Katriina Aalto-Setälä

Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA.

The low‐molecular‐mass insulin‐like growth factor‐binding protein (IGF‐BP) and placental protein 12 (PP12) are identical proteins that are present in human serum, amniotic fluid, secretory endometrium and decidua. IGF‐BP/PP12 is believed to act as an autocrine or paracrine regulator of cell growth. A cDNA clone encompassing the entire protein coding region of this protein was isolated from a human decidual cDNA library. The authenticity of the cDNA was verified by in vitro transcription/translation experiments and by the identity of the 10 N‐terminal amino acids deduced for the mature peptide with those obtained by direct protein sequencing. The amino acid sequence indicates that pre‐IGF‐BP/PP12 consists of 259 amino acid residues. The putative signal peptide is 25 residues long, and the mature protein thus contains 234 amino acids and has a molecular mass of 25 293 Da. The sequence is very cysteine‐rich at the N‐terminus after which there are regions of clustered Pro, Glu, Ser and Thr residues (so‐called PEST regions), which exist in proteins with short half‐lives. The amino acid sequence also includes an Arg‐Gly‐Asp tripeptide that may function as a cell recognition signal. The IGF‐BP/PP12 gene encodes a single 1.6 kb mRNA species that is expressed in decidua, secretory endometrium, liver and a human hepatoma cell line (HepG2). Southern blot analysis suggests that there is a single IGF‐BP/PP12 gene in the human genome.

Lipoproteins and their genetic variation in subjects with and without angiographically verified coronary artery disease.

To examine the concentration of serum lipoproteins and the association of their genetic variation with the occurrence of coronary artery disease (CAD), composite serum lipoprotein profiles including lipoprotein(a) (Lp[a]), apolipoprotein (apo) E phenotypes, and apo B Xba I genotypes were determined in patients with angiographically verified CAD (CAD+ group, n = 111) and in subjects with no angiographic evidence of CAD (CAD- group, n = 46). In addition, we determined the concentrations of serum lipids, lipoproteins, and apolipoproteins in 96 healthy controls. Both CAD- and CAD+ groups had lower concentrations of apos A-I and A-II but higher concentrations of serum total and very low density lipoprotein triglyceride and very low density lipoprotein cholesterol than did healthy controls. The mean concentrations of serum total and low density lipoprotein cholesterol and the median values of Lp(a) were similar in the CAD+ and CAD- groups, both having higher concentrations of low density lipoprotein cholesterol and apo B than the healthy controls. Irrespective of gender, patients with CAD had significantly lower serum high density lipoprotein cholesterol than did those without CAD (1.48 +/- 0.40 versus 1.16 +/- 0.29 mmol/l, p less than 0.001). In women, the mean serum total and very low density lipoprotein triglyceride concentration was also higher in the CAD+ than in the CAD- group. The frequency of the apo E4 allele (epsilon 4) was significantly higher in the CAD+ group (0.293) than in the CAD- group (0.174; p less than 0.001). The frequencies of the two apo B alleles, X1 (Xba I restriction site absent) and X2 (Xba I restriction site present), were similar in the two groups. Stepwise discriminant analysis revealed that in men, serum high density lipoprotein cholesterol had the highest power to discriminate for CAD. In addition, the concentration of plasma apo B levels and the occurrence of apo E phenotypes were independently associated with CAD in men. In women, the only independent factor associated with CAD after adjustment for beta-blocker and diuretics usage was the concentration of serum triglycerides.

XbaI and c/g polymorphisms of the apolipoprotein B gene locus are associated with serum cholesterol and LDL-cholesterol levels in Finland

Several restriction fragment length polymorphisms (RFLPs) have been identified within or adjacent to the gene locus for apolipoprotein B (apo B), the major protein component of serum low density lipoprotein (LDL). One of these, detected with the restriction enzyme XbaI, has been suggested to be involved in the determination of serum lipid levels in some but not all populations. We determined the XbaI genotypes and serum lipoprotein levels of 176 apparently healthy unrelated Finns. Subjects homozygous (genotype X2X2) or heterozygous (genotype X1X2) for the presence of the XbaI restriction site within the apolipoprotein B gene (n = 113) had, on the average, an 11% higher serum total cholesterol (P = 0.01) level than those homozygous for the absence of this site (genotype X1X1, n = 63). In addition, the X2 allele was significantly associated with apo B(c), another allele reportedly associated with elevated serum cholesterol levels. The combined genotype (both X2 and apo B(c) alleles present) resulted in a greater elevation of total cholesterol (P = 0.004, when compared to subjects with neither allele) and LDL-cholesterol (P = 0.02) than the presence of either allele alone. The results suggest that both the XbaI and apo B(c/g) sites are in linkage disequilibrium with a functionally important DNA alteration within or adjacent to the apo B gene but the XbaI locus may be in stronger linkage disequilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of apolipoprotein E polymorphism and Xbal polymorphism of apolipoprotein B on response to lovastatin treatment in familial and non‐familial hypercholesterolaemia

Abstract. Despite the well‐documented efficacy of lovastatin, a wide inter‐individual variation in treatment responses has been observed. The aim of the present study was to investigate the possible roles of apolipoprotein E (apo E) phenotype and apolipoprotein B (apo B) Xbal genotype on this variation. The apo E phenotype was determined in 232 subjects (78 cases of familial hypercholesterolaemia [FH] and 154 cases of non‐familial hypercholesterolaemia [non‐FH]) and the apo B Xbal genotype was determined in 211 subjects (67 cases of FH, 144 cases of non‐FH). Depending on their baseline total serum cholesterol levels, these patients used a starting dose of lovastatin of either 20 or 40 mg nightly. After 6 weeks of therapy, slightly but significantly smaller reductions in LDL‐cholesterol were observed in patients with the E4/3 phenotype compared with those with the E3/3 phenotype in non‐FH with lovastatin 20 mg (–20 vs. –28%; P = 0.043) and in total cholesterol in FH with lovastatin 40 mg (–23 vs. –27%; P = 0.023). No significant differences were found in non‐FH patients starting with lovastatin, 40 mg. After doubling of the lovastatin doses, all treatment responses became similar among apo E phenotypes. Moreover, when all patients using lovastatin 40 mg either at 6 or 12 weeks were pooled (n = 224), no differences in treatment responses were observed between the E3/2, E3/3, E4/3 and E4/4 phenotypes. The apo B Xbal genotype did not affect the hypocholesterolaemic efficacy of lovastatin in any of the patient groups. Thus our results indicate that inter‐individual variation in the treatment response to lovastatin in both familial and non‐familial hypercholesterolaemia is mainly due to factors other than the apo E phenotype or apo B Xbal genotype.

Polymorphisms of the gene encoding cholesterol ester transfer protein and serum lipoprotein levels in subjects with and without coronary heart disease

SummaryWe determined TaqI-A, TaqI-B and EcoNI genotypes at the cholesteryl ester transfer protein (CETP) locus in 111 healthy volunteers and in 187 hyperlipidemic men of whom 72 had suffered a myocardial infarction. There were no significant differences in the allele distributions at these polymorphic loci either between the population sample and the hyperlipidemic subjects, or between patients with and without previous myocardial infarction. To detect the associations between the CETP polymorphisms and serum lipid and apoprotein levels, we determined the serum concentrations of total cholesterol, triglycerides, high density lipoprotein (HDL)-cholesterol, apoA-I, apoA-II and apoB in the subjects studied and correlated them to the 3 RFLPs. No significant differences were observed in the serum levels of apoproteins and lipid parameters between subjects with different genotypes in any of these polymorphic CETP loci, either in the population sample or in hyperlipidemic men. Multivariate analyses did not reveal a significant independent role for any of the 3 polymorphisms in determining serum HDL-cholesterol or apoA-I levels after adjusting for triglyceride and low density lipoprotein cholesterol concentrations. This was evident for the group of healthy volunteers and for hyperlipidemic subjects, including those who had survived a myocardial infarction. We conclude that, in Finns, the CETP RFLPs are not useful markers for the risk of coronary heart disease.

Absence of familial defective apolipoprotein B-100 in Finnish patients with elevated serum cholesterol.

Familial defective apolipoprotein B-100 is a genetic disorder which is associated with elevated plasma LDL levels. It appears to result from a G----A mutation at nucleotide 10,708 in exon 26 of the apolipoprotein B-100 gene leading to a substitution of glutamine for arginine at amino acid residue 3500. We explored the possible role of this point mutation as a cause of elevated plasma cholesterol among the Finns, a genetically isolated population in which both hypercholesterolemia and coronary heart disease are common: 552 hyperlipidemic patients from Western and Southern Finland were screened either by assaying patient sera with monoclonal antibody MB47 or by amplifying the region of the apo B gene containing the nucleotide 10,708 followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes. Not a single individual with this particular mutation could be found. We conclude that familial defective apo B-100 is not a common cause of elevated plasma cholesterol in this population.

Genetic polymorphism of the apolipoprotein B gene locus influences serum LDL cholesterol level in familial hypercholesterolemia

SummaryAn XbaI restriction fragment length polymorphism (RFLP) within the coding region of the apolipoprotein B (apoB) gene has been found to be associated with serum cholesterol and triglyceride levels in several populations. Mutations in another genetic locus, the low density lipoprotein (LDL) receptor gene, give rise to familial hypercholesterolemia (FH), a disease characterized by hypercholesterolemia, tendon xanthmas and atherosclerosis. We determined the XbaI genotypes and serum lipoprotein levels of 120 unrelated patients with the heterozygous form of FH. A non-parametric analysis of variance showed a significant association between elevated serum total cholesterol concentration (P<0.05), serum LDL-choleterol concentration (P<0.025) and the presence of the XbaI restriction site (X2 allele). Thus, patients homozygous for the presence of the XbaI restriction site (genotype X2X2, n=28) had on average a 14% higher serum total cholesterol level and a 21% higher serum LDL-cholesterol level than those homozygous for the absence of this site (genotype X1X1, n=29); patients heterozygous for the XbaI restriction site (genotype X1X2, n=63) had intermediate serum total and LDL-cholesterol levels. No significant differences were seen in serum triglyceride or high-density lipoprotein (HDL)-cholesterol values between these patient groups. These data demonstrate that genetic polymorphism of the principal ligand for the LDL receptor, apoB, may contribute to serum cholesterol regulation, even in patients with grossly distorted cholesterol homeostasis.

Identification of a deletion in the LDL receptor gene A Finnish type of mutation

A cDNA probe for the low density lipoprotein (LDL) receptor gene was used to screen DNA samples from 52 unrelated Finnish patients with the heterozygous form of familial hypercholesterolemia (FH) and 51 healthy controls. Southern blot analysis using the restriction enzyme PvuII revealed an abnormal 11 kb (kilo base‐pair) restriction fragment in 16 (31%) of the patients but none of the controls. A more detailed restriction enzyme analysis of the DNA from patients revealed a mutation which apparently is due to an 8 kb deletion extending from intron 15 to exon 18 of the LDL receptor gene. Co‐segregation of FH with the mutated gene was demonstrated in three families. These data are consistent with a ‘founder gene effect’ and support the assumption that recombinant DNA methods may have great impact on the diagnostics of FH in genetically homogeneous populations.

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.

Variability gene effects of DNA polymorphisms at the apo B, apo A I/C III and apo E loci on serum lipids : the Cardiovascular Risk in Young Finns Study

We studied the influence of selected genetic markers on the intra‐individual long‐term variability in serum lipid levels. The study cohort consisted of a sub‐sample from a large follow‐up study of atherosclerosis precursors in children and young adults. A total of 320 subjects had determinations of apo B XbaI RFLP genotypes, 305 subjects had apo AI/CIII SstI RFLP genotype determinations and 1581 subjects had their apo E phenotypes determined. Complete data on serum lipids were available at 3‐year intervals over a 6‐year follow‐up period. The subjects were healthy and aged 3–18 years at baseline. Intra‐individual variability was assessed with a nested analysis of variance procedure. Each of the genetic markers studied here significantly affected intra‐individual variability of serum lipid levels. No clear sex influence was observed, although the differences in variability tended to be more significant in males. Apo B XbaI genotypes significantly influenced intra‐individual variability of total and LDL‐cholesterol levels in both sexes. A marked effect of the XbaI geno‐type was also found on triglyceride variability. In males the standardized intra‐individual triglyceride variances were 0.71 and 0.34 in genotypes X1X1 and X2X2, respectively (p < 0.001), with a clear gene dosage effect. The apo AI/CIII genotype had an influence only on the variability of total cholesterol and LDL‐cholesterol levels and only in males. The apo E phenotypes were associated with intra‐individual variability in total and LDL‐cholesterol levels but again, only in males. The lowest variability was observed in the phenotype E4/3 where high mean values were also observed. We also examined the effect of combined genetic markers. Up to 7 times greater variability was found in the combination E3/2 + S1S1 compared to combination E4/3 + S1S2 (p < 0.001). In addition, mean levels of, e.g., LDL‐cholesterol were 70% greater in the combination of E4/3 +S1S2 compared to E3/2 + S1S1. This implies that subjects with both these genetic markers have high LDL‐cholesterol values that also tend to remain constantly elevated. In conclusion, it is evident that many of the presently known DNA polymorphisms of the coronary heart disease candidate gene loci also influence intraindividual variability of serum lipid or lipoprotein levels. These findings can be used to further refine our ability to predict the risk of a cardiovascular event.