Anna M. Kessling

Apolipoprotein B gene variants are involved in the determination of serum cholesterol levels: a study in normo- and hypelipidaemic individuals☆

We have investigated the frequencies of 3 restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene in normo- and hyperlipidaemic individuals. In individuals with type III hyperlipidaemia, the allele frequency for the RFLP detected with XbaI was significantly different from the allele frequency in normolipidaemic individuals and in those with other types of hyperlipidaemia. No significant difference in allele frequency was found among these groups for the RFLPs detected with MspI or EcoRI. Within a sample of 62 normolipidaemic individuals, homozygotes for the X2 allele (cutting site) of the XbaI RFLP had a significantly higher serum cholesterol level than homozygotes for the XI allele, with individuals of the genotype X1X2 having an intermediate value (X2X2 mean 5.71 mmol/l, X1X1 mean 4.81 mmol/l, X1X2 mean 5.30 mmol/l). There were also significant differences in serum triglyceride levels in individuals with different XbaI genotypes. In these normolipidaemic individuals there was no correlation between the EcoRI and MspI RFLP genotypes and levels of any serum lipid variable. Information from the XbaI and EcoRI RFLPs was used in conjunction to define apo B haplotypes. These haplotypes are a more precise measure of the genotypic variation, and they explain a greater fraction of the serum cholesterol and triglyceride levels than the single-site polymorphisms considered separately. This study suggests that variations in the gene for apo B are associated with the determination of serum cholesterol and triglyceride levels both in patients with type III hyperlipidaemia and in the normal population.

Influences of common variants of apolipoprotein E on measures of lipid metabolism in a sample selected for health.

Five-hundred seventy-five white-collar workers (374 men; 99% Caucasians) aged 20-59 years were selected on the basis of their being healthy and clinically free from cardiovascular risk factors (except smoking and family history). We have observed a higher relative frequency of the epsilon 3 allele in this population, as is true of populations with a low prevalence of coronary heart disease. Each of the 11 plasma lipid and lipoprotein traits studied was adjusted for age, weight, height, body mass index, plasma glucose, and uric acid in men and women separately. The influence of each of the three common apo E alleles on each adjusted trait was evaluated by use of the average excess statistic. We established that in a population selected for health, the epsilon 2 allele is associated with lower plasma levels of total cholesterol, low density lipoprotein (LDL) cholesterol, and apolipoprotein B associated with LDL cholesterol in both men and women. Conversely, the epsilon 4 allele is associated with higher levels of these traits in women only. In contrast to the findings in populations not selected for health, the presence of the epsilon 2 allele in our subjects tended to be associated with lower and the epsilon 4 allele with higher plasma triglyceride levels. Finally and of particular note, the influence of the apolipoprotein E polymorphism on plasma measures of LDL metabolism is different in men and women. Specifically, the influence of the epsilon 4 allele is of greater magnitude in women. A part of this gender difference in allele effects on LDL metabolism in women is associated with the use of oral contraceptives and postmenopausal hormone replacement therapy.

A study of DNA polymorphisms around the human apolipoprotein Al gene in hyperlipidaemic and normal individuals

We have used a 2.2 kb fragment of the human apolipoprotein AI (apo AI) gene to screen a number of unrelated individuals for common restriction fragment length polymorphisms (RFLPs) of the gene. As well as the previously reported SstI RFLP (allele frequencies in normolipidaemic individuals 0.94 and 0.06) we have detected RFLPs with the enzymes PstI and XmnI (allele frequencies in normolipidaemic individuals 0.88 and 0.12 for both polymorphisms). In the population studied, the RFLPs appear to be in linkage equilibrium and can be used in conjunction as a haplotype, with a PIC value (polymorphism information content) of 0.5. Significant differences in allele frequency were observed between subgroups of hyperlipidaemic patients and normolipidaemic controls. There is no strong population association in our patient group between any allele of the RFLPs studied and hypertriglyceridaemia.

MTHFR association with arteriosclerotic vascular disease

Abstract Complex diseases are far more common than diseases that follow simple Mendelian patterns of inheritance. Difficulties are experienced in the designing of experiments to dissect out the contribution of a single allele to a complex phenotype. We review the literature regarding a point mutation in methylenetetrahydrofolate reductase, a candidate gene for susceptibility to vascular diseases.

A common DNA polymorphism of the low-density lipoprotein (LDL) receptor gene and its use in diagnosis.

A cloned gene probe coding for the low-density lipoprotein (LDL) receptor was used to detect a restriction fragment length polymorphism with the enzyme Pvu II. The frequency of the rare allele is approximately 0.2 both in normal controls and individuals with familial hypercholesterolaemia (FH). About 30% of individuals are heterozygous for the polymorphism, and are potentially informative for family studies and for early diagnosis of FH. This polymorphism was used to follow the inheritance of the LDL receptor gene in two families with FH. In these families, the polymorphism co-segregates with the disease unambiguously, and therefore can be used for early diagnosis.

Association of the TNF-α-308 (G→A) polymorphism with self-reported history of childhood asthma

Asthma is a complex disease involving genetic and environmental aetiology. The tumour necrosis factor-alpha (TNF- α) and angiotensin-converting enzyme (ACE) genes have been implicated in asthma pathogenesis. This study investigated the association of a G-308A variant of TNF- α and an insertion/deletion (I/D) variant of ACE with a self-reported history of childhood asthma, in two population groups. At Northwick Park Hospital, London, 1,811 pregnant women attending for antenatal care were recruited. Participants with a self-reported history of childhood asthma, determined by a researcher-administered questionnaire, and controls with no personal or family history of asthma, of UK/Irish (cases n=20; controls n=416) and South Asian (cases n=6; controls n=275) origin were used in this study. Participants were genotyped for the TNF-α-308 and ACE I/D variants by a PCR-RFLP and PCR approach. The TNF-α-308 allele 2 (−308A) was significantly associated with self-reported childhood asthma in the UK/Irish (Odds ratios (OR): 2.6; 95% confidence intervals (CI): 1.1–6.2; P=0.03) but not in the South Asian population. The ACE DD genotype was not associated with childhood asthma in either population group. Gametic phase disequilibrium between the TNF-α-308 and ACE I/D variants was significantly different from zero in UK/Irish cases (Δ=0.09; P=0.034). The TNF-α-308 allele 2 or a linked major histocompatibility complex (MHC) variant may be a genetic risk factor for childhood asthma in the UK/Irish sample.

Identification of a second "French Canadian" LDL receptor gene deletion and development of a rapid method to detect both deletions.

Hobbs et al. (N. Engl. J. Med. 317: 734–737, 1987) reported a large deletion of approximately 10 kilobases in the 5′ portion of the human low‐density lipoprotein (LDL) receptor gene. This deletion affects about 60% of familial hypercholesterolemia (FH) heterozygotes in the French Canadian population. We have developed a rapid, convenient method for the detection of the deletion using double digestion with the restriction enzymes Xbal and EcoRV, or triple digestion with Xbal, EcoRV and XmnI, and a 650 bp cDNA probe, radio‐labeled using a random oligonucleotide primer technique. Eighty French Canadian FH heterozygotes were screened for the presence of the deletion. Forty‐seven (59%) of them were found to carry the 10 kb deletion. Using the same method, we also identified a new mutation which was found in four of the 80 (5%) FH patients. This mutation has been found to be a 5 kb deletion removing exons 2 and 3 of the LDL receptor gene, which correspond to the first two repeats of the LDL receptor binding domain. Cosegregation of the 5 kb deletion and the FH phenotype was observed in one family. Possible structure‐function relationship is discussed.

EXPRESSION OF THE DEVELOPMENTAL AND ONCOGENIC PAX2 GENE IN HUMAN PROSTATE CANCER

PURPOSE In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma. MATERIALS AND METHODS PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue. RESULTS Using reverse transcription-polymerase chain reaction combined with Southern hybridization PAX2 expression was detected in 52% of primary cancers and all 3 cell lines. PAX2 expression in these samples was confirmed at a protein level using immunoblotting and immunofluorescence. PAX2 messenger RNA was not detected in any benign or normal prostatic samples. Immunoblotting of protein from benign prostatic hyperplasia samples confirmed the lack of expression of PAX2 protein. CONCLUSIONS The expression of PAX2 in prostate cancer compared to nonmalignant prostates is statistically significant (Fishers exact test p = 0.0004). These results suggest a possible role for PAX2 in prostate cancer. Although previous studies have suggested a role for PAX2 for supporting proliferation in undifferentiated cells, no correlation of PAX2 expression with Gleason score was found in prostate cancer.

Molecular genetic evidence for a founder effect in familial hypercholesterolemia among French Canadians

SummaryFamilial hypercholesterolemia (FH), at a prevalence of about 1 in 200 in the French-Canadian population, is caused by a 10-kb deletion in the low-density lipoprotein (LDL) receptor gene in 60% of French-Canadian FH heterozygotes. We genotyped 159 FH patients who carry this common mutation and 221 healthy French-Canadian controls for five DNA restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene. The allele numbers for four of the five RFLPs differed significantly (P < 0.001) between FH patients and control subjects. Our results suggest that the 10-kb deletion carrier allele is associated with a single haplotype (called the B haplotype). In a family study including a patient homozygous for the 10-kb deletion, we showed that the B haplotype cosegregates with the deletion in affected members and that the B haplotype is also associated with the normal allele in some members of the family. We identified 15 different haplotypes for the normal allele in 10-kb deletion carrier FH heterozygotes. These results offer strong support, at a molecular level, for the hypothesis of a founder effect for the 10-kb deletion in the French-Canadian population.

Variations in the apolipoprotein AI-CIII-AIV gene region and in lecithin: cholesterol acyltransferase concentration are determinants of plasma cholesterol concentrations

We have examined the effects of variation in the region of the apolipoprotein (apo) AI-CII-AIV genes, and in plasma lecithin: cholesterol acyltransferase (LCAT) concentration, on plasma cholesterol concentration in 109 unrelated men aged 52-67 yrs. Restriction fragment length polymorphisms (RFLPs) were detected using the restriction enzymes XmnI, PstI and SstI and individuals were divided into groups using information from all three RFLPs in conjunction. Mean plasma concentrations of both total cholesterol and estimated low density lipoprotein-cholesterol differed significantly (P less than 0.0125) among groups of men with different genotypes. Thus, variation in this gene region may be one of the polygenetic factors involved in determining cholesterol levels in the normal population. In the same subjects, plasma cholesterol was also positively correlated with plasma LCAT concentration (r = 0.55, P less than 0.001), due mainly to an increase in the cholesteryl ester content of apo B-containing lipoproteins with increasing LCAT concentration. Since apolipoproteins AI, CIII and AIV have each been shown to modify the activity of LCAT in vitro, the associations of the RFLPs with plasma cholesterol concentration may reflect changes in LCAT activity secondary to qualitative or quantitative changes in one or more of these apolipoproteins.