with respect to distribution of genotypes and gene frequencies in the A1166C polymorphism at the angiotensin II type 1 receptor (AT1R) locus. No differences in allele frequencies or genotype distribution were observed when all patients were compared with all controls. When comparing CC homozygotes with the combined group of CA heterozygotes and AA homozygotes (CA/AA), a difference in borderline significance between the MI group and controls was observed (p=0.05). In males alone, this difference was much more pronounced because of the larger proportion of males with the CC genotype in MI cases than in male controls (p=0.01). No significant differences were observed between female cases and controls. No interaction between the insertion/deletion (I/D) polymorphism at the angiotensin I-converting enzyme (ACE) locus and the polymorphism at the AT1R locus was detected. When subdividing the subjects into a "low-risk" and a "high-risk" group, based on levels of apolipoprotein B (apoB) and body mass index (BMI), and whether or not the person used lipid-lowering drugs, the frequency of CC homozygotes in male cases of the "low-risk" group differed significantly compared to the frequency in male controls of the "low-risk" group (p<0.001). No differences were observed in females, but the number of "low-risk" group female cases was low (n=3). Thus, CC homozygosity appears to be associated with MI in Norwegian males, especially among those with a "low-risk" phenotype.

Analyses of mutations in the human renal kallikrein (hKLK1) gene and their possible relevance to blood pressure regulation and risk of myocardial infarction.

The kallikrein-kinin system is involved in the maintenance of blood pressure (BP), and studies have shown an inverse correlation between BP and urinary kallikrein levels. These and other effects, make the human tissue kallikrein (hKLK1) gene a candidate gene with respect to BP regulation as well as risk of myocardial infarction (MI). By analysis for single-stranded conformation polymorphisms (SSCPs), patterns consistent with four different variants of the gene were detected and further characterized by DNA sequencing. Three of the variants have not been described before. Two of the polymorphisms changed the codon for an amino acid. Methods based on the polymerase chain reaction (PCR) were developed to analyze these polymorphisms at the hKLK1 locus. We found no evidence of association between any genotype in the polymorphisms and normal BP level in two series of healthy, unrelated individuals. In a third series, diastolic BP exhibited a weak association with genotypes in three of the four polymorphisms. Since no such association was detected in the other two series, we conclude that no effect on normal BP level is exerted by variants in the hKLK1 as expressed in these polymorphisms. In two series of monozygotic (MZ) twin pairs, there were no differences between genotypes in within-pair difference in systolic BP or diastolic BP. Finally, no differences in allele frequencies or genotype frequencies in the four polymorphisms at the hKLK1 locus were found between a series of young MI survivors and a series of controls. Thus, genes in the four polymorphisms at the hKLK1 locus detected by SSCP and DNA sequencing did not exhibit associations with MI, and had neither "level gene" nor "variability gene" effects on normal blood pressure.

Studies on effects of Lp(a) lipoprotein on gene expression in endothelial cells in vitro.

The reason(s) for the atherogenic properties of Lp(a) lipoprotein is still unclear, and several mechanisms have been studied. Alterations in gene expression in endothelial cells (ECs) could be important with respect to risk for coronary heart disease (CHD). We have tested the effects of Lp(a) lipoprotein or the apolipoprotein of Lp(a) lipoprotein (apo(a)) on cultured human umbilical vein endothelial cells (HUVECs) with respect to: (1) the level of endothelin-1 (ET-1) mRNA; (2) release of ET-1 into the culture medium; (3) plasminogen activator inhibitor-1 (PAI-1) secretion into the culture medium and; (4) total gene expression in HUVECs, examined by a polymerase chain reaction (PCR)-based technique, differential display-reverse transcription-PCR (DD-RT-PCR). Lp(a) lipoprotein reduced the level of ET-1 mRNA as well as the release of ET-1. The reduction of ET-1 in the medium was even more pronounced when HUVECs were incubated with apo(a), but we found no effect of apo(a) on ET-1 mRNA level. Neither Lp(a) lipoprotein nor apo(a) had a significant influence on PAI-1 secretion. DD-RT-PCR revealed 11 fragments that could represent differences between cells exposed or not exposed to Lp(a) lipoprotein. Following subcloning and sequencing, 18 sequences that differed between exposed and unexposed cultures were obtained. Four of the subcloned fragments have up to now been used as a probe for northern blot analyses, and one fragment was confirmed to be regulated by Lp(a) lipoprotein. In conclusion, Lp(a) lipoprotein is shown to control ET-1 mRNA levels and the function of at least one more gene, the nature of which is unknown.

Cardiovascular risk factors in people with different genotypes in the insertion/deletion (I/D) polymorphism at the locus for angiotensin I-converting enzyme (ACE).

The deletion (D) allele of an insertion/deletion (I/D) polymorphism at the locus for angiotensin I-converting enzyme (ACE) has been reported to be an independent risk factor for myocardial infarction (MI), particularly in people lacking traditional risk factors. Furthermore, a borderline association between Lp(a) lipoprotein level and the I/D polymorphism at the ACE locus was reported in one study. We have searched for possible "level gene" or "variability gene" effects of ACE genes on Lp(a) lipoprotein, total cholesterol (TC), high density lipoprotein (HDL) cholesterol (HDLC), low density lipoprotein (LDL) cholesterol (LDLC), triglycerides (TG), apolipoprotein B (apoB), apolipoprotein A-I (apoA-I), and body mass index (BMI). None of these variables differed significantly between genotypes in the I/D polymorphism in any of three population samples. A single population sample created by combining the three series, exhibited an insignificant trend towards individuals carrying the D-allele having a higher level of Lp(a) lipoprotein than those lacking it, and DD homozygotes had a significantly higher Lp(a) lipoprotein level than the combined group of ID/II individuals (p = 0.03). These results may indicate that the D-allele of the I/D polymorphism at the ACE locus could influence the level of Lp(a) lipoprotein.

Vitronectin modulates the expression of complement components of the terminal pathway synthesized by human umbilical vein endothelial cells in vitro.

In this study we demonstrate that human endothelial cells (EC) synthesize mRNA for vitronectin by using techniques based on reverse transcriptase (RT) reaction and polymerase chain reaction (PCR). The identification of vitronectin mRNA, shown by sequence analysis of PCR-amplified RT product of RNA extracted from EC, clearly demonstrates that these cells synthesize mRNA for vitronection. We further investigated whether vitronectin in serum-free EC cultures regulates the net expression of the terminal complement pathway, measured as the terminal complement complex (TCC) bound to co-cultured agarose beads which activate the alternative pathway. Presence of polyclonal F(ab')2 anti-human vitronectin (VN) antibodies, regardless of concentration (10-80 micrograms/ml), significantly reduced the binding of monoclonal anti-C3c antibodies to co-cultured beads, whereas the binding of monoclonal anti-TCC antibodies was unaltered or significantly increased compared with controls. Despite some interexperimental variation in the results, addition of vitronectin (10-80 micrograms/ml) to the EC resulted in an inversely related pattern compared with experiments using anti-VN antibodies. The binding indices of anti-C3c are comparable to the controls. On the other hand, there is a steady concentration-dependent (10-80 micrograms of vitronectin added) reduction in binding of anti-TCC up to approximately 60%. The results indicate that vitronectin regulates the expression of synthezised and surface-bound TCC in serum-free EC cultures, comparable to previous findings in serum.

Interleukin-1 alpha, interleukin 6 and tumor necrosis factor alpha increase the synthesis and expression of the functional alternative and terminal complement pathways by human umbilical vein endothelial cells in vitro.

The proinflammatory cytokines interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) modulate the synthesis of complement factors B and C3 by endothelial cells (EC), and are considered to play an important role in the development of sepsis. By using agarose beads activating the alternative pathway of complement, we wanted to study the net effect of these cytokines on EC synthesis of the alternative and terminal pathways, measured by binding of anti-C3c and anti-TCC (terminal complement complex) antibodies to beads kept with the EC. Addition of IL-1 alpha and TNF alpha at concentrations of 50 and 100 U/ml resulted in a significant increase in binding of these antibodies to co-incubated beads, most pronounced for anti-C3c. IL-6 from 50-200 U/ml resulted in a stronger (two to fourfold) binding for both antibodies compared to experiments with IL-1 alpha and TNF. However, increased concentrations of IL-1 alpha (200 U/ml) and IL-6 (400 U/ml) resulted in a strong reduction in binding of anti-C3c and anti-TCC antibodies to the co-cultured beads. This study indicates that proinflammatory cytokines upregulate the synthesis by EC of the functional alternative and terminal pathways of complement.

No effect on blood pressure level or variability of polymorphisms in DNA at the locus for atrial natriuretic factor (ANF).

We have examined healthy Norwegians with respect to two restriction fragment length polymorphisms at the locus for atrial natriuretic factor, detectable with the restriction enzymes XhoI and BglI, respectively. No association with systolic or diastolic blood pressure level or variability was found. Thus, the normal genes detected by examination of these restriction fragment length polymorphisms have neither "level gene" nor "variability gene" effects on normal blood pressure.