Progress in Determining the Genes for Hypertension, Insulin Resistance,

and DysIipidemia" MARK CAULFIELD,b PIERRE-MARC BOULOUX,C

AND PATRICIA MUNROE~ b Department of Clinical Pharmacology

St. Bartholomavk Hospital London EClA 7BE, United Kingdom

CDepartment of Endocrinology Royal Free Hospital School of Medicine

London Nw3 ZQG, United Kingdom dDepartment of Pediatrics

University College London Medical School llte Rape Institute

London WCIE UJ, United Kingdom

ESSENTIAL HYPERTENSION

In the general population, blood pressure adopts a normal distribution, with es- sential hypertension being defined on the basis of thresholds for pharmacological inter- vention to reduce blood pressure and thus risk of stroke and heart attack. Family studies imply that approximately 30% of blood pressure variation is due to genes, with the remainder arising from environmental influences such as sodium, alcohol, and obesity. Since populations cannot be completely demarcated into hypertensives or normotensives, it is likely that several genes are involved in the genetic basis of this trait.

GENETIC STRATEGIES FOR HYPERTENSION

During the early nineties, there have been considerable advances in molecular ap- proaches available to investigate the genetic basis of complex diseases such as human essential hypertension. The strategy that has been most widely employed thus far to identify the major genes for hypertension involves investigation of candidate genes arising from systems that are physiologically implicated in blood pressure regulation.

a This work was supported by the Joint Research Board of St. Bartholomew's Hospital and by funds from the Medical Research Council of Great Britain to M. Caulfield and P. Munroe.

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However, there are many potential candidates that could alter blood pressure levels and evaluation of the role of all of these genetic loci could be extremely time-consuming.

An alternative and complimentary strategy is to screen the whole genome utilizing a dense map of highly polymorphic markers spread throughout the human genome to test for linkage of regions to blood pressure. Using this approach, it may be possible to identify chromosomal regions that harbor susceptibility genes that we do not even suspect contribute to the hypertensive phenotype. However, this still leaves the task of refining the region down to a specific causative gene for which no function may yet have been described and this may be quite laborious.

LINKAGE STUDIES IN HYPERTENSION

The most-powerful approach to understanding the genetics of complex traits will probably prove to be family-based linkage studies.’ In many cases, these studies are based on affected sibling pairs from nuclear families because raised blood pressure may have a variable age of onset. In addition, it may be possible for apparently un- affected family members to possess a susceptibility variant for hypertension, but not manifest the trait because of the absence of an environmental stimulus.2 Equally, the manifestation of elevated blood pressure may rely on gene-gene interaction and may require the inheritance of genetic variants at more than one locus.2

With linkage studies, the aim is to observe the tracking of a particular allele at a genetic locus with hypertension through a family. This can be based on computing a logarithmic likelihood ratio (Lod score), which simply expresses the likelihood of linkage divided by the likelihood of nonlinkage.2 It has previously been accepted that support for linkage has been demonstrated if Lod scores are greater than 3.0, which means that data at the genetic locus under evaluation are lo00 to 1 in favor of linkage.2 However, in complex traits, large data sets may be required to offer adequate power to generate such support for linkage. Recently, some workers have proposed that the stringency accepted for such Lod scores should be even greater in genomewide screens.2 This would require identification of large numbers of families to offer any chance of being able to refute or accept involvement of a region in raised blood pressure.

A complimentary statistical method of linkage analysis employed in hypertension genetics research is to ask the question of whether hypertensive affected siblings share alleles at a genetic locus more often than would be expected by chance in the popu- lation.* This allele-sharing method uses a t statistic to measure excess allele sharing in affected sibling pairs.2 An advantage of such methods is that they can take account of variable penetrance of susceptibility variants and do not require specification of Mendelian modes of inheritance, which makes them particularly useful in complex traits like hypertension.

POPULATION ASSOCIATION STUDIES

This study design has been widely employed to compare the distribution of genetic variants of a known candidate gene in unrelated cases and controls. A positive asso-

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ciation implies that an allele of the variant studied exhibits linkage disequilibrium with the complex trait under investigation.2 However, this does not mean that the asso- ciated variant causes hypertension. Since linkage disequilibrium only operates over short chromosomal distances, it is possible that the association may be merely high- lighting the presence of a causative mutation close by, but probably within, the same gene.2 When evaluating reports of association studies, it is important to check that there has not been selection bias in the ascertainment of cases and controls2-for ex- ample, by comparing people of different ethnic origin or recruiting study groups from highly selected clinic populations rather than the general population.2

PROGRESS TOWARD THE GENETIC BASIS OF HYPERTENSION

“Simple Mendelian Forms of Hypertension”

Lifton and Shimkets have approached the genetic investigation of hypertension in a novel way by investigating rare Mendelian hypertensive syndromes and demon- strating genetic influences that may have extremely large effects on blood p re~su re .~ .~ This approach could provide important insights into mechanisms that may hold wider importance when investigated in the entire hypertensive population or in defined ho- mogenous subsets.

Glucocorticoid-suppressible Hyperaldosteronism

This syndrome is characterized by autosomal dominant transmission of hyperten- sion with variably elevated aldosterone levels, suppressed plasma renin activity, and high levels of abnormal adrenal steroids, 18-hydroxycortisol and 18-oxocortisol.~ It appears that the aberrant steroids and the aldosterone produced in this syndrome are under direct adrenocorticotrophic hormone control since they are suppressible by ex- ogenous glucocorticoids such as dexamethasone. Families with this syndrome have early onset hypertension, which may be severe, and there appears to be an excess of cerebral hemorrhage in some families.

The genes encoding llp-hydroxylase, which is normally expressed in the zona fasicdata and glomerulosa, and aldosterone synthase, which is normally only expressed in the zona glomerulosa, have 95% homology in nucleotide sequence and lie in close physical proximity on chromosome 8.6

In 1992, Lifton ef al. described a chimeric gene duplication arising from unequal crossing over between 11 p-hydroxylase regulatory sequences that became fused to the aldosterone synthase gene.3 This results in ectopic aldosterone production under adrenocorticotrophic hormone control. Subsequently, several crossover sites ranging from intron 2 to 4 have been described.’ Although these appear to be independent mutations, there is certainly a preponderance of Celtic ancestry in the families with this form of hyperten~ion.~ Interestingly, some families with this “Simple Mendelian Form of Hypertension” have individuals who possess the chimeric aldosterone syn-

CAULFIELD et al.: GENES 113

thase gene, but are not hypertensive. It is possible that this incomplete penetrance of the chimeric gene is due to the requirement of coexisting environmental factors such as sodium ingestion or indeed other genes to permit the hypertensive phenotype to be manifest. While this is a rare hypertensive trait, it illustrates the benefit of specific intermediate phenotypes in demarcating hypertensive subgroups and focusing research for genetic markers. It also underscores the complexity of apparently simple hyper- tensive traits.

Liddle’s Syndrome

Liddle’s syndrome is a rare form of hypertension described initially in 1963 within a single kindred with multiple siblings having severe hypertension.8 Some of these siblings manifest hypokalemia with suppressed plasma renin activity and low aldos- terone levels even when challenged by a low sodium diet.8 Absence of response of these features to an aldosterone antagonist, but reduction in blood pressure and cor- rection of hypokalemia by triamterene suggested that an abnormality of the distal epi- thelial sodium channel might underlie this condition.8

Recently, mutations within subunits of the epithelial sodium channel have been described in the original Liddle’s kindred.4 Functional studies reveal that these mu- tations could lead to constitutive overactivity of this channel with excess reabsorption of sodium in the kidney.4

Hypertension and Brachydactyly

An autosomal dominant form of severe hypertension associated with brachydactyly has been documented in a large Turkish kindred.9 In studying this form of hyperten- sion, there was no specific phenotypic clue as to chromosomal localization of the causative gene or genes and therefore a genomewide screen was used to link a region on 12p to this trait.9 While the precise nature of the defect remains to be determined, this demonstrates the potential value of genome screening.

STUDIES IN HUMAN ESSENTIAL HYPERTENSION

The Renin Angiotensin System

The close involvement of this system in blood pressure regulation and cardiovas- cular disease has prompted researchers to focus on candidate genes from within the system.Io Cleavage of angiotensinogen by renin is a key rate determinant of this cas- cade. 10 The observation that plasma angiotensinogen may be elevated with increased blood pressure and indeed track with hypertension through families makes this an attractive candidate gene for hypertension. 11 Indeed, three studies have reported sup- port for the linkage of the angiotensinogen gene to hypertension in affected sibling

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pairs of white European origin and African-Caribbeans.12-14 In addition, in one of these studies, two genetic variants within the angiotensinogen molecule were asso- ciated with hypertension. 12 Furthermore, one of the variants, encoding threonine instead of methionine at position 235 in the final molecule, was associated with raised plasma angiotensinogen levels.12 However, there have been a number of studies that have not found an association of these variants of angiotensinogen with hyper- tension and therefore it is important to remain cautious about the role of this gene in hypertension. 13,14

Hypertension as a Metabolic Syndrome

Hypertension commonly clusters with dyslipidemia and insulin resistance, which raises the possibility that genetic factors predisposing to these coexistent features might play a primary role in the development of raised blood pressure. 15 From the San An- tonio Heart Study, it has been reported that glucose intolerance and raised insulin levels may predate hypertension by eight years.16 However, it could be that insulin sensitivity is altered by a general membrane defect as part of the metabolic syndrome of hypertension. 15 Evidence from strong familial aggregation of non-insulin-dependent diabetes mellitus (NIDDM) where insulin resistance is prevalent suggests that, in some phenotypes, sensitivity to insulin may be affected by genes in some individuals. Re- cently, a preliminary report from a genome screen in Mexican-American families with NIDDM suggests that there is a potential locus on chromosome 2.17 However, this will require confirmation in further studies and, interestingly, the locus linked to NIDDM is not near any potential candidates for this trait.I7

Znsulin Receptor Gene in Hypertension

The insulin receptor mediates the actions of insulin, enhances glucose transport, and may represent an end-organ site for insulin resistance, but studies investigating a role for this receptor in NIDDM have reported conflicting results.18J9 An associa- tion study on Australian hypertensives reported an association of an Rsa 1 restriction fragment length polymorphism with high blood pressure. 19 Subsequently, a linkage study on 31 affected hypertensive pairs reported very borderline support for linkage using RFa 1 and Sst 1 restriction fragment length polymorphisms with analysis using an allele-sharing method.20 However, this was not confirmed by the Lod score, which was zero, suggesting there is no support for linkage in this data set.20 It must be em- phasized that none of the studies on the insulin receptor gene in hypertension have characterized the subject’s status with regard to insulin sensitivity nor offered adequate power to refute a role for this gene in hypertension.

Dyslipidemia and the Genetics of Hypertension

Several reports have noted the association between hypertension and dyslipidemia in the form of raised cholesterol and triglycerides and low levels of high-density

CAULFIELD et a!.: GENES 115

lipoprotein. 16 Indeed, familial clustering of lipid anomalies and hypertension has been documented in families from Utah and has led to the proposal that familial dyslip- idemic hypertension may occur in as many as 12-20% of hypertensives.21 It has been suggested that dyslipidemia may affect endothelial function and consequently may contribute to impaired vasodilatation and thus to elevated blood pressure.

The Lipoprotein Lipase Genetic Locus and Systolic Blood Pressure

Recently, a study on 48 Taiwanese families selected by a proband with NIDDM rather than hypertension were analyzed using quantitative trait locus analysis for the relationship of a variety of loci to blood pressure.22 There was support for linkage of the region chromosome 8, which contains lipoprotein lipase, to systolic blood pres- sure, which was also supported by data from flanking markers.22 This does not mean that lipoprotein lipase causes raised systolic blood pressure since other loci in the area may be important. It is unclear how an enzyme like lipoprotein lipase, which hydrolyzes triglyceride to free fatty acids and transfers particles to high-density lipo- protein, might influence blood pressure. Further studies to define the role of this locus in blood pressure are required and it remains possible that this locus is really im- plicated in NIDDM.

Apolipoprotein B Gene and Hypertension

Apolipoprotein B is the predominant protein of low-density lipoprotein and a com- ponent of lipoprotein (a) constituting atherogenic lipid fractions.23 Genetic associa- tions of restriction fragment length polymorphisms and cardiovascular disease and plasma apolipoprotein B levels have been o b ~ e r v e d . ~ ~ . ~ ~ In a study on 56 hyperten- sive sibling pairs of white European origin, there was borderline support for linkage of a highly polymorphic marker in the flanking region of the apolipoprotein B gene.26 This was strengthened if the data were partitioned according to cholesterol level, and 20 hypertensive sibling pairs had cholesterol levels greater than 6.5 mmoVL.26 There was support for linkage in these families with elevated cholesterol, but not among families with low cholesterol, even though both sibships were similarly hypertensive.26 These data need confirmation and must be treated with caution. However, there is some suggestion that the apolipoprotein B gene may influence dyslipidemia in hypertensives.

SUMMARY

For the first time, we have techniques available that may enable us to determine cause and effect in common cardiovascular diseases. The observations presented herein require cautious interpretation until replicated. There are currently several large pro- grams under way that seek to establish substantial family resources for investigation of the genetic basis of hypertension. When interpreting the results of genome screens,

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it will be necessary to consider that we may link genetic loci for coexistent features such as dyslipidemia and insulin resistance to hypertension. Therefore, careful pheno- typic data will be necessary to dissect out the causes of hypertension.

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