Josep Oriola

Renin-Angiotensin System Genetic Polymorphisms and Salt Sensitivity in Essential Hypertension

We evaluated the association between salt-sensitive hypertension and 3 different genetic polymorphisms of the renin-angiotensin system. Fifty patients with essential hypertension were classified as salt sensitive or salt resistant, depending on the presence or absence of a significant increase (P<0.05) in 24-hour ambulatory mean blood pressure (BP) after high salt intake. The insertion/deletion (I/D) angiotensin-converting enzyme (ACE) gene, the M235T angiotensinogen (AGT) gene, and the A1166C angiotensin II type 1 (AT1) receptor gene polymorphisms were determined with the use of standard polymerase chain reaction methods. Twenty-four (48%) patients with significantly increased (P<0.05) 24-hour mean BP with high salt intake (from 107.3+/-9.4 to 114.8+/-10.6 mm Hg) were classified as salt sensitive. In the remaining 26 patients (52%), high salt intake did not significantly modify 24-hour mean BP (from 107.6+/-10 to 107. 8+/-9 mm Hg), and they were classified as having salt-resistant hypertension. We did not find any significant association between either M235T AGT or A1166C AT1 receptor genotypes and the BP response to high salt intake. However, patients with essential hypertension homozygous for the insertion allele of the ACE gene (II) had a significantly higher BP increase with high salt intake (9. 8+/-8.1 mm Hg for systolic BP and 5.2+/-4.2 mm Hg for diastolic BP) than that observed in patients homozygous for the deletion allele (DD) (1.2+/-5.9 mm Hg for systolic BP; P=0.0118 and -0.2+/-4.2 mm Hg for diastolic BP; P=0.0274). Heterozygous patients (ID) exhibited an intermediate response. The prevalence of salt-sensitive hypertension also was significantly higher (P=0.012) in II (67%) and DI patients (62%) compared with DD hypertensives (19%). We conclude that a significant association exists between the I/D polymorphism of the ACE gene and salt-sensitive hypertension. Patients with II and DI genotypes have significantly higher prevalence of salt sensitivity than DD hypertensives.

Mspl identifies a biallelic polymorphism in the promoter region of the α2A‐adrenergic receptor gene

Candidate gene: An increased activity of the sympathetic nervous system has been suggested to play a role in the pathophysiology of essential hypertension ( EHT). However, plasma catecholamine levels are not increased in these patients. An increased density of a,,-adrenergic receptors (a,,-AR) has been reported in patients with EHT (Mores et al. 1990, Calls et al. 1995). The activation of these receptors by epinephrine or norepinephrine promotes peripheral vasoconstriction and salt and water retention. The human cc,,-AR gene is located at 10q23-q25. The complete nucleotide sequence of this gene (HUMADRA2R) has been previously published (Fraser et al. 1989), and two restriction fragment length polymorphisms (DraI and Bsu36I RFLPs) have been reported to date (Hoehe et al. 1988, Sun et al. 1992). A significant association between DraI RFLP and essential hypertension has been described (Lockette et al. 1995, Svetkey et al. 1996), although there is no general agreement on these findings.

Endothelin 1 does not play a major role in the homeostasis of arterial pressure in cirrhotic rats with ascites

BACKGROUND/AIMS Patients with cirrhosis and ascites have increased plasma levels of endothelin, a powerful vasoconstrictor peptide. This study assessed the mechanisms underlying this phenomenon. METHODS Plasma endothelin was measured in control rats and cirrhotic rats with and without ascites. In addition, the tissue concentration of endothelin and endothelin 1 messenger RNA (mRNA) and the effect of an endothelin A receptor antagonist on arterial and portal pressure were assessed in cirrhotic rats with ascites and control rats. RESULTS Plasma endothelin levels were significantly higher in cirrhotic rats with ascites (24.5 +/- 2.8 pg/mL; P < 0.001) than in cirrhotic rats without ascites and control rats (7.9 +/- 2.0 and 5.8 +/- 0.9 pg/mL, respectively). In animals with ascites, endothelin and endothelin 1 mRNA content in the lung, kidney, and aorta was similar to that of the controls. In contrast, higher endothelin content (0.567 +/- 0.217 vs. 0.045 +/- 0.002 pg/mg protein; P < 0.05) and endothelin 1 mRNA was observed in hepatic tissue of rats with cirrhosis and ascites. Endothelin A receptor blockade was not associated with significant changes in arterial and portal pressure in any group of animals. CONCLUSIONS Increased endothelin 1 mRNA and endothelin production occurs in the livers of cirrhotic rats with ascites. In addition, our findings suggest that endothelin is not involved with the homeostasis of arterial or portal pressure in cirrhosis with ascites.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (<or=9% normal forms) compared with other patients (4.5%; P < .05) or compared with controls (2.97%; P < .005). The allelic frequency of the PRM1 -190 C-->A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Association of the G protein β3 subunit T allele with insulin resistance in essential hypertension

A polymorphism (C825T) in the gene encoding the G protein β3 subunit (GNB3) has recently been associated with hypertension and obesity in several populations. The aim of the study was to analyse the relationship between this polymorphism and insulin sensitivity, an hypothesised unifying factor for hypertension and obesity. One hundred thirty unrelated patients with essential hypertension, 70 female and 60 male, aged 58±1 years with systolic blood pressure of 173±2 mm Hg and diastolic blood pressure of 105±1 mm Hg, were genotyped for the GNB3 polymorphism by PCR and restriction digestion with BseDI, and classified in two groups according to the genotypes CC and CT+TT. Body mass index (BMI) was significantly higher in patients with the T allele as compared with patients without the T allele (29.3±0.4 vs. 26.7±0.6 kg/m2, p<0.001). On the contrary, there were no differences in the level of systolic or diastolic blood pressure among the genotypes. Insulin sensitivity was measured in a subgroup of 35 patients by means of an euglycemic hyperinsulinemic clamp test. In this subgroup, patients with the T allele displayed lower insulin sensitivity index (1.6±0.3 vs. 2.7±0.3 mg/kg/min, p=0.022), higher fasting serum insulin (121±16 vs. 77±11 pmol/L, p=0.032), higher serum glucose 120 min after 75 g load (9.8±1.2 vs. 7.0±0.5 mmol/L, p=0.038), and higher glycosilated haemoglobin (5.7±0.4 vs. 4.7±0.2%; p=0.042) as compared with patients without the T allele. A regression analysis showed that the association between the T allele and insulin sensitivity was independent of BMI (β coefficient −0.386, p=0.022). These results suggest a relationship between the 825T allele of GNB3 and insulin resistance in the essential hypertensive patients studied, which seems to be independent of BMI.

Identification of a Novel Modulator of Thyroid Hormone Receptor-Mediated Action

Background Diabetes is characterized by reduced thyroid function and altered myogenesis after muscle injury. Here we identify a novel component of thyroid hormone action that is repressed in diabetic rat muscle. Methodology/Principal Findings We have identified a gene, named DOR, abundantly expressed in insulin-sensitive tissues such as skeletal muscle and heart, whose expression is highly repressed in muscle from obese diabetic rats. DOR expression is up-regulated during muscle differentiation and its loss-of-function has a negative impact on gene expression programmes linked to myogenesis or driven by thyroid hormones. In agreement with this, DOR enhances the transcriptional activity of the thyroid hormone receptor TRα1. This function is driven by the N-terminal part of the protein. Moreover, DOR physically interacts with TR α1 and to T3-responsive promoters, as shown by ChIP assays. T3 stimulation also promotes the mobilization of DOR from its localization in nuclear PML bodies, thereby indicating that its nuclear localization and cellular function may be related. Conclusions/Significance Our data indicate that DOR modulates thyroid hormone function and controls myogenesis. DOR expression is down-regulated in skeletal muscle in diabetes. This finding may be of relevance for the alterations in muscle function associated with this disease.

Angiotensinogen gene M235T and T174M polymorphisms in essential hypertension: relation with target organ damage.

The molecular variants M235T and T174M of the angiotensinogen gene have been linked to essential hypertension in some populations, but there are discrepancies about this association in other studies. We studied 75 patients with essential hypertension (BP > 160/100 mm Hg) from our outpatient clinic, aged 55+/-1 years, 30 men, systolic BP 182+/-2.5, diastolic BP 109+/-1 mm Hg (mean +/- SEM), and a family history of the disease. Target organ damage was evaluated by measuring urinary albumin excretion rate, left ventricular hypertrophy, and fundoscopy. As a control group, 75 healthy subjects with BP < 130/85 mm Hg and with no family history of cardiovascular disease were selected. M235T and T174M angiotensinogen genotypes were determined by PCR and subsequent digestion of the products with SfaNI and NcoI, respectively. The frequency (q) of genotypes of the variant M235T in the patients with essential hypertension was MM 0.31, MT 0.41, and TT 0.28, not significantly different (P = .93) from that of the controls (MM 0.28, MT 0.44, and TT 0.28). For the variant T174M, the genotype frequencies in hypertensives were TT 0.83, TM 0.15, and MM 0.02, which was not significantly different (P = .89) from that of the controls (TT 0.86, TM 0.12, and MM 0.02). Similarly, there was no evidence for association between angiotensinogen genotypes and hypertension in subjects aged < or = 40 years old (n = 24) or with severe (stage III) hypertension (n = 31). Within the group of patients with essential hypertension, there were no differences in genotype distribution between patients with and without retinopathy (n = 31), left ventricular hypertrophy (n = 37), or microalbuminuria (n = 14). This study shows that M235T and T174M variants are not associated either with essential hypertension or with target organ damage in a Spanish sample.

Polymorphisms, haplotypes and mutations in the protamine 1 and 2 genes

Protamines are the most abundant nuclear proteins and alterations in their expression have been described in infertile patients. Also, protamine haplo-insufficient mice have been described as infertile. Therefore, the protamine 1 and 2 genes have been considered important candidates in different mutational studies. In this article, we review all published articles related to protamine gene mutations and report new data on mutations from patients and controls drawn from the Spanish and Swedish populations. Sequencing of the protamine 1 and 2 genes in a total of 209 infertile patients and 152 fertility-proven controls from the Spanish and Swedish populations identified two novel and rare non-pathogenic missense mutations (R17C and R38M) in the protamine 1 gene and several additional polymorphisms. Furthermore, we have identified and we report for the first time five novel rare haplotypes encompassing the protamine 1 and 2 genes. A review of all available protamine gene mutational studies indicates that none of the reported missense mutations can be considered of proven pathogenicity. However, it is interesting to note that rare protamine 1 promoter variants have been reported only in infertile patients, but not in fertile control groups. Pathogenic high penetrance protamine gene missense mutations, if any, must be extremely rare. However, the detected presence of rare variants and haplotypes in infertile patients deserves further investigation.

Lack of association between ACE gene polymorphism and left ventricular hypertrophy in essential hypertension.

The possible association between the insertion/deletion (I/D) polymorphism of the angiotensin I converting enzyme (ACE) gene and left ventricular hypertrophy (LVH) was investigated in a group of essential hypertensive patients. Seventy-one essential hypertensive patients (35 men and 36 women), aged 51 ± 1 years, were genotyped by PCR for the I/D polymorphism of the ACE gene. Cardiac morphology and function were assessed by means of M-mode echocardiography. The relative frequencies of the three genotypes, DD, DI, and II, were respectively: 24%, 55%, and 21%. Mean values of left ventricular mass index were 145, 144, and 150 g/m2 for DD, DI, and II genotypes, without significant differences among them (P = 0.82). Likewise, the prevalence of LVH (76%, 64%, and 87%) was not significantly different among the three genotypes (P = 0.23). We conclude that the ACE gene I/D polymorphism is not associated with LVH in essential hypertension.

Methodological advances in sperm proteomics

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims to identify the proteins that compose the sperm cell and the study of their function. Recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and study sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. A wide range of MS techniques are also rapidly becoming available for researchers. The present review summarises the different methodological options to study the sperm cell using MS and to provide a summary of some of the ongoing proteomic studies.