Helena E. Miettinen

Aging and genetic variation of plasma apolipoproteins. Relative loss of the apolipoprotein E4 phenotype in centenarians.

We determined the common polymorphism of apolipoprotein E (E2, E3, and E4), apolipoprotein B Xba I polymorphism, and apolipoprotein C-III Sst I polymorphism in almost all Finnish centenarians alive in 1991 (n = 179/185). Plasma lipid and lipoprotein levels in different apolipoprotein genotypes were also measured. In comparison with younger Finnish populations studied previously, the frequency of the apolipoprotein E epsilon 2 allele was almost twice as high (7.0% versus 4.1%; P < .05) and that of the epsilon 4 allele only approximately one third as high (8.4% versus 22.7%; P < .001) in the centenarians. Plasma cholesterol and high-density lipoprotein cholesterol levels tended to be lowest in the group with the epsilon 2 allele (4.33 mmol/L and 1.41 mmol/L, respectively), intermediate in those with the epsilon 3 allele (4.57 mmol/L and 1.48 mmol/L, respectively), and highest in those with the epsilon 4 allele (4.82 mmol/L and 1.60 mmol/L, respectively). The frequencies of the apolipoprotein B X1 and X2 alleles (Xba I restriction site absent or present, respectively) among the centenarians and among the young Finns were not significantly different, whereas the apolipoprotein C-III S2 allele (Sst I restriction site present) occurred more often in the centenarians (frequency, 12.9%) than in the youngest reference population (frequency, 8.8%; P < .05). Centenarians with the apolipoprotein B X2X2 genotype and apolipoprotein E4 phenotype had a higher mean plasma cholesterol level than those with the X1X1 genotype and E2 phenotype (5.24 versus 3.43 mmol/L; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)–deficient mice

Mammalian female fertility depends on complex interactions between the ovary and the extraovarian environment (e.g., the hypothalamic-hypophyseal ovarian axis). The role of plasma lipoproteins in fertility was examined using HDL-receptor SR-BI knockout (KO) mice. SR-BI KO females have abnormal HDLs, ovulate dysfunctional oocytes, and are infertile. Fertility was restored when the structure and/or quantity of abnormal HDL was altered by inactivating the apoAI gene or administering the cholesterol-lowering drug probucol. This suggests that abnormal lipoprotein metabolism can cause murine infertility--implying a functional hepatic-ovarian axis--and may contribute to some forms of human female infertility.

Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5–6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between ≈3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease.

Polymorphisms of the apolipoprotein and angiotensin converting enzyme genes in young North Karelian patients with coronary heart disease

The genes encoding apolipoproteins (apos) A-I, B, C-III and E as well as that encoding the angiotensin converting enyzme (ACE) have been proposed as candidate genes for coronary heart disease (CHD). We determined the common polymorphisms of the apo genes, previously found to influence serum lipid levels at the population level, and the insertion/deletion polymorphism of the ACE gene, recently reported to reflect the risk of myocardial infarction, in 82 very young (mean, 41 years) North Karelian Finns with symptomatic CHD and 50 controls of similar age. Patients with familial hypercholesterolemia had been excluded from this material. None of the polymorphisms examined, including the apo A-I promoter MspI, apo C-III SstI and apo B XbaI restriction fragment polymorphisms, a common variation of apo E (ɛ2, ɛ3 and ɛ4 alleles) and an ACE insertion/deletion (I/D) polymorphism, was significantly associated with the risk of premature CHD. Patients with CHD had a higher mean serum LDL cholesterol/HDL cholesterol ratio than controls (3.15±1.30 vs 2.72±0.98, P < 0.05), but no significant associations between the common apo gene polymorphisms and serum lipid levels were disclosed in either group. It is possible that other genetic loci than those proposed to be associated with accelerated atherosclerosis may be more important as risk factors of symptomatic CHD at the age of 40 years.

Common variants of the beta and gamma subunits of the epithelial sodium channel and their relation to plasma renin and aldosterone levels in essential hypertension

BackgroundRare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddles syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system.MethodsInitially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests.ResultsTwo commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males (2.9%, p = 0.007) and blood donors (3.0%, p = 0.001). βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001). When expressed in Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean (of supine and upright values) plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048).ConclusionsAt least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels.

Apolipoprotein A-IFin Dominantly Inherited Hypoalphalipoproteinemia Due to a Single Base Substitution in the Apolipoprotein A-I Gene

We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-IFin). The T-to-G substitution destroys an Fsp I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-IFin mutation. Isoelectric focusing revealed the presence of the mutant apo A-IFin protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-IFin, causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-I Fin ). The T-to-G substitution destroys an Fsp I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-I Fin mutation. Isoelectric focusing revealed the presence of the mutant apo A-I Fin protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-I Fin , causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.

Apolipoprotein A-IFin

We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-IFin). The T-to-G substitution destroys an Fsp I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-IFin mutation. Isoelectric focusing revealed the presence of the mutant apo A-IFin protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-IFin, causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.We have identified a large kindred with severe serum HDL cholesterol deficiency. The proband, a 65-year-old woman, had greatly diminished concentrations of serum HDL cholesterol (0.19 mmol/L) and apolipoprotein (apo) A-I (21.9 mg/dL). HDL cholesterol and apo A-I levels were similarly reduced in all affected family members, while apo A-II levels were about half of those in the nonaffected family members. Pedigree analysis suggested a dominant inheritance pattern of the phenotype. Sequence analysis of the exons and exon-intron boundaries of the apo A-I gene revealed heterozygosity for a single T-to-G point mutation substituting arginine for leucine at residue 159 of the mature apo A-I protein (apo A-I Fin ). The T-to-G substitution destroys an Fsp I cleavage site, permitting direct polymerase chain reaction/restriction enzyme analysis of the mutation. All the affected family members were shown to be heterozygous for the apo A-I Fin mutation. Isoelectric focusing revealed the presence of the mutant apo A-I Fin protein in both serum and HDL of the affected subjects. Functional consequences of the mutation were examined by expressing the mutated and wild-type apo A-I cDNAs in COS-7 cells. The mutant apo A-I mRNA had a size similar to that of the normal mRNA, and both mutant and wild-type apo A-I proteins were secreted into the cell media. In vivo kinetic studies of apo A-I revealed increased catabolism in affected subjects. In conclusion, we describe a novel point mutation of the apo A-I gene, apo A-I Fin , causing a dominantly negative phenotype as regards serum HDL levels, possibly due to increased catabolism of apo A-I.

Polymorphisms of the Genes Encoding Apoproteins A-I, B, C-III, and E and LDL Receptor, and Cholesterol and LDL Metabolism During Increased Cholesterol Intake Common Alleles of the Apoprotein E Gene Show the Greatest Regulatory Impact

Genetic and dietary factors regulate serum cholesterol level, but detailed investigations into their interactions have not been established. We assessed the effects of apoprotein (apo) E phenotype and polymorphic alleles of the apo A-I, apo B, apo C-III, and LDL receptor genes, separately and together, on regulation of serum LDL cholesterol level. The study group consisted of 29 middle-aged men, and cholesterol absorption, bile acid, and cholesterol synthesis and LDL apo B kinetics were studied in these men during low- and high-cholesterol diets. The six apo B alleles were identified on the basis of Xba I, EcoRI, and Msp I restriction fragment length polymorphism (RFLP), the apo A-I alleles with the Msp I RFLP, and the apo C-III and LDL receptor alleles corresponded to the Sst I and PvuII RPLPs of these genes, respectively. During low cholesterol intake, LDL cholesterol levels were similar in all of the genetic groups except for men with apo E2 phenotype. They had significantly (P < .05) lower levels of LDL apo B and cholesterol than men without the epsilon 2 allele. The low values were caused by a significantly higher removal of LDL apo B (apo E2, 0.453 +/- 0.03 versus apo E3, 0.312 +/- 0.01 pools per day, P < .05). High cholesterol intake increased LDL cholesterol levels in all genetic categories except in the apo E2 phenotype irrespective of the combinations with other polymorphisms. Carriers of the apo B R+ allele (EcoRI site present) presented with the most prominent LDL cholesterol rise (from 2.71 +/- 0.14 to 3.37 +/- 0.29 mmol/L). In multiple stepwise regression analysis, apo B EcoRI RFLP and apo E phenotypes were the only variables that explained the variability of high cholesterol intake-induced change in LDL cholesterol levels. In summary, in any genetic combination, individuals with the epsilon 2 allele had the lowest LDL cholesterol values and were nonresponders to dietary cholesterol, whereas subjects with the apo B R+ allele had marked LDL elevations, especially in combination with the epsilon 4.

Molecular Genetic Study of Finns With Hypoalphalipoproteinemia and Hyperalphalipoproteinemia

In an attempt to identify genetic factors underlying extreme alterations of serum HDL cholesterol (HDL-C) concentrations, we examined two probands with HDL-C levels A mutation of cholesterol ester transfer protein (CETP) in the high-HDL-C group and subsequently demonstrated cosegregation of the mutant allele with elevated HDL-C levels in the probands family. In conclusion, we have identified a novel LCAT gene Gly230Arg mutation (LCAT[Fin]), which, together with the LPL Asn291Ser mutation, represents a relatively common genetic cause of diminishing HDL-C levels, at least among Finns. This article also reports occurrence of a CETP mutation in subjects having non-Japanese roots.

Genetic risk factors and ischaemic cerebrovascular disease: role of common variation of the genes encoding apolipoproteins and angiotensin-converting enzyme

DNA polymorphisms in genes encoding apolipoproteins (apo) A-I, C-III, B and E and angiotensin-converting enzyme (ACE) have been proposed to be associated with the risk of coronary artery disease (CAD). We studied whether the same genetic markers would also be associated with the occurrence and extent of atherosclerosis in cervical arteries. DNA samples from 234 survivors of stroke or a transient ischaemic attack aged 60 years or less were examined. The presence of atherosclerosis was assessed using aortic arch angiograms. The SstI polymorphism of apoA-I/C-III gene locus, the XbaI polymorphism of apoB gene, common apoE phenotypes and the insertion/deletion polymorphism of the ACE gene were analysed. The allele frequencies of the apoA-I/C-III, apoB, apoE or ACE gene did not differ between the groups with (n = 148) or without (n = 85) cervical atherosclerosis. However, when patients with at least one apoE4 allele and one X2 allele of apoB were combined and compared with those without either of them (E2E3 or E3E3 and X1X1), a significant association with the presence of cervical atherosclerosis was found (P = 0.03). The patients having the E2E3 phenotype had a significantly elevated serum triglyceride level compared with those with the E3E3 phenotype (P = 0.03). Serum high-density lipoprotein (HDL) cholesterol was lower in the patients with the E2E3 phenotype than in those with the E3E3 and E3E4 (P = 0.01 and P = 0.06, respectively). The apoB or ACE genotypes were not significantly associated with serum lipid or lipoprotein levels. There was no association between the ACE gene polymorphism and the occurrence of hypertension. In conclusion, the interaction of common apoB and apoE alleles may increase the risk of atherosclerosis in cervical arteries.