GENOTYPING AND GENETIC DIAGNOSIS UNIT

Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased low-density lipoprotein (LDL)-cholesterol levels, leading to high risk of premature cardiovascular disease. More than 900 mutations in LDL receptor, six in APOB and 10 in PCSK9 have been identified as a cause of the disease in different populations. All known mutations in PCSK9 causing hypercholesterolemia produce an increase in the enzymatic activity of this protease. Up to now, there are data about the implication of PCSK9 in ADH in a low number of populations, not including a Spanish population.

To assess the relationship of the lipid profile to coronary heart disease in a group of heterozygous familial hypercholesterolaemic subjects with similar age, sex, body mass index, prevalence of angiotensin converting enzyme DD genotype and type of low density lipoprotein receptor mutation.

Methods presently employed for detection of large rearrangements have several drawbacks, such as the amount of sample and time required, technical difficulty, or the probability of false-negative carriers. Using the low-density-lipoprotein receptor (LDLR) gene, whose mutations are responsible for familial hypercholesterolemia (FH), we have developed a procedure to detect large rearrangements in this gene based on semiquantitative PCR, with important improvements as compared to previous methods. Our method covers the complete LDLR gene and introduces an internal control in the reaction. The procedure discriminates the four different large rearrangements (two deletions and two insertions) that we have used as positive mutation controls (Valencia-1 to -5). All altered exons from each rearrangement are identified. Furthermore, when families from probands carrying these large rearrangements (34 members) were analyzed, our results agreed with those obtained previously with Southern blot. We have also analyzed a sample of 110 unrelated FH probands and the method has correctly identified the two different large rearrangements present and insertions or deletions as small as 1 bp. In conclusion, the method we present allows the identification of large rearrangements affecting exons of the gene, including small insertions or deletions or complete gene deletion. In addition, it constitutes a first characterization step of rearrangements, and is easy to carry out fast, and can be applied to the analysis of any gene. Hum Mutat 27(8), 822–828, 2006. © 2006 Wiley-Liss, Inc.

Objectives Oxidative stress can modulate blood pressure levels in different models. Xanthine oxidoreductase is one of the enzymes producing free radicals in the cardiovascular system, and it can contribute to the increment of the oxidative stress and, consequently, blood pressure. We analyzed the association between the -337GA and 565 + 64CT polymorphisms of the xanthine oxidoreductase gene with blood pressure and oxidative stress levels.

Hypertension is frequently associated with oxidative stress caused by high production of reactive oxygen species and compromised antioxidant defenses. Humans with essential hypertension, with or without treatment, and controls were examined (35 hypertensive and 30 normotensive). We noted a discordant response of the glutathione and thioredoxin systems in essential hypertension and to antihypertensive treatment. Further studies examining the significance of these thiols in hypertension outcomes are warranted. Antioxid. Redox Signal. 9, 0000-0000.

The vascular effects induced by chronic testosterone (TS) deficiency in men is not totally clear. Thus, we assessed vascular reactivity in the forearm microcirculation of congenital hypogonadal patients (HP), at baseline and after 6-month TS replacement therapy (TS enanthate, 100-250 mg i.m. every 3 weeks). In 8 HP (28.6Ϯ2.8 years, with hypogonadotropic, nϭ6, or hypergonadotropic, nϭ2, hypogonadism; blood pressure, BP, 123Ϯ6/78Ϯ4 mmHg) and 9 male matched control subjects (CS, 29.0Ϯ1.7 years; BP 122Ϯ2/78Ϯ1 mmHg) we studied forearm blood flow (strain-gauge plethysmography) changes induced by intraarterial acetylcholine (ACH, 0.15-15 mg/100 ml/min), an endotheliumdependent agonist, or sodium nitroprusside (SNP, 1-4 mg/100 ml/min), utilized as direct smooth muscle cell vasodilator. ACH was repeated during intrabrachial L-NMMA infusion (100 g/100 ml/min), a nitric oxide (NO) inhibitor. At baseline, in HP the percent maximal vasodilation (VD) to ACH (387Ϯ79%) was significantly (pϽ0.01) reduced as compared to CS (753Ϯ106%). As expected, in CS the VD to ACH was significantly blunted by L-NMMA (268Ϯ47%, inhibition: 64.4%). In contrast, in HP the inhibitory effect of L-NMMA on maximal response to ACH was reduced (297Ϯ35%, inhibition: 23.3% pϽ0.001 vs CS). At baseline, the VD to SNP also resulted to be lower in HP (347Ϯ54%) compared with CS (574Ϯ59%, pϽ0.05). TS therapy did not modify BP levels (114Ϯ5/78Ϯ5 mmHg). In contrast, hormone replacement therapy increased (pϽ0.05) plasma free TS (41.3Ϯ12.8 vs 9.7Ϯ3.1 pmol/L) and total TS (11.3Ϯ3.1 vs 2.8Ϯ1.1 nmol/L), without modifying 17-␤ estradiol and estrone values. TS therapy reduced HDL cholesterol (1.11Ϯ0.06 vs 1.3Ϯ0.07 mmol/L, pϽ0.01) and total cholesterol (4.1Ϯ0.3 vs 4.7Ϯ0.4 mmol/L), while not modifying plasma triglycerides and LDL cholesterol. TS therapy further reduced the vascular response to ACH (187Ϯ29%, pϽ0.01 vs baseline) and abrogated the inhibition by L-NMMA on ACH (180Ϯ37%; inhibition: 3.2%) compared with pre-intervention results. TS totally unaffected the VD to SNP (355Ϯ47%). Congenital HP are characterized by a reduced vascular reactivity, including an endothelial dysfunction due to a reduced NO availability. Chronic TS administration selectively impairs NO availability in these patients.

The objective of the present study was to analyze the impact of metabolic syndrome (MS) and its individual components on oxidative stress (OX) and on the activity of antioxidant enzymes of patients with essential hypertension. One hundred and eighty-seven hypertensives, 127 (61.9%) of them having criteria for MS according to the International Diabetes Federation criteria and 30 healthy normotensive subjects were included. OX status was assessed by measuring glutathione oxidized/glutathione reduced and reactive oxygen species-induced byproducts of lipid peroxidation, malondialdehide, and DNA damage, 8-oxo-dG genomic and mitochondrial. Antioxidant enzymatic activity of Cu/Zn extracellular-superoxide dismutase (SOD) and catalase (CAT) was measured in plasma and glutathione peroxidase 1 in hemolysad erythrocytes. In mononuclear cells, total-SOD activity, CAT and glutathione peroxidase 1, were assessed as well. The OX state in both blood and peripheral mononuclear cells observed in hypertensives were not enhanced by the addition of components of the so-called MS. Likewise, the reduction in the activity of antioxidant enzymes, both extracellular and cytoplasmic, was not affected by the presence of additional components of the MS. Neither the number of components nor the individual addition of each of them, low high-density lipoprotein, triglycerides, abdominal obesity or fasting glucose, further impact in the OX abnormalities observed in those with only hypertension in absence of other components. In conclusion, the present data indicates that contribution of MS components to the OX burden generated by high blood pressure is minimal.

Aims: There is evidence of an excess of acute cardiovascular (CV) events in marathon runners. High plasma total homocysteine (tHcy) concentrations are a recognised risk factor for CV events. Therefore, we investigated the changes in plasma tHcy concentrations 24 h before and after a marathon race. Methods and results: Twenty-two non-professional male athletes, mean age 35.6 (6.6), range 23e49 years, were studied the day before and 24 h after finishing a marathon race. None of the athletes was a carrier of the MTHFR 677TT genotype and no ingestion of supplements of vitamins (B12, B6, folic acid) was allowed. Results: Changes in plasma folate and plasma vitamin B12 concentrations were not detected post-race, but a significant increase in plasma tHcy concentrations was demonstrated. Plasma tHcy increased 19% 24 h after the race. Before the race 20% of the subjects had a plasma tHcy concentration O 10 mmol/l (cut-off point for ischaemic heart disease risk), while after the race 50% had plasma tHcy concentrations O 10 mmol/l. Conclusion: An increase in plasma tHcy concentrations was observed after a marathon race in non-professional not well-trained male athletes performing strong physical activity. The potential physiological or pathological implications of this finding are unknown. ª

Objective Oxidative stress is implicated in hypertension and the NADPH oxidase systems constitute the main source of superoxide in vascular wall. We searched for new polymorphisms within the CYBA promoter, the human gene that encodes the p22phox protein, and studied their potential association with essential hypertension.

Untreated hypertensive patients show increased oxidative stress and decreased antioxidant enzyme activity in mononuclear cells. Therefore, the objective of this study was to determine whether or not the low antioxidant enzyme activity observed in mononuclear cells of hypertensive subjects is in part dependent on a defective activity of antioxidant mechanisms. Activity and mRNA level of antioxidant enzymes, CuZn-and Mn-superoxide dismutases, catalase, glutathione peroxidase type 1, and glutathione reductase were simultaneously measured in mononuclear cells of controls (n ϭ 38) and hypertensive subjects (n ϭ 35), in the absence of and during antihypertensive treatment. An increase in oxidative stress and a decrease in the activity of cytoplasmic enzymes were observed in untreated hypertensive patients. Concurrently, CuZn-superoxide dismutase and glutathione reductase mRNA levels were significantly reduced, and glutathione peroxidase type 1 mRNA was slightly reduced. In contrast, increased activity and mRNA levels of the mitochondrial Mn-super-oxide dismutase were observed. Antihypertensive treatment, nonpharmacologic with or without a drug regimen of ␤-blocker or angiotensin AT1 receptor blocker was administered for a 3-month period. Afterward, after the improvement in oxidative stress during treatment, a recovery of the cytoplasmic antioxidant enzymatic activity and a more profound decrease in mRNA levels were observed for CuZn-superoxide dismutase, glutathione peroxidase type 1, and glutathione reductase. Meanwhile mitochondrial enzymatic activity decreased, as did the mRNA level. The inadequate response of the main cytoplasmatic antioxidant systems, as well as of the enzymes participating in the maintenance of glutathione levels, may contribute to the vulnerability of hypertensives to oxidative stress. Am J Hypertens 2007;20:62-69

The aim of the present study was to assess the effects of angiotensin coverting enzyme inhibitor (ACE-I) perindopril and angiotensin receptor blocker (ARB) candesartan on the survival of Dahl salt-sensitive (DS) hypertensive rats. In addition, we also studied the effects of ACE-I and ARB on LV contractile dysfunction and LV fibrosis for exploring to the mechanism contributing to the improvement of the survival.

Background and Objectives: Even though the gene polymorphisms in renin-angiotensin system (RAS) had been known as the predisposing factors for the cardiovascular diseases, the precise mechanism and interactions in the confounding factors have not been clarified yet. We investigated whether the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene (ACE) and A1166C polymorphism of angiotensin II type 1 receptor gene (AT1R) were involved in insulin sensitivity using a Japanese large epidemiological study, the Tannno-Sobetsu Study.

Background: The elongase of long chain fatty acids family 6 (ELOVL6) is an enzyme that specifically catalyzes the elongation of saturated and monounsaturated fatty acids with 12, 14 and 16 carbons. ELOVL6 is expressed in lipogenic tissues and it is regulated by sterol regulatory element binding protein 1 (SREBP-1).

Hypoxia increased LDH release by 4 folds. Exposure to ET-1 (0.1 M) before hypoxia resulted in a 6-fold increase in LDH release (pϽ0.05 vs hypoxia alone). Pretreatment with BQ-123 before hypoxia significantly attenuated LDH release. Morphological assays revealed a severe cytotoxic effect induced by ET-1. Pretreatment with BQ-123 (5 M) prevented the cytotoxic effects. The results were confirmed by electron microscopy. Exposure of myocytes to ET-1 (10-100 nM) caused an increase in the concentration of intracellular free Ca2ϩ in a concentration-dependent manner. The effect was completely inhibited by pretreatment with the ET A antagonist BQ-123 at a concentration of 5 M. Conclusions: ET-1 augments cytotoxic damage in cardiac cells 24 h after exposure to simulated hypoxia. The effect is mediated through activation of the ET A receptor.

Abstract—The objective of the present study was to analyze the influence of the I/D polymorphism of the ACE gene on the outcome of microalbuminuria in essential hypertensive patients who were receiving antihypertensive treatment. One hundred thirty-six essential hypertensive ...

Background: ABCG1 mediates cellular cholesterol transport, but there is very little known about the influence of ABCG1 polymorphisms on human plasma lipoprotein cholesterol concentrations or on the interactions of these polymorphisms with diet. Objective: Our objective was to investigate whether interactions between PUFA intake and ABCG1 polymorphisms modulate associations with plasma total cholesterol (TC), LDL-and HDL-cholesterol in two Spanish populations. Methods: We grounded our investigation on two general population-based studies: the Hortega study (population A) and the Pizarra study (population B). Participants included 1178 individuals (50.0% women, age range 21-85 years) and 763 individuals (66% women, age range 23-73 years) from populations A and B, respectively, without lipid lowering drugs. Subjects were genotyped for ABCG1 variants. Biochemical measurements were taken by standard procedures. Dietary intakes were estimated with a validated questionnaire. Results: In population A, the A allele homozygotes of SNP rs4148102 had higher TC and LDLc concentrations in subjects on a high PUFA diet than did the carriers of the G allele (242.1 ± 38.9 vs. 198.0 ± 36.0 mg/dL, p = 0.003, and 149.8 ± 37.9 vs. 111.4 ± 32.1 mg/dL, p = 0.005, respectively), and significant gene-diet interactions were observed (p = 0.020 and p = 0.013, respectively). In population B, similar differences in TC and LDLc concentrations were also found in association with this SNP under a high PUFA diet (253.2 ± 24.9 vs. 197.7 ± 39.9 mg/dL, p = 0.009, and 171.8 ± 20.5 vs. 120.4 ± 34.2 mg/dL, p = 0.004, respectively), but the gene-diet interactions observed were not significant (p = 0.379 and p = 0.422, respectively). In the pooled populations, differences in the TC and LDLc concentrations increased (246.8 ± 32.9 vs. 198.0 ± 37.5, p = 6 × 10 −5 , and 159.0 ± 32.6 vs. 114.3 ± 33.1, p = 3 × 10 −5 , respectively), and significant gene-diet interactions were maintained (p = 0.006 and p = 0.003, respectively). Conclusion: In two Spanish populations, the ABCG1 polymorphism rs4148102 was associated with variations in plasma lipoprotein cholesterol concentrations in subjects with high PUFA intakes. Carriers of the AA genotype consuming high PUFA diet showed higher plasma LDLc concentrations.

The aim of the study was to determine the influence of twenty single nucleotide polymorphisms (SNPs) of the ABCA1, ABCG1, ABCG5 and ABCG8 genes on the plasmatic concentrations of total cholesterol (TC), HDL and LDL cholesterol (HDLc, LDLc) in the postprandial state with a representative Spanish Caucasian population (1473 individuals, 50.0% women, ages ranging 21-85 years). In men, subjects with the AA genotype of the ABCA1 rs2230806 (R219K) polymorphism were associated with increased plasma LDLc levels, while the ABCA1 haplotype, which included the rs2230806 A allele, was associated with higher TC and LDLc plasma concentrations. In women, significant relationships were found between rs1893590 polymorphisms (ABCG1 gene) and HDLc plasma concentrations (subjects with the AA genotype had lower HDLc levels). For the ABCG8 gene, the rs4148211 polymorphism was associated with higher plasma TC and LDLc concentrations in the total population. Moreover, an ABCG5-G8 haplotype, which included the rs6544718 T allele, was associated with higher HDLc plasma concentrations in women. In conclusion, different SNPs of the ABCA1, ABCG1 and ABCG5-ABCG8 genes were associated, some under gender-specific analysis, with variations in the plasma lipid levels under postprandial conditions in a representative Spanish population.