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Hypertension. 2007;49:740-747
Published online before print February 12, 2007, doi: 10.1161/01.HYP.0000259105.09235.56
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(Hypertension. 2007;49:740.)
© 2007 American Heart Association, Inc.

Brief Reviews

Positional Cloning of Quantitative Trait Loci for Blood Pressure: How Close Are We?

A Critical Perspective

Alan Y. Deng

From the Research Centre, Centre Hospitalier de l’Université de Montréal, l’Université de Montréal Montréal, Québec, Canada.

Correspondence to Alan Y. Deng, Research Centre, Centre Hospitalier de l’Université de Montréal, Technopôle Angus, 2901 Rachel St East, Room 312, Montréal, Québec H1W 4A4, Canada. E-mail alan.deng{at}umontreal.ca


*    Introduction
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 *Introduction
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Blood pressure (BP) in any human population exhibits as a continuous variable that fits a bell-shaped curve. Hypertensive individuals are those whose BP is maintained at one extreme of the curve and above a defined cutoff. Despite progress made in identifying the mechanisms underlying certain rare monogenic forms of hypertension,1,2 the etiology and pathogenesis of essential hypertension remain poorly understood.

Because existing human populations are genetically heterogeneous, and because environmental factors impacting on the pathogenesis of hypertension cannot be controlled in a given population, it is difficult to identify the molecular mechanisms that transduce the sequela of essential hypertension via direct human studies.3 To alleviate the drawbacks of human investigations, animal models, especially inbred rodents, have been developed and experimentally manipulated to identify quantitative trait loci (QTLs) for BP, because major confounding environmental factors, such as diet and genetic background, can be systematically controlled. Once identified in animal models, the molecular basis may be translated into physiological understandings of essential hypertension in humans.

It is with this expectation that efforts have been launched to identify the molecular basis of BP QTLs in animal models. Because the identification of individual QTLs is primarily based on their chromosome locations unbiased by, or unrestricted to, their physiological roles, positional cloning is believed to be the most efficient strategy.

Before we embark on discussions regarding QTL discovery, a definition is in order. Semantic arguments abound as to exactly what a QTL, that is, a locus,4 entails. Is it 1 gene or a collection of genes? As genetic mapping progresses from a large chromosome segment to an interval of submegabase, several regions initially thought to contain 1 BP QTL5 appear to harbor >1 in each of them,6–10 whereas several other regions turned out to harbor 1 QTL as expected.11,12 This outcome has precipitated a conceptual conflict as to what a QTL consists of, that is, a congregation of multiple QTLs versus the view that a QTL can still be considered as 1 locus represented by 1 gene, and a chromosome segment can contain >1 of them.

To shed some light on the concept of a locus, a historic visit seems appropriate. The first and classical genetic locus to be defined is the white locus, w+, which, when mutated, changes the wild-type red eye color to white in Drosophila.13 From its initial chromosome assignment to final molecular identification,14 w+ has been considered as 1 gene, and positional cloning has confirmed it.14 Similarly, rare monogenic forms of human hypertension are attributed to single loci, and each is determined by a single gene.1,2 The only difference between them and a BP QTL seems to be that multiple QTLs are located adjacent to one another, and all can influence the same BP phenotype. As such, there is no compelling reason to reject the definition of a BP QTL as 1 gene, even in the absence of its molecular identity.

Despite the varying definitions of a QTL with differing conceptual bend, the major drive in understanding the molecular basis of a QTL is to identify the gene responsible for it. For this purpose, I attempt to adhere to the classical definition of 1 locus, including 1 QTL, as 1 causative gene, and a chromosome segment, large or small, can be designated as a region harboring it. This being said, when localizing a BP QTL genetically or physically before its molecular identity is known, it is imperative to bear in mind that >1 QTL can be closely situated to each other in 1 chromosome region.

A little over a decade ago, in the early stage of genetic analysis, the question of the day was, "Is it possible to detect and positionally clone BP QTLs?"15 Since then, a large number of BP QTLs have been detected using a variety of inbred rat16 and mouse17–19 strains. Moreover, a new concept governing the genetic architecture of polygenic hypertension has emerged, and the significant progress has been made toward the identification of individual QTLs. Within this conceptual framework and prompted by the tactical achievement approaching positional cloning of BP QTLs, the time has come to reassess the current status and to look toward to the future of genetic hypertension research, especially using animal models.


*    Experimental Tools
up arrowTop
 up arrowIntroduction
 *Experimental Tools
down arrowGenetic Architecture: Individual...
 down arrowOrganization of QTLs According...
 down arrowProspective Research in Gene...
 down arrowReferences
 
To experimentally investigate the genetic basis of polygenic hypertension, 2 sets of tools must be available, that is, adequate phenotyping and informative genetic models. The benchmark phenotype for hypertension has always been BP, but the link between a gene to BP, except for certain cases, such as the Liddle’s syndrome, seems rather remote, and, thus, researchers have moved to identifying "intermediate phenotypes,"20,21 which connect a gene to an organ function that appears to contribute to BP. The basis for these studies is that BP does not arise directly from a gene function but, rather, is created via physiological mechanisms that are regulated at organ levels. Thus, there is a much shorter distance between a gene and an "intermediate phenotype" than between a gene and BP. Therefore, etiologically, these phenotypes seem simpler to identify than BP. However, there is evidence that certain probable intermediate phenotypes, such as cardiovascular and renal functions, have their own complex etiologies, which can be independent from BP.22–24 Hence, BP remains the only appropriate final phenotype for hypertension research,25 but intermediate phenotypes, such as cardiovascular, renal, neuronal, and hormonal functions, may play an important role in facilitating BP QTL discovery. These phenotypes can be valuable when dealing with genes of unknown functions. A large amount of physiological data on BP has been accumulated in the rat, and its large size makes it highly suitable for accurate physiological measurements of functions that impact BP. Thus, the rat has remained the most widely used model for biomedical research also comprising hypertension.26 Similar to mice, rats have a short breeding cycle and a large litter size. Inbred rat strains have also been developed. Thus, the rat is an invaluable organism for genetic studies. Empowered by recent advances in genome information27 and cloning technology,28 the exploration of rats as a model organism for hypertension research has gained renewed momentum.

The established inbred rat models and BP QTLs detected using them up until 1999 have been reviewed in detail elsewhere.16 The basic principles and experimental strategies for QTL detection have been addressed extensively,15,16,29–31 and comparative reviews of the chromosome locations of BP QTLs among rats, mice. and humans have also been presented.16,32–34 There is evidence that gene–environment interactions and epigenetic factors can influence the development of hypertension.32,35 Hence, these aspects will not be reiterated here.

The present review focuses on 3 complementary facets of genetic hypertension research, using inbred experimental models of polygenic hypertension. First, some conceptual insights into the genetic architecture will be discussed briefly. Second, an integrative organizational hierarchy of BP QTLs will be concisely formulated and probable mechanistic interpretations speculated. Finally, critical discussions of strategies will be coalesced that enable positional cloning of BP QTLs to take place.


*    Genetic Architecture: Individual BP QTLs Resemble "Monogenic" Determinants
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 *Genetic Architecture: Individual...
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A quantitative trait such as BP is believed to be molded by cumulative aggregations of multiple QTLs, each possessing a minor or weak "average" effect.3,31,36 The concept implies that a phenotypic "threshold" needs to be overcome by amassing multiple QTLs before a BP effect is detectable. This explanation has been given to rationalize the observations that, in a broad spectrum of populations, no single QTL has been detected to have a "major" BP effect.36,37

However, animal model research has yielded an opposite conclusion, namely, each QTL alone, isolated via a congenic strain, appears to be sufficient to influence BP, and no combination with other QTLs seems necessary to demonstrate a BP effect. The use of congenic strains as a strategy of BP QTL identification will be compared with linkage analysis later on in the Prospective Research in Gene Discovery of BP QTLs section.

Table 1 lists the BP QTLs that have been localized to subcentimorgan levels or limited to <20 genes via congenic strains. The QTLs are limited to those specified by congenic strains, but not by linkage, for 2 reasons. First, a congenic strain establishes an approximate cause–effect relationship between a chromosome interval harboring a QTL and its direct BP effect. Second, each congenic strain consists of genetically homogeneous rats that are identical in their genomes and, consequently, yields results specific for the QTL interval in question. Because of these fundamental differences, conclusions drawn from linkage analysis are considered more or less tentative and probable, whereas those from congenic strains are more proven.


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  		TABLE 1. BP QTLs in Rats That Have Been Restricted to <1 cM or to Intervals Containing <20 Genes by Congenic Strains Based on a Cause–Effect Relationship

An examination of these congenic strains (Table 1) indicates that, assuming that a chromosome interval harbors 1 BP QTL, each of these intervals alone is sufficient to exert an effect and capable of doing so independent of other QTLs. Its effect on BP does not require a combination with that of another QTL, although it remains remotely possible that 2 yet unidentified QTLs might require a combination to achieve a BP effect. Specifically, as an example, the congenic strain C10S.L20 restricts C10QTL1 to an interval of 1.17 Mb that contains 16 possible genes.8 Consequently, the possibility is minimized that >1 BP QTL can be present in the QTL region on chromosome (Chr) 10. And yet, C10QTL1 alone without the involvement of another QTL is adequate to influence BP, and no combination with another QTL is required to achieve this effect.8

Studies of heterozygotes for 10 representative QTLs, including C10QTL1, C10QTL4, and C16QTL, further support the interpretation of their "monogenic" behavior.38 The heterozygote carrying 1 Dahl salt-sensitive (DSS) and 1 normotensive allele for each of 8 QTLs exhibits a complete normotensive dominance, indicating that 1 copy of the normotensive allele is sufficient to change BP to the same extent as 2 copies. These results can be interpreted to mean that the DSS alleles for these 8 QTLs may represent loss of function alleles.

The DSS allele for C2QTL3 shows complete dominance, that is, 2 copies of normotensive alleles are required to change BP, and 1 copy has virtually no effect. The DSS allele for C17QTL demonstrates incomplete dominance, that is, it increases BP in a dose response to the number of its copies. Therefore, both sets of data combined, that is, the independent action of a BP QTL from others and the strength of QTL alleles as (complete or incomplete) dominance, illustrate the Mendelian behavior of individual BP QTLs and suggest them to resemble monogenic determinants.

A caveat to the above interpretation is that, because the strength of the heterozygote for not all of the BP QTLs has been analyzed, the possibility cannot be excluded that another BP QTL might not follow the pattern of monogenic inheritance, nor does it dictate the mode of inheritance of the same QTL in another rodent model of hypertension.


*    Organization of QTLs According to Epistatic Modules and Participation in Pathways/Cascades
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Two types of complex QTL organizations are apparent, that is, redundancy of QTLs and the existence of QTLs with paradoxical effects. First, there appear to be more QTLs than necessary to account for the total difference between a hypertensive and a normotensive strain. For example, >10 QTLs have been found in a single contrast between the DSS and Lewis strains,24 and yet C10QTL1 alone seems sufficient to account for 45% of the total BP difference between DSS and Lewis. What role, then, do the remaining QTLs play in the overall maintenance of BP?

Second, QTLs with opposing BP effects exist in both hypertensive or normotensive strains, that is, the genomes of the hypertensive DSS and normotensive Lewis strains are actually composed of QTL alleles that increase as well as decrease BP.6,9,39 Thus, to regulate BP in each strain, a counterbalanced equilibrium of both BP-increasing and BP-decreasing alleles must be achieved at different QTLs.

One way to achieve a balanced equilibrium and to solve the issue of QTL redundancy can be via epistatic interactions. Epistasis40 refers to the effect of 1 QTL (ie, 1 gene) hiding or suppressing that of another. An example of epistasis is illustrated by the study of interactions between 2 adjacent QTLs, C10QTL1 and C10QTL4.8 The combined BP effect is equal to C10QTL4 alone, indicating that these 2 QTLs act epistatically to each other.

The hierarchical relationship between 2 epistatic QTLs is exemplified by the relationship between 2 QTLs on Chr 3.9 Within a given strain, the allele at 1 QTL possesses a BP-lowering effect (ie, –BP QTL), whereas at another QTL, the allele possesses a BP-raising effect (ie, +BP QTL). As the combined effect of both of them was the same as that of –BP QTL alone, –BP QTL acts epistatically to +BP QTL; that is, –BP QTL stands higher on the epistatic hierarchy. Based on epistatic relationships, BP QTLs can be categorized preliminarily into epistatic modules. Therefore, the epistatic phenomenon appears to play an important regulatory role governing how multiple QTLs can function together.

Mechanistically, QTLs belonging to the same epistatic module can conceivably participate in a common pathway/cascade of reactions leading to BP determination. For example, –BP QTL1 and +BP QTL could be involved in the sequential steps. Because phenotypic differences are distinguishable between –BP QTL and +BP QTL9 and with the former being epistatic to the latter, –BP QTL would function in the latter part of the pathway/cascade.

Another mechanism by which 2 QTLs function together is by the mode of additivity, not epistasis. For example, C2QTL2 and C2QTL3 act additively, that is, the 2 QTLs augment each other’s effect.41 In this case, each QTL could use separate yet parallel pathways/cascades, and, when combined, C2QTL2 and C2QTL3 can cause an additive effect.

In summary of the genetic architecture of polygenic hypertension, a single QTL seems to act as if it controls a monogenic trait and, by itself, to be sufficient to change the BP phenotype without the participation of other QTLs. While functioning together, certain QTLs can be modularized according to their epistatic relationships, whereas others possess additive relationships.


*    Prospective Research in Gene Discovery of BP QTLs
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 up arrowOrganization of QTLs According...
 *Prospective Research in Gene...
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So far, few BP QTLs shown in Table 1 have been provisionally identified, and even fewer have been causatively authenticated. With the exception of a QTL on Chr 7 being probably the gene encoding 11ß-hydroxylase,11 the remaining QTL intervals that have been resolved to contain a limited number of candidates harboring genes or unidentified loci (ie, Locs) that have unknown functions or have functions unrelated to BP regulation. Consequently, these QTLs will likely represent novel genes controlling BP. If so, such a discovery will contribute to our understanding of the new etiologies of polygenic hypertension and will potentially have novel clinical applications. However, to achieve such a gene discovery also poses daunting challenges. It is of paramount importance to uncover a mutation(s) that alters either the function(s) of a gene product(s) or the level of gene expression. Furthermore, the functional relevance of the discovered mutation must be authenticated in vivo, that is, the mutation has to be able to change BP by itself. The following discussions will focus on integrating these 2 aspects of QTL discovery.

Congenic Strains Versus Linkage
A comparison between these 2 approaches is given in Table 2. First, regardless of experimental design (eg, using F2 or backcross populations16 or recombinant inbred strains29), linkage analysis has certain advantages and limitations. It suggests a correlative correspondence and cannot establish a cause–effect relationship between a chromosome region containing a QTL and BP phenotype. In contrast, a congenic strain tends to establish such a cause–effect relationship.


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  		TABLE 2. Comparisons Between Linkage and Congenic Approaches in BP QTL Identification

Second, linkage studies can lead to false-negatives or false-positives. As an example of false-negative, linkage analysis showed no QTL carrying BP-lowering alleles from DSS rats on Chr 8.5 It was only after the suspected region was dissected by 2 nonoverlapping congenic strains that this QTL appeared.6 A false-positive can be illustrated by the gene coding for angiotensin-converting enzyme. The initial linkage of BP to angiotensin-converting enzyme was strong,5 but congenic studies have excluded it as a BP QTL.42

Third, linkage analysis can be limited by the resolution of QTLs. For example, the region that initially appeared to contain 1 QTL in a broad region on Chr 2 shown by linkage43 actually harbors 3 QTLs,41,44 which were resolved only by congenic strains.

A variation on the same theme as congenic strains is the use of consomic strains.45–48 Instead of a chromosome segment replaced in a congenic strain, a consomic strain engages an entire chromosome. The advantage of a consomic strain is that the BP QTL in question on a chromosome is sure to be "trapped" in the manipulation. The disadvantage is the large size of the area under study. To approach the resolution of a congenic strain, further microdissection is required, but the effectiveness of this strategy is yet to be proven for hypertension research.

Fine Congenic QTL Definition
Ideally, a congenic strain should both lower BP and carry only 1 gene with functionally contrasting alleles in its QTL interval, thus proving that this gene is the QTL in question. This outcome relies on generating crossovers flanking solely 1 gene of interest, because 2 closely linked markers are subjected to the frequency of chromosome exchanges and are constrained by local chromosome configurations. Depending on whether the interval is gene rich and/or located at a crossover hotspot, crossovers flanking only 1 gene can be a challenge. Nevertheless, congenic fine resolution can restrict the number of genes in a QTL interval to a "minimum," that is, 100 to 200 kb.11,12,49

Gene Profiling
Microarray technology has opened up new vistas for analyzing the expression patterns of a large quantity of genes simultaneously.50 Based on this approach, investigators identified a gene responsible for insulin resistance.51 Microarray application to discovering BP QTLs could provide a shortcut to directly and rapidly identify gene candidates with expression differences.52 A critical issue dealing with genes found in this way is to distinguish those impacting on BP functions from those that are irrelevant.53

In gene profiling carried out thus far in hypertension research,12,54–59 2 general types of results have been obtained. First, in comparing congenic to progenitor strains, a number of differentially expressed genes (DEGs) were found, and all of them fell outside the intervals containing BP QTLs identified in congenic strains.12,56,58 Some DEGs were located even on chromosomes that appeared identical between 2 comparing strains. These DEGs could be a compensatory genome response or results of regulations in trans from the genes present in chromosome intervals introgressed in given congenic strains.53

Second, although certain DEGs remain as QTL candidates, their locations defined by congenic strains are still so large that their status needs further verification.55,56,59 One DEG is supported as a candidate BP QTL by linkage only.54 Because linkage analysis does not seem to be sufficient to define a BP QTL region to the submegabase level, its status as a candidate gene for a BP QTL remains provisional.

There are other examples of DEGs that have been proven to be false-positives for BP QTLs. The Sa gene is a case in point. Although it was differentially expressed and linked to BP,60,61 physical definition has excluded it as a QTL, because the congenic strains harboring it failed to demonstrate any BP effect.62,63 In contrast, an adjacent and nonoverlapping congenic strain alone exhibited a BP effect,64,65 indicating that a real BP QTL is near Sa and does not require a combination with other QTLs.

Here, it is important to make a distinction between a BP phenotype and a phenotype represented by the differential expression of a gene, that is, the difference between a BP QTL and an expression QTL.66 Expression QTLs are regulated by elements in cis and trans,66 but BP QTLs can function independently of trans elements (Table 1). Although a phenotype determined by an expression QTL may be shown only in combination with a regulatory element,66 BP determined by QTLs does not require such a combination (Table 1). Thus, a gene shown by a congenic strain as lacking a BP effect is definitely ruled out as a candidate gene for a BP QTL, whereas the same gene can still be a candidate for an expression QTL determining a phenotype other than BP.

Therefore, DEGs must be functionally verified in vivo to truly have effect on BP. The use of heterozygotes may be a means of performing functional tests on DEGs.38 If a DEG is a QTL, a heterozygote is expected to produce a BP intermediate between 2 homozygotes, unless the DEG is the result of a null allele. Other genetic verification approaches include fine congenic QTL definition and transgenesis.

Transgenesis
This technology can be potentially used for validating the function(s) of a gene that is expressed at different levels and testing a phenotype specified by a segment of DNA.

The transgenic strategy has been used, as a complement to fine congenic resolution, to authenticate QTLs in the rat in ≥2 instances, that is, the rescue of insulin resistance by a Cd36 transgene67 and the rescue of susceptibility to type 1 diabetes mellitus.68 To verify whether a gene is a BP QTL, it could be expressed at a lower level in animals with low (higher) BP, and, when overexpressed in transgenesis, it should cause higher (lower) BP.

There are several important considerations in adapting this approach to BP QTL discovery. First, because a majority of genes are expressed in a tissue-specific manner, a transgene composed of a full cDNA construct may lack a tissue-specific regulatory element, and, thus, it may not be suitable for making appropriate transgenics. In its place, overexpression of a candidate gene contained in a rat bacterial clone (BAC) is promising. The BAC likely contains tissue-specific genomic element(s) required for regulating gene expression. Resulting from the rat genome project, BACs were generated from the genome of Brown Norway rats.69 Before selecting an appropriate BAC, it is important to determine the identity of the gene allele between the Brown Norway rat and donor strains. The drawback in introducing a transgene by BAC is its large size. The efficiency of its incorporation into the host genome is expected to be low.

Second, the choice of the recipient strain is crucial in that it should express the tested gene at a low level. For generating DSS-based transgenics, either DSS or a congenic strain harboring the BP QTL in question can be used. If the level of gene expression is higher in the DSS strain than in the congenic strain, the transgene should be introduced into the congenic strain and vice versa. In addition to validating a DEG, there is another class of genes to be authenticated. They are the genes that contain mutations that potentially change the function of gene products that they encode. Because most of the BP QTL alleles in DSS rats may cause a loss of function,38 the dominant allele of such a QTL can be transgenically introduced into the recipient strain that carries the recessive allele. Because of full dominance, a phenotypic rescue is expected to be achieved.

Transgenesis has 3 inherent drawbacks to be considered: random site(s) of insertion into the host genome, varying copy numbers (from 1 to many), and dosage effects.70 A transgene usually gets integrated into the host genome in an unspecified location and in tandem copies. Although transgenes can be targeted by chimerical promoters to specified tissues,71 methodologies have yet to be developed for controlling how much a transgene can be expressed. Moreover, integration of a piece of DNA into the host genome could disrupt its function at the site of insertion and/or introduce unwanted positional effects on genes adjacent to, and even distant from, the site of insertion.72 The dosage effect may also lead to posttranscriptional gene silencing.73 To overcome these drawbacks, multiple transgenic lines have to be studied for 1 transgene.

Gene Targeting
Affirmative proof of a candidate gene as a BP QTL may require that a specific mutation(s) be replaced (ie, a knock-in) or eliminated (ie, a knockout) in vivo. Although homologous recombination could not yet be achieved for the rat, the mouse can conceivably be used to meet this objective. The homologous mouse sequence for a candidate BP QTL can be identified, and a specific mutation(s) can be knocked in or out of the mouse by gene targeting. Nevertheless, a recent breakthrough in somatic nuclear transfer28 opens new possibilities by which gene knockouts and knock-ins will be facilitated in the rat.

Interference RNAs
MicroRNAs (miRNAs) are a class of RNA molecules that do not encode protein products and appear to be involved in the regulation of expression of other genes with which they share sequence similarity.74 Their capability in silencing the expressions of other genes offers 2 research potentials: as a powerful tool in gene regulation technology (ie, small interference RNA) and as possible candidates for genes, including QTLs.

Interference RNAs could serve to test the function of almost any gene of interest or a QTL candidate in cell culture.71,75,76 They are also potentially useful to silence gene expression in mice and rats by transgenics.77 Moreover, miRNAs can have physiological functions in vivo. Both developmental fates78 and mammalian cell differentiation79 have been shown to be affected by miRNAs. It is also possible that an miRNA gene might be a BP QTL. As the rat genome sequence becomes available,27 miRNAs present in rat candidate QTL intervals should gain importance. Because BP QTLs behave in the Mendelian fashion in full and partial dominance,38 any miRNA, if suspected to be a candidate, has to follow this mode of inheritance as well.

Mutation Screenings for Candidate Genes
It is informative to detect mutations that have potential functional consequences in all of the genes present in a QTL interval, without any functional bias, for example, renal versus cardiovascular functions. The genes chosen for mutation screenings are those that are either known genes or Locs residing in a QTL interval (Table 1). Several methods can ensure that Locs are genuine genes and not pseudogenes. Their expressions can be verified by hybridization on a multitissue Northern blot (ie, a commercially available membrane with RNAs extracted from several representative organs) or by real-time RT-PCRs. It is only after this verification that a detailed study of a Loc can be successfully undertaken. This requires, first, cloning of the entire cDNA sequence and regulatory regions from strains that have functionally contrasting alleles for the BP QTL in question (eg, DSS and Lewis). Second, their sequences must be compared with reveal significant mutations.

Other Genetic Approaches
Linkage followed by the use of congenic strains in the QTL definition may not be the only approach to QTL identifications. Ethylnitrosourea mutagenesis provides another means of achieving gene identification.80 Nevertheless, so far, ethylnitrosourea mutagenesis has not uncovered genes underlying hypertension. Therefore, the potential of ethylnitrosourea mutagenesis has yet to be realized for gene identification in hypertension.

Perspectives
Studies using animal models have provided valuable insights into probable genetic architecture, QTL–QTL interactions, and genome modulations in polygenic hypertension. BP QTL discovery remains a challenge that must incorporate mutational screening with functional verification in vivo. It appears that a majority of BP QTLs may represent either unidentified genes or genes with previously unknown functions in BP control. The payoff seems considerable, because their discovery will probably lead to novel antihypertension targets, even for humans.


*    Acknowledgments
 
Sources of Funding

This work was supported by grants from the Canadian Institutes of Health Research.

Disclosures

None.

Received December 7, 2006; first decision January 10, 2007; accepted January 18, 2007.


*    References
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 *References
 

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