Kevin M. O’Shaughnessy

A Common Variant of the Endothelial Nitric Oxide Synthase (Glu298→Asp) Is a Major Risk Factor for Coronary Artery Disease in the UK

BACKGROUND Endothelium-derived nitric oxide (NO) is synthesised from L-arginine by endothelial nitric oxide synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because reduced NO synthesis has been implicated in the development of coronary atherosclerosis, which has a heritable component, we hypothesised that polymorphisms of NOS 3 might be associated with increased susceptibility to this disorder. METHODS AND RESULTS Single-strand conformation polymorphism analysis of NOS 3 identified a G-->T polymorphism in exon 7 of the gene which encodes a Glu-->Asp amino acid substitution at residue 298 of eNOS. We investigated the relationship between this Glu(298)-->Asp variant and atherosclerotic coronary artery disease (CAD) using 2 independent case-controlled studies. In the first study (CHAOS), cases consisted of 298 unrelated patients with positive coronary angiograms and controls were 138 unrelated healthy individuals ascertained through a population health screen. In the second study (CHAOS II), the cases were 249 patients with recent myocardial infarction (MI), and a further 183 unrelated controls. There was an excess of homozygotes for the Asp298 variant among patients with angiographic CAD, and among patients with recent MI when compared with their respective controls (35.9% versus 10.2%, P<0.0001 in CHAOS, and 18.1% versus 8.7%, P<0.02 in CHAOS II). In comparison to Glu(298) homozygotes, homozygosity for Asp(298) was associated with an odds ratio of 4.2 (95% CI, 2.3 to 7.9) for angiographic CAD and 2.5 (95% CI, 1.3 to 4.2) for MI. CONCLUSIONS Homozygosity for a common NOS 3 polymorphism (894 G-->T) which encodes a Glu298-->Asp amino acid substitution in eNOS is a risk factor for angiographic CAD and recent MI in this population.

Salt Handling and Hypertension

The kidney plays a central role in our ability to maintain an appropriate sodium balance, which is critical for the determination of blood pressure. The kidneys capacity for salt conservation may not be widely appreciated, and in general we consume vastly more salt than we need. Here we consider the socioeconomics of salt consumption, outline current knowledge of renal salt handling at the molecular level, describe some of the disease entities associated with abnormal sodium handling, give an overview of some of the animal models and their relevance to human disease, and examine the evidence that lowering our salt intake can help combat hypertension and cardiovascular disease.

Variation in the Human Matrix Metalloproteinase-9 Gene Is Associated With Arterial Stiffness in Healthy Individuals

Background—Arterial stiffness is an important determinant of cardiovascular risk. Elastin is the main elastic component of the arterial wall and can be degraded by a number of enzymes including serine proteases and matrix metalloproteinases (MMPs). Serum MMP-9 levels correlate with arterial stiffness and predict cardiovascular risk. Polymorphisms in the MMP-9 gene are also associated with large artery function in subjects with coronary artery disease. Therefore, we investigated the influence of known MMP-9 (−1562C>T, R279Q) polymorphisms on arterial stiffness in a large cohort of healthy individuals (n=865). Methods and Results—Aortic pulse wave velocity (PWV) and augmentation index were assessed. Supine blood pressure, biochemical markers, MMP-9 levels, and serum elastase activity (SEA) were also determined. Genomic DNA was extracted and genotyping performed. Aortic PWV, serum MMP-9, and SEA were higher in carriers of the rare alleles for the −1562C>T and R279Q polymorphisms. These polymorphisms were also associated with aortic PWV after correction for other confounding factors. Stepwise regression models with known or likely determinants of arterial stiffness revealed that ≈60% of the variability in aortic PWV was attributable to age, mean arterial pressure, and genetic variants (P<0.001). Conclusions—We have demonstrated for the first time that aortic stiffness and elastase activity are influenced by MMP-9 gene polymorphisms. This suggests that the genetic variation in this protein may be involved in the process of large artery stiffening.

Factor V Leiden and Thermolabile Methylenetetrahydrofolate Reductase Gene Variants in an East Anglian Preeclampsia Cohort

Preeclampsia is a heritable condition that develops as a result of widespread vascular endothelial dysfunction. The thrombotic tendency in this condition has suggested a number of candidate genes, and there have been recent reports of positive association with the Leiden variant of factor V and the thermolabile variant of methylenetetrahydrofolate reductase. We attempted to reproduce these results in a large cohort of well-characterized women with preeclampsia, recruited prospectively within the East Anglian region of the United Kingdom. Women in the preeclampsia cohort (n=283) were genotyped for both the Leiden variant (G1691A) of factor V and the thermolabile variant (C677T) of methylenetetrahydrofolate reductase. Genotype and allele frequencies were compared with those of 2 control groups, one consisting of women recruited prospectively (n=100) from the same maternity hospital as the subjects and another consisting of normotensive women (n=100) from East Anglia. No significant differences were detected. Specifically, the carrier rate for the Leiden variant was 5.3% in the preeclampsia group and 5. 5% in the combined control group. T677 homozygotes for methylenetetrahydrofolate reductase were 11% and 11.5% in the 2 groups, respectively. We conclude that there is no evidence of association of preeclampsia with either of these 2 polymorphisms in our study population.

A new kindred with pseudohypoaldosteronism type II and a novel mutation (564D>H)in the acidic motif of the WNK4 gene.

We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon’s syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690G>C) that causes a missense mutation within the acidic motif of WNK4 (564D>H). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22Na+ flux in Xenopus oocytes expressing NCCT by ≈90% (P<0.001), whereas the 564D>H mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced >50% inhibition of steady-state current through ROMK at a +20-mV holding potential (P<0.001). The 564D>H mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein–NCCT) and ROMK (enhanced green fluorescent protein–ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564D>H mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564D>H causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564D>H, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.

Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within the KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na+ currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Detection of mutations in KLHL3 and CUL3 in families with FHHt (familial hyperkalaemic hypertension or Gordon's syndrome)

The study of families with rare inherited forms of hypo- and hyper-tension has been one of the most successful strategies to probe the molecular pathophysiology of blood pressure control and has revealed dysregulation of distal nephron Na+ reabsorption to be a common mechanism. FHHt (familial hyperkalaemic hypertension; also known as Gordons syndrome) is a salt-dependent form of hypertension caused by mutations in the regulators of the thiazide-sensitive Na+–Cl− co-transporter NCC [also known as SLC12A3 (solute carrier family 12 member 3)] and is effectively treated by thiazide diuretics and/or dietary salt restriction. Variation in at least four genes can cause FHHt, including WNK1 [With No lysine (=K) 1] and WNK4, KLHL3 (kelch-like family member 3), and CUL3 (cullin 3). In the present study we have identified novel disease-causing variants in CUL3 and KLHL3 segregating in 63% of the pedigrees with previously unexplained FHHt, confirming the importance of these recently described FHHt genes. We have demonstrated conclusively, in two unrelated affected individuals, that rare intronic variants in CUL3 cause the skipping of exon 9 as has been proposed previously. KLHL3 variants all occur in kelch-repeat domains and so probably disrupt WNK complex binding. We have found no evidence of any plausible disease-causing variants within SLC4A8 (an alternative thiazide-sensitive sodium transporter) in this population. The results of the present study support the existing evidence that the CUL3 and KLHL3 gene products are physiologically important regulators of thiazide-sensitive distal nephron NaCl reabsorption, and hence potentially interesting novel anti-hypertensive drug targets. As a third of our non-WNK FHHt families do not have plausible CUL3 or KLHL3 variants, there are probably additional, as yet undiscovered, regulators of the thiazide-sensitive pathways.

Gordon Syndrome: a continuing story

Gordon Syndrome (GS) is a rare familial hypertension syndrome with a characteristic hyperkalaemia which distinguishes it from other syndromic forms of hypertension that typically cause hypokalaemia. Patients with GS respond to aggressive salt-restriction or relatively small doses of thiazide diuretics, which suggests that activation of the thiazide-sensitive Na/Cl cotransporter (NCC) in the distal nephron is to blame. However, the mechanism has proved to be complex. In 2001, mutations in genes encoding two serine/threonine kinases, WNK1 and WNK4, were identified as causing GS. However, it took several years to appreciate that these kinases operated in a cascade with downstream serine/threonine kinases (SPAK and OSR1) actually phosphorylating and activating NCC and the closely related cotransporters NKCC1 and NKCC2. The hyperkalaemia in GS arises from an independent action of WNK1/WNK4 to reduce cell-surface expression of ROMK, the secretory K-channel in the collecting ducts. However, mutations in WNK1/4 are present in a small minority of GS families, and further genes have emerged (CUL3 and KLHL3) that code for Cullin-3 (a scaffold protein in an ubiquitin–E3 ligase) and an adaptor protein, Kelch3, respectively. These new players regulate the ubiquitination and proteasomal degradation of WNK kinases, thereby adding to the complex picture we now have of NCC regulation in the distal nephron.

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK

WNK kinases have rapidly emerged as important regulators of Na+ and K+ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K+ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordons syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (DeltaAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, DeltaAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.

An inducible transgenic mouse model for familial hypertension with hyperkalaemia (Gordon's syndrome or pseudohypoaldosteronism type II).

Mutations in the novel serine/threonine WNK [With No lysine (=K)] kinases WNK1 and WNK4 cause PHAII (pseudohypoaldosteronism type II or Gordons syndrome), a rare monogenic syndrome which causes hypertension and hyperkalaemia on a background of a normal glomerular filtration rate. Current animal models for PHAII recapitulate some aspects of the disease phenotype, but give no clues to how rapidly the phenotype emerges or whether it is reversible. To this end we have created an inducible PHAII transgenic animal model that expresses a human disease-causing WNK4 mutation, WNK4 Q565E, under the control of the Tet-On system. Several PHAII inducible transgenic mouse lines were created, each with differing TG (transgene) copy numbers and displaying varying degrees of TG expression (low, medium and high). Each of these transgenic lines demonstrated similar elevations of BP (blood pressure) and plasma potassium after 4 weeks of TG induction. Withdrawal of doxycycline switched off mutant TG expression and the disappearance of the PHAII phenotype. Western blotting of microdissected kidney nephron segments confirmed that expression of the thiazide-sensitive NCC (Na⁺-Cl⁻ co-transporter) was increased, as expected, in the distal convoluted tubule when transgenic mice were induced with doxycycline. The kidneys of these mice also do not show the morphological changes seen in the previous transgenic model expressing the same mutant form of WNK4. This inducible model shows, for the first time, that in vivo expression of a mutant WNK4 protein is sufficient to cause the rapid and reversible appearance of a PHAII disease phenotype in mice.