Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer.

The C825T polymorphism in the G-protein beta3 subunit gene and diabetic complications in IDDM patients.

Complications of insulin-dependent diabetes mellitus (IDDM) are a major cause of morbidity and mortality; however, the mechanisms of their development are still to be elucidated. Genetic susceptibility contributes to the pathogenesis of nephropathy in IDDM. Enhanced G-protein activation, a cellular phenotype observed in cultured cells from patients with essential hypertension, was recently documented in IDDM subjects with nephropathy. A C825T polymorphism was recently described in GNB3, the gene encoding the beta 3 subunit of heterotrimeric G-proteins. This genetic variant has been associated with enhanced G-protein activation. The 825T allele was observed more frequently in a group with essential hypertension. We analyzed the role of the C825T polymorphism in the predisposition to diabetic complications in IDDM. In this study, we investigated the frequency of this polymorphism in a large case-control study and found no association of the 825T allele with diabetic nephropathy, retinopathy, and neuropathy.

Paraoxonase 1 Met--Leu 54 polymorphism is associated with Parkinson's disease.

Two up-to-date known paraoxonase 1 (PON1) polymorphisms (Gln--Arg 191 and Leu--Met 54) affect the hydrolysis of toxic oxons and might intensify effects of pollutants, organophosphates and other environmental chemicals in development of Parkinson's disease (PD). We reported previously that PON1 G1n--Arg 191 polymorphism did not influence on the susceptibility to PD. In the present study we have investigated the PON1 Leu--Met 54 polymorphism in 117 patients with sporadic idiopathic PD. A new approach for Leu--Met 54 polymorphism genotyping has been developed. We have showed the frequency of the Met 54 allele of PON1 to be significantly increased in patients with PD compared with the controls (chi(2)=8.63, df=1, P<0.003). The relative risk of PD in the Met 54 allele carriers has been estimated to be 2.3 fold higher than in homozygotes for the L allele. Moreover it appeared to be even 5.15 higher in the subgroup of patients with early-onset PD. We suggest that the Met 54 allele may be considered to be an independent risk factor for PD. This mutation could probably cause PON1 impaired metabolism of environmental neurotoxins and might be responsible for neurodegeneration.

Age-associated accumulation of the apolipoprotein C-III gene T-455C polymorphism C allele in a Russian population.

Apolipoprotein C-III (apoC-III) is the major component of triglyceride-rich lipoproteins. One of six identified polymorphisms in the apoC-III 5'-untranslated region (T-455C) is located within a functional insulin-response element. In a group of 137 elderly individuals (70-106 years old), the allele distribution was analyzed using restriction fragment length polymorphisms. Statistical analysis of allele frequencies was performed on subgroups selected by age and in elderly patients with arterial hypertension or ischemic heart disease. A greater frequency of the apoC-III -455C allele was demonstrated with aging (p < .005). No statistically significant difference in allele distributions was detected between healthy subjects and groups of elderly patients of the same age with either ischemic heart disease or arterial hypertension. The increased incidence of the C allele with advanced age indicates that this variant promoter is associated with longevity. The greater incidence of this allele is detectable only in adults older than 80 years of age.

Gene-gene interaction in the RAS system in the predisposition to myocardial infarction in elder population of St. Petersburg (Russia).

The aim of this study was to estimate the frequencies of some DNA polymorphisms of two genes of the renin-angiotensin system (RAS), M235T angiotensinogen gene and insertion-deletion polymorphism in angiotensin-converting enzyme gene, in older (>55 years old) myocardial infarction survival and control groups. For this purpose 198 myocardial infarction (MI) patients and 152 randomly selected healthy persons have been analyzed. We have not found any differences in allele and genotype distribution in the above-mentioned genes for either group. However, statistical research showed a significant increase of double homozygotes IITT in the group of MI patients as compared with those in the control group. In this respect we suggested that gene-gene interaction in the RAS system may be considered to be a predisposing factor for MI development.

Methylenetetrahydrofolate reductase gene polymorphism as a risk factor for diabetic nephropathy in IDDM patients.

A missense mutation in the methylenetetrahydrofolate reductase gene (MTHFR), C677T, results in a thermolabile variant with reduced activity. Elevated levels of homocysteine have been recognized as a risk factor for vascular disease. Insulin-dependent diabetes mellitus (IDDM) is characterized by a higher prevalence of vascular complications. We analyzed the frequency of C677T MTHFR in IDDM and control groups. The genotype distribution did not differ between control subjects (n = 297) and IDDM patients (n = 392) (chi(2) = 5.413, df = 2, P > 0.05). The MTHFR T677T genotype was found significantly more frequently in IDDM patients with diabetic nephropathy (0.216) compared with the IDDM patients without nephropathy (0.056); the odds ratio was 2.635 (95% CI 1.768-3.927). Thus, we suggest that the T677T genotype of the MTHFR gene is an independent risk factor for diabetic nephropathy in IDDM.

Distribution of insulin-dependent diabetes mellitus (IDDM)-related HLA alleles correlates with the difference in IDDM incidence in four populations of the Eastern Baltic region.

The high incidence of insulin-dependent diabetes mellitus (IDDM) in Finland contrasts strikingly with the low rates in the neighbouring populations of countries in the Eastern Baltic region: Estonia, Latvia and Russia. To evaluate the possible contribution of genetic factors to these differences, the frequencies of HLA-DQB1 alleles and relevant DQB1-DQA1 or DQB1-DRB1 haplotypes associated with IDDM risk or protection were analysed among IDDM patients and control subjects from these four populations. An increased frequency of HLA-DQB1*0302, DQB1*02-DQA1*05 and DQB1*0302-DRB1*0401 was observed in subjects with IDDM in all studied populations, whereas the prevalence of DQB1*0301 and DQB1*0602 and/or *0603 was decreased among patients. The degree of IDDM risk associated with HLA alleles analysed here did not differ significantly between the populations. Comparisons of the distribution of IDDM-related HLA alleles and haplotypes in the background populations revealed its consonance with IDDM incidence. The combined frequency of high risk genotypes was significantly higher among Finns than in other populations studied. Our data support the hypothesis that variance in the dispersion of HLA alleles is the genetic basis of variation of IDDM incidence observed in the Eastern Baltic region.

Two novel slavic point mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia from St. Petersburg, Russia.

Using PCR-single-strand conformation polymorphism analysis, followed by sequencing of the abnormal samples, two novel point mutations in the 5' end of the fourth exon of the low-density lipoprotein receptor gene were found in two Russian families with familial hypercholesterolemia. These missense mutations consist of C127W and C139G transitions and result in a loss of one of three disulfide bonds in the fourth cysteine-rich repeat of the ligand-binding domain of the low-density lipoprotein receptor. Hypercholesterolemia segregated with the identified mutations.

Two novel low-density lipoprotein receptor gene mutations (E397X and 347delGCC) in St. Petersburg familial hypercholesterolemia.

Familial hypercholesterolemia (FH), a monogenic disease known to be caused by low-density lipoprotein receptor (LDLR) gene mutations, results in the development of premature atherosclerosis and coronary artery disease in affected individuals. The spectrum of LDLR gene mutations in Russia is poorly known. Using polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis, followed by DNA sequencing, we have screened selected exons of the LDLR gene in 80 unrelated St. Petersburg FH patients for the presence of mutations. Two new LDLR gene mutations, 347delGCC and E397X, were characterized among individuals with familial hypercholesterolemia in St. Petersburg. The carriers of both mutations possessed highly elevated blood serum cholesterol. Cosegregation of E397X mutation and LDLR gene RFLP haplotypes with hyperlipidemia was demonstrated by family study. Both mutations seem to be specific to Slavic patients.

The effect of HLA-B allele on the IDDM risk defined by DRB1*04 subtypes and DQB1*0302.

The genes encoding the HLA-DQ heterodimer molecules, DQB1 and DQA1, have been found to have the strongest association with IDDM risk, although there is cumulative evidence for the effect of other gene loci within the major histocompatibility complex gene region. After the HLA-DQ locus, the HLA-DR locus has been suggested most often as contributing to the disease susceptibility. In this study we analyzed at the population level the effect of DR4 subtypes and class I, HLA-B alleles, on IDDM risk when the influence of the DQ locus was stratified. In all three populations studied (Estonian, Latvian, and Russian), DQB1*0302 haplotypes most frequently carried DRB1*0401 or DRB1*0404. DRB1*0401 was the most prevalent subtype in IDDM patients, whereas DRB1*0404 was decreased in frequency. DRB1*0402 was also prevalent among Russian haplotypes, but was not associated with IDDM risk. When HLA-B alleles were analyzed, strong associations between the presence of specific B alleles and DRB1*04 subtypes were detected. The HLA-B39 allele was found significantly more often in DRB1*0404-DQB1*0302-positive patients than in healthy control subjects positive for this haplotype: 27 of 54 (50%) vs. 4 of 49 (8.2%) (P < 0.0001). The results demonstrate that DQ and DR genes cannot explain all of the HLA-linked susceptibility to IDDM, and that the existence of a susceptibility locus telomeric to DR is probable.

Comparative analysis of apo(a) gene alleles: distribution of pentanucleotide repeats in position -1373 and C/T transition in position +93 among patients with myocardial infarction and a control group in St. Petersburg, Russia.

To evaluate whether polymorphisms in the 5' region of the apolipoprotein(a) gene alter the risk for myocardial infarction, 289 Russian male patients with myocardial infarction (MI) and 284 subjects in a control group were investigated regarding the distribution of pentanucleotide repeats (PNRs) at position -1373 and a C/T transition at position +93. For detection of the C/T (+93) allele, we developed a rapid, nonisotopic method by mismatch PCR-mediated site-directed mutagenesis and restriction enzyme digestion. We observed significant differences in prevailing alleles with over eight (TTTTA) repeats among MI patients, including those with MI younger than 55 years of age. We observed the prevalence of the T (+93) allele in children without a family history of CHD compared to young MI patients. These findings support the notion that PNR alleles with over eight (TTTTA) repeats may play a pathogenic role, and the T (+93) allele may have a protective effect for the inherited predisposition to heart disease.

HLA-DQB1*0304-DRB1*0408 haplotype associated with insulin-dependent diabetes mellitus in populations in the eastern Baltic region.

The rare HLA-DQB1*0304 allele was found increased among IDDM patients in the populations of the eastern Baltic region. Its frequency among IDDM patients was 4.5% (20/443) compared to 1.1% (9/853) in healthy controls in the combined series of Estonian, Latvian and St. Petersburg Russian populations (P=0.0001). HLA-DQB1*0304 in these populations was associated with DRB1*0408, and the haplotype was further characterized by a B35 allele and a typical combination of microsatellite markers from the TNF gene region. The result is compatible with the significance of the 57th amino acid in the DQ beta-chain but also emphasizes the importance of alleles in other HLA loci adjacent to DQ in the determination of IDDM susceptibility.

CYP2D6 genotyping in a Russian population using a novel approach for identification of the CYP2D6A mutation.

The frequency of 29A and 29B mutations in the CYP2D6 gene, the most common mutations among Caucasoid PM (debrisoquine 4-hydroxylase defficient) individuals, has been analyzed in the Russian population. For the detection of the 29A mutation, a new one-step ARMS PCR approach has been developed. The frequency of the 29B mutant allele in Russians appeared to be significantly lower than in other Caucasoid populations, and this observation can be explained by the mixed origin of the population inhabiting Russia. Comparative analysis of the mutation frequencies among individuals of various ages showed no age-related differences.

Short-term changes in calcium but not protein intake alter the rate of bone resorption in healthy subjects as assessed by urinary pyridinium cross-link excretion.

This study was conducted to determine whether the markers of bone resorption, pyridinium cross-links of collagen, are sensitive to changes in dietary protein and calcium intake. Fifteen young healthy subjects (7 males and 8 females) participated in three 5-d diet periods. Dietary intake during each dietary period consisted of: 1) low nitrogen and low calcium [0.49 +/- 0.11 g protein/ (kg.d), 429 +/- 190 mg calcium/d]; 2) low nitrogen and high calcium [0.44 +/- 0.08 g protein/(kg.d), 1643 +/- 171 mg calcium/d]; and 3) a high nitrogen and high calcium [2.71 +/- 0.75 g protein/(kg.d), 1589 +/- 633 mg calcium/d] diet, and this was compared with subjects' baseline dietary intake [0.99 +/- 0.51 g protein/(kg.d), 589 +/- 152 mg calcium/d]. The order of these diets was randomly assigned. Twenty-four-hour and 3-h urine samples were collected before and during each dietary period and were analyzed for pyridinium cross-links (pyridinoline, deoxypyridinoline), nitrogen and creatinine. The rate of pyrdinium cross-link excretion did not vary with protein intake but was approximately 33% lower (P < 0.01) during periods of high compared with low calcium intake. These data indicate that a short-term increase in calcium intake is accompanied by a reduced rate of bone resorption and that this effect is independent of dietary protein intake.

Frequency of a specific cytochrome P4502D6B (CYP2D6B) mutant allele in clinically differentiated groups of patients with Parkinson disease.

The frequency of the cytochrome P4502D6B CYP2D6B (29B) mutant allele has been determined in three clinically distinct groups of patients with Parkinson disease. No differences in mutation frequency among the patients with the rigidity-akinetic and monosymptomatic tremor forms has been observed compared to the healthy control group, while in the group with akinetic-rigidity tremor symptoms the frequency of heterozygous wt/29B individuals was significantly increased. Therefore, individuals bearing the CYP2D6B mutation appear to be predisposed to the development of this particular form of Parkinson disease, and the presence of the 29B mutation in the genotype may serve as an additional diagnostic criteriaum for the clinical differentiation of patients with Parkinson disease.

Recurrence of the R408W mutation in the phenylalanine hydroxylase locus in Europeans.

The relative frequency of the common phenylalanine hydroxylase (PAH) mutation R408W and its associations with polymorphic RFLP, VNTR, and short-tandem-repeat (STR) sites in the PAH gene were examined in many European populations and one representative North American population of defined European descent. This mutation was found to cluster in two regions: in northwest Europe among Irish and Scottish peoples, and in eastern Europe, including the Commonwealth of Independent States. This allele was significantly less frequent in intervening populations. In eastern European populations, the R408W mutation is strongly associated with RFLP haplotype 2, the three-copy VNTR allele (VNTR 3), and the 240-bp STR allele. In northwestern European populations, it is strongly associated with RFLP haplotype 1, the VNTR allele containing eight repeats (VNTR 8), and the 244-bp STR allele. An examination of the linkage between the R408W mutation and highly polymorphic RFLP, VNTR, and STR haplotypes suggests that recurrence is the most likely mechanism to account for the two different major haplotype associations of R408W in Europe.