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From linkage to genes in human hypertension

Melander, Olle

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In the current issue of the journal, Yan-Qing Ma et al. report on the association between systolic blood pressure and the microsatellite marker, D8S282, near the lipoprotein lipase locus in Hong Kong Chinese individuals [1]. This particular locus previously showed evidence of linkage to systolic blood pressure [2,3]. The article by Yan-Qing Ma et al. [1] therefore raises questions about two central questions in the field of genetics of hypertension: when and how should we proceed in order to identify the responsible disease gene when promising linkage data of the locus in question are available?

Available linkage study results are not utilized enough

To date, a clear majority of the scientific publications in the field of genetics of primary hypertension have focused on searching for an association between polymorphisms in a very limited number of genes for components in known blood pressure regulatory systems, and blood pressure levels or hypertension. The narrow target for association studies is surprising. A number of well-performed linkage studies of hypertension and blood pressure variation have been performed, many of which have scanned the entire genome for linkage (genome wide scans). In spite of the rapid increase in general accessibility of the human genomic sequence, association studies of ‘new’ positional candidate genes (genes residing in genetic regions that have shown evidence of linkage) have been much less popular than the study of polymorphisms of genes in well-known blood pressure regulatory systems. The latter approach has been fruitful to a certain degree [4]; however, the complex nature of primary hypertension necessitates other strategies. Sceptical voices have expressed pessimism as to whether it is possible to identify hypertension genes ‘as this research field now after more than 10 years still have not resulted in the identification of any hypertension genes which could be of help for the clinician in the diagnostic and therapeutic routine'. To expect that the enormously great and important task of dissecting the genetic background of primary hypertension would have been completed after this relatively short time frame is nothing other than unrealistic. As only a tiny fraction of the genome has been tested for allelic association with hypertension to date, there is great reason to be optimistic, provided that we broaden the search to include linkage disequilibrium mapping and association studies of variants in positional candidate genes in genetic regions that have shown promising linkage results.

Proceeding from linkage to disease gene discovery seems difficult, but not impossible

Identification of gene variations responsible for linkage results is indeed difficult as the genetic region in question often spans over a genetic distance of more than 10 cM (1 cM corresponds to approximately one million base pairs). Such a region could thus harbour hundreds of genes, many of which have an unknown function. This may appear to be a deterrent. However, one of the greatest possibilities of genetics as a tool to better understand the pathophysiology of primary hypertension is just that; to identify genes that no one would guess would be of importance for blood pressure regulation. Genome wide scan linkage studies followed by positional cloning and linkage disequilibrium mapping have proven fruitful in finding disease genes in two other complex diseases: Crohn's disease [5] and type 2 diabetes mellitus [6]. After previously having been unknown to the diabetologist, genetic variation at the calpain 10 gene was found to be responsible for the earlier established linkage between type 2 diabetes and the ‘NIDDM 1’ locus on chromosome 2 [6], and this has opened up new and exciting views of mechanisms in the pathogenesis of type 2 diabetes. In the case of primary hypertension, there are several genome wide scans and other linkage studies in which regions showing evidence of linkage overlap. One promising example is the genetic region between 60 and 70 cM (Marshfield distances) from the beginning of chromosome 17 (chromosome 17p11-q21), which has shown linkage to blood pressure variation and hypertension in three independent studies [7–9]. Interestingly, a syntenic region on rat chromosome 10 is of importance for blood pressure variation in this species [10]. Hopefully, the chromosome 17p11-q21 locus will be subject to linkage disequilibrium mapping and association studies of variants in positional candidate genes in the near future. By the end of 2003, the entire human genomic sequence, as well as a genome wide map of existing single nucleotide polymorphisms, is expected to be available on the internet for the general scientific community. This will simplify the work considerably, as the heavy positional cloning efforts will no longer be necessary. Of course, the same is true for all genetic regions that show promising evidence of linkage. Presently, the chromosome 17p11-q21 region appears especially interesting as it contains a gene, ‘with no lysine kinase 4’ (WNK4), in which variance was recently shown to be the cause of pseudohypoaldosteronism type 2B, a monogenic form of hypertension [11]. WNK4 thus represents a promising candidate gene also for primary hypertension.

The lipoprotein lipase locus

In the present issue of the journal, Yan-Qing Ma et al. [1] aim to investigate whether there is an association between the microsatellite marker, D8S282, near the lipoprotein lipase (LPL) locus on chromosome 8p22 and blood pressure variation (as well as with other variance components of the metabolic syndrome) in 229 healthy Chinese subjects from Hong Kong. This particular locus was chosen for study because it previously showed evidence of linkage to systolic blood pressure variation in Taiwanese [2] and hypertension in Mexican–American [3] families. The D8S282 marker was selected because it provided the strongest evidence of linkage to systolic blood pressure in a previous linkage study in Taiwanese families [2]. The present study is thus an example of an attempt to bring the earlier linkage results one step closer to gene identification by establishing an association between blood pressure and alleles of the genetic marker in question. After dividing their material into tertiles of systolic blood pressure, the main finding is that presence of one of the D8S282 alleles, the 278 base pair allele (`D-allele'), was more common among subjects in the upper tertile than among those in the lower tertile of systolic blood pressure. It is concluded that the D8S282 marker contributes to variation in systolic blood pressure in the healthy Hong Kong Chinese subjects studied, and that the data support the earlier finding of linkage to systolic blood pressure in Taiwanese [2].

The report by Yan-Qing Ma et al. [1] is interesting. However, there is reason to be cautious in the interpretation of the presented data. In contrast to family-based association studies, such as the transmission disequilibrium test [12] and the genotype discordant sibling analysis [13], non-family-based association studies such as the present one run a risk of showing a difference in allele- or genotype frequencies due to different population substructures in the subgroups, rather than being the result of a true genetic association between the marker and the phenotype. This risk can be minimized, or at least moderated, if large population-based materials are studied, or if clearly unrelated individuals (e.g. one member per family from family studies) are studied. The material in the present study was recruited from ‘hospital staff or their family members and friends'. Therefore, there is the risk that some of the subjects were relatives. As blood pressure is heritable, subjects belonging to the same tertile of systolic blood pressure are more likely to be relatives. This could lead to allele and genotype frequency differences between the subgroups, which are not due to allelic association between the genetic marker and the disease gene, as relatives share alleles at any locus more often than unrelated individuals. This potential problem is especially important given the small size of the study sample (n = 229). Furthermore, the experience from the field of genetics of hypertension is that very few positive association studies with less than 250 subjects have been possible to replicate. As the D8S282 marker was tested for an association with many different components of the metabolic syndrome without correcting for multiple comparisons, there is an additional risk that the association with systolic blood pressure is a false-positive.

The fact that the D8S282 marker showed strongest linkage to systolic blood pressure in the earlier linkage study in Taiwanese families [2] does not necessarily mean that this particular marker is in linkage disequilibrium with the hunted ‘blood pressure gene', neither in the Taiwanese study population [2], nor in the Hong Kong subjects studied in the present report. Furthermore, the D8S282 marker is unlikely to have any functional significance itself. Therefore, it would have been informative to study more markers in the vicinity of D8S282 and variants within the LPL gene or other positional candidate genes in this locus. This would allow for analysis of haplotypes in order to search for blocks of linkage disequilibrium. However, the small sample size in the present study does not allow such an analysis because this would require an even larger number of tests and, subsequently, an even greater risk of false-positive results.

It is extremely important for the field of genetics of hypertension to follow-up available genome wide scans and linkage studies with promising results, to a much greater degree than has been done so far, with positional candidate gene studies and tests for linkage disequilibrium. For example, for the validation of the present study, it would be interesting to know whether there is linkage disequilibrium between the D8S282 and other nearby markers and systolic blood pressure in the Taiwanese material which originally showed linkage to the LPL locus [2].

Which linkage results are so promising that they are worth following up?

Following up linkage results in order to find responsible disease genes is a huge and time-consuming work. A critical review of the available linkage data is essential before deciding to further dissect a genetic region. It is impossible to give an exact answer to which linkage results are worth proceeding with. However, without underestimating the complexity of the pathogenesis of primary hypertension, it seems fair to say that the current attitude on this issue is too conservative. Promising follow-up attempts have been made [14] but they are rare.

In genome wide scans, hundreds of markers are tested for linkage without any à priori hypothesis of where in the genome the disease gene or genes are located. If conventional significance levels are applied, there is a great risk of obtaining false-positive linkage results. By simulating data, Lander and Kruglyak [15] suggested guidelines for the interpretation of linkage results. A nominal P-value of 2 × 10–5 was determined to be the threshold for ‘genome wide significant evidence of linkage at the level of 0.05’ (5% probability that this statistical evidence would occur by chance in a genome wide scan) and a nominal P-value of 7 × 10–4 for ‘genome wide suggestive evidence of linkage’ (statistical evidence that would be expected to occur one time at random in a genome wide scan). However, these thresholds assume complete informativeness and an infinite marker map density, which never is the case in genome wide scans. Therefore, if these significance thresholds are used to determine when it is worthwhile to follow-up genome wide scan results, there is a risk of overlooking true hypertension susceptibility loci. It therefore seems preferable to perform simulations for each particular genome wide scan in order to obtain appropriate significance thresholds, taking into account the actual marker density and degree of informativeness. In my opinion, genetic regions which, according to these simulations, show genome wide significant evidence of linkage, and which remain or increase after fine mapping with at least a 5 cM map, are definitely worthwhile taking on to positional candidate gene association studies or linkage disequilibrium mapping. Regions showing suggestive evidence of linkage may also be worthwhile following up. This is especially the case if the 1 – LOD interval of the linkage peak in question overlaps with genetic regions that have shown significant or suggestive evidence of linkage in other studies with a comparable design and phenotype.

Apart from significance levels, a number of additional issues should be taken into account when evaluating genome wide scans and other linkage data. It is practically impossible to perform a meaningful power analysis for genome wide scan linkage studies of complex diseases as the number of genes involved in the disease is unknown, as is the magnitude of the effect of each gene. However, the power increases with increasing study sample, provided that expansion of the study sample is not at the expense of the quality of the phenotyping.

Accurate phenotyping is extremely important. As office blood pressure measurements display a certain degree of intra-individual variability [16], it is essential to document the individual blood pressure phenotype or the diagnosis of hypertension with several blood pressure measurements at different occasions. Longitudinal blood pressure phenotypes [9] and 24-h ambulatory blood pressure measurements are also likely to be more accurate than both single and repeated office blood pressure measurements taken during one visit.

The power and reliability of a linkage study are intimately dependent on the heritability of the studied phenotype. Longitudinal blood pressure has a heritability close to 60% for both systolic and diastolic blood pressure [9], whereas the heritability of office blood pressure is between 30 and 50% [17]. Furthermore, the genetic component of the aetiology of primary hypertension depends heavily on the age at onset of the disease. Whereas the genetic component of primary hypertension with an age at diagnosis below the age of 50 years is considerable, it decreases with increasing age at diagnosis and is negligible if the disease makes its debut after the age of 70 years [18]. Therefore, when reviewing linkage studies that use hypertensive status as the phenotype, it is important to examine the age at diagnosis of the patients.

Conclusion

In spite of several published genome wide scans and other linkage studies of primary hypertension and blood pressure variation, very few studies have been followed up with attempts to perform linkage disequilibrium mapping or positional candidate gene association studies. Doing so would increase the vitality of an already interesting field and justify optimism for the future for both patients with hypertension and hypertension-interested physicians and scientists.

References

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© 2002 Lippincott Williams & Wilkins, Inc.