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Hypertension. 1995;25:1121-1128

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(Hypertension. 1995;25:1121-1128.)
© 1995 American Heart Association, Inc.

Articles

Detection and Positional Cloning of Blood Pressure Quantitative Trait Loci: Is It Possible?

Identifying the Genes for Genetic Hypertension

John P. Rapp; Alan Y. Deng

From the Department of Physiology and Molecular Medicine, Medical College of Ohio, Toledo.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowLinkage Analysis: Candidate...
 down arrowPopulation Specificity in QTL...
 down arrowFine Mapping of BP...
 down arrowPositional Cloning
 down arrowConclusions
 down arrowReferences
 
Abstract Identification of the quantitative trait loci that influence blood pressure and cause genetic hypertension is a major challenge. Several genetically hypertensive rat strains exist and can be used to locate by linkage analysis broad chromosomal regions containing blood pressure quantitative trait loci. Such broad chromosomal regions, and then narrower subregions, can be moved among strains (ie, production of congenic strains and congenic substrains) to identify small chromosomal regions containing the blood pressure quantitative trait loci. However, ultimate positional cloning of the quantitative trait loci presents a major difficulty because the genetic variants involved are likely to result in subtle changes in function rather than the blatant loss of function characteristic of all mendelian disease genes discovered so far by positional cloning.


Key Words: genes • rats, inbred strains • rats, inbred SHR • hypertension, genetic


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowLinkage Analysis: Candidate...
 down arrowPopulation Specificity in QTL...
 down arrowFine Mapping of BP...
 down arrowPositional Cloning
 down arrowConclusions
 down arrowReferences
 
Many human genetic diseases, such as cystic fibrosis or Duchenne's muscular dystrophy, are caused by mutations at a single genetic locus that result in major disruption in function of the gene product. On the other hand, some of the more common human afflictions, such as hypertension, atherosclerosis, diabetes mellitus, and mental disorders, are likely to be influenced more subtly by multiple genetic loci. When appropriate phenotypes are available in animals, animal models of human disease provide an opportunity for gaining fundamental insight into genetic causes of the disease.

Blood pressure (BP) in humans and animals is well known to be a quantitative trait under polygenic control.1 Essential hypertension (high BP with no obvious cause) has an important genetic component1 2 and afflicts 15% to 20% of the human population.3 In the 1960s and 1970s several investigators developed animal models of hypertension by selectively breeding rats4 5 6 7 8 9 and mice10 for increased and in some cases decreased BP.

An unidentified genetic locus influencing a quantitative genetic trait such as BP is called a quantitative trait locus (QTL). Presumably, many QTLs are scattered throughout the genome. This article emphasizes the synthesis of an overall strategy for detection and identification of BP QTL by (1) identification by linkage analysis of large chromosomal regions containing a QTL, (2) production of congenic strains for isolating each QTL in a separate strain, (3) fine genetic mapping of each QTL by a series of congenic substrains containing progressively smaller chromosomal regions, (4) biochemical/physiological identification of functional changes in the congenic strains, and (5) cloning of candidate genes from the appropriate small chromosomal regions identified in a congenic substrain to search for functional genetic variants.


*    Linkage Analysis: Candidate Genes Versus Genome Scanning
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Linkage Analysis: Candidate...
down arrowPopulation Specificity in QTL...
 down arrowFine Mapping of BP...
 down arrowPositional Cloning
 down arrowConclusions
 down arrowReferences
 
The main tool for identifying the effects of individual BP QTL is linkage analysis.11 12 13 Typically, a hypertensive and normotensive rat strain are crossed to produce F1 progeny that are then crossed (F1xF1) to produce a segregating F2 population. In the F2 population alleles at a marker locus identical to, or closely linked to, a QTL must remain associated with a BP component. Alleles at a marker locus not genetically linked to a QTL will segregate independently of the QTL, and there will be no statistical association of the marker genotypes with BP.

There are two approaches to identifying chromosomal regions genetically linked with BP: the candidate gene approach and random genome scanning. As will be seen, the two methods basically are converging to be the same approach in genetic hypertension and only represent initial screening procedures that direct more precise studies.

The candidate gene approach draws on the wealth of biochemical and physiological data on how BP is regulated through the nervous, endocrine, and cardiovascular-renal systems. It makes sense to examine genes in known pathways controlling BP for cosegregation with BP. The objective is to find which components of these biochemical/physiological systems harbor genetic variants causing quantitatively important genetic variation in BP.

For example, previously we found that alleles at the candidate locus for atrial natriuretic peptide receptor A (also called guanylyl cyclase A, GCA) cosegregated (P=.0027) with BP in an F2 population derived from inbred Dahl salt-sensitive hypertensive (SS/Jr) rats and Wistar-Kyoto rats (WKY)14 ; the population is designated F2(SS/JrxWKY). The critical question arises, is it really the candidate GCA gene causing differences in BP, or is it a linked gene? Chromosomal mapping studies are somewhat helpful in providing an answer.

Fig 1 shows a reasonably detailed map of the region of rat chromosome 2 containing GCA, along with the results of linkage analysis of BP at each locus. The data illustrate the principle that the quantitative effect of a QTL associated with a marker locus decreases (because of chromosomal crossing over) as the distance between the QTL and the marker increases. The exact relationship15 is that the effect decreases as a function of (1-2r), where r is the recombination fraction (proportion of individuals with recombination caused by chromosomal crossing over between the marker and the QTL). As seen in Fig 1, GCA lies at the edge of the chromosomal region likely to contain the actual QTL. Statistically, the most probable location for the QTL is between Na+,K+-ATPase {alpha}1 isoform (NAK{alpha}1) and calmodulin-dependent protein kinase II-delta (CAMK) based on the maximal logarithm of the odds (LOD) score obtained from the MAPMAKER/QTL program16 17 and similar data from an additional F2 population.18 Although GCA is still a good candidate, NAK{alpha}1 is statistically better. It is also noteworthy that in just the region of rat chromosome 2 mapped in Fig 1, there are five loci (angiotensin type 1B receptor [AT1B], neutral endopeptidase/enkaphalinase [NEP], GCA, NAK{alpha}1, CAMK) that could be rationalized as candidate loci for effects on BP. Our experience is that this region is not unique; rather, many chromosomal regions we have examined that cosegregate with BP contain multiple candidates. This is bound to be the case because there are so many candidates.



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  		Figure 1. Diagram shows rat chromosome 2 linkage map and blood pressure quantitative trait locus (QTL) localization for an F2 population derived from Dahl salt-sensitive (SS/Jr) and Wistar-Kyoto rats (WKY) raised on a high salt (8% NaCl) diet. The distances between markers on the left side of the linkage map are in centimorgans, with the Haldane correction. Blood pressure effect is the difference in blood pressure between rats homozygous for the SS/Jr allele and rats homozygous for the WKY allele at each locus. The probability from one-way ANOVA tests whether blood pressure differs significantly among the three genotypes (homozygous for SS/Jr allele, heterozygous, homozygous for WKY allele) segregating at each locus. The bar at the right shows the location of the blood pressure QTL predicted by the MAPMAKER/QTL program16 17 ; the arrowhead indicates the position of maximal LOD score of 3.42; the heavy bar is ±1 LOD unit and the thin bar is ±2 LOD units. Data are from Deng et al.18 D2N35 indicates an anonymous locus; CAMK, calmodulin-dependent protein kinase II-delta; NAK{alpha}1, Na+,K+-ATPase {alpha}1; HSD, 3ß-hydroxysteroid dehydrogenase/delta 5 isomerase; GCA, guanylyl cyclase A/atrial natriuretic peptide receptor A; FGG, fibrinogen gamma; NEP, neutral endopeptidase/enkephalinase (EC 3.4.24.11); CPB, carboxy peptidase B; AT1B angiotensin II type 1B receptor; and PRLR, prolactin receptor.

Thus, in the absence of other compelling data, merely finding a candidate that cosegregates with BP only identifies a chromosomal region that is genetically linked with BP. Additional data can have an effect on the interpretation given to the candidate. For example, alleles at the renin locus in segregating populations derived from SS/Jr and Dahl salt-resistant (SR/Jr) rats cosegregate with BP.19 20 21 An adequate map of the renin region on rat chromosome 13 is only beginning to emerge.22 It is unclear, however, whether the renin locus is actually causing the BP difference, because sequencing of SS/Jr and SR/Jr renin alleles revealed no differences in the coding or immediate 5'-regulatory region.23 Thus, the data at present do not establish renin as "the" QTL on chromosome 13, although the data do support the existence of a QTL on chromosome 13 in the region of renin.

An example of a possibly successful candidate gene is steroid 11ß-hydroxylase,24 which cosegregates with a modest component of BP in Dahl rats.25 26 Base changes in the coding region give rise to amino acid changes,26 27 and the amino acid changes at positions 381, 384, or both are likely to account for the known altered steroidogenic pattern.27 This pattern is highly characteristic, is inherited as a mendelian trait,25 and results in excess production of 18-hydroxydeoxycorticosterone, which could account for the associated BP changes.28

The other approach to identifying the genes regulating BP is random genome scanning with DNA markers scattered throughout the genome. Two groups29 30 have done such a genome scan for BP using the same F2 population derived from stroke-prone spontaneously hypertensive rats and WKY. The strongest BP effect in this population was seen with markers on rat chromosome 10. The markers were near the angiotensin-converting enzyme (ACE) locus, and ACE is a logical candidate. But because candidates are so numerous, some known candidate is almost always likely to be in a region cosegregating with BP.

Obviously, the result that a region on chromosome 10 cosegregates with BP and contains a candidate gene is analogous to the result that a region on chromosome 2 cosegregates with BP and contains a candidate gene, even though the results were arrived at by different approaches (candidate gene versus genome scanning). If enough candidate genes are tested, the data take on the properties of a genome scan. Since there are also presumably many QTLs, evaluation of enough candidates eventually will lead to a QTL region. For example, in evaluating five loci of the endothelin system, two chromosomal regions cosegregating with BP on rat chromosomes 5 and 17 were found.31

When BP QTLs are being sought by either the candidate gene approach or genome scanning, numerous loci will be evaluated. If the probability for significance of linkage of loci to BP is too high (eg, P<=.05), many false positives will be obtained. Thus, in general, such experiments should set a significance level in the range of P<=.001,17 and more recently it has been argued that the significance level should be, for example, set in the range of 5x10-5 for a genome the size of the mouse and using F2 populations.32 In contrast, many articles using the single candidate gene approach accept much less stringent criteria.33 34 35 Accepting a criterion less stringent than P<=.001 in the absence of other supporting data is ultimately a poor procedure. Increasing the size of the study population to improve statistical power is obviously useful if it is feasible.


*    Population Specificity in QTL Detection
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowLinkage Analysis: Candidate...
 *Population Specificity in QTL...
down arrowFine Mapping of BP...
 down arrowPositional Cloning
 down arrowConclusions
 down arrowReferences
 
There are six well-known inbred rat strains selectively bred for hypertension from outbred stock. These hypertensive strains also have normotensive contrasting strains developed concomitantly by selection for low BP or developed as an afterthought from the original outbred stock. These hypertensive strains are genetically hypertensive rats,4 SS/Jr rats,5 36 spontaneously hypertensive rats (SHR),6 Sabra hypertensive rats,7 Lyon hypertensive rats,8 and the Milan hypertensive strain.9

Various inbred strains are likely to have genetically fixed (ie, be homozygous for) different subsets of QTL alleles causing BP differences. Thus, it has been our expectation as well as our observation that the detection of the effect of a given QTL is highly dependent on the two strains (usually a hypertensive strain and a normotensive strain) initially crossed to produce a segregating population. This cross-specific phenomenon has been observed for chromosomal regions marked by the anonymous SA locus on rat chromosome 1,37 the GCA locus on rat chromosome 2,14 the endothelin-3 locus on rat chromosome 3,31 38 the endothelin-2 locus on rat chromosome 5,31 the ACE locus on rat chromosome 10,14 and the renin locus on rat chromosome 13.21

Some of the complexity involved in causing the strain specificity of QTL detection in different crosses is illustrated in Fig 2. Consider a marker locus, M, with alleles M1 and M2, and a QTL, with alleles QTL1 and QTL2, that do alter BP. The M locus and the QTL are on the same chromosome and close enough such that crossovers between them are minimal. The marker and QTL alleles can be coupled differently into four haplotypes shown in Fig 2, each haplotype being homozygous in each of four different inbred strains that are to be used as parental strains in the production of segregating populations. Assume that only the M locus can be genotyped and is used to make inferences about the existence of the BP QTL linked to it. There are six ways to cross the four parental strains. Each pairwise cross is represented by a vertical line on the right side of Fig 2. Of the six crosses, two are not informative because the marker is not variant, two are not informative because the QTL is not variant, and two are informative because both the marker and QTL are variant.



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  		Figure 2. Diagram shows possible outcomes of cosegregation analysis between blood pressure and a marker locus linked to a quantitative trait locus (QTL) that actually does influence blood pressure. Four different chromosomal arrangements (haplotypes) of two marker alleles and two QTL alleles are shown, and each haplotype is assumed to be carried in the homozygous state in a different inbred strain. Strains are crossed pairwise (shown by brackets at the right), and F2 populations are produced from the cross. The diagram indicates in which pairwise combinations the marker is informative and will allow a cosegregation test between the marker and blood pressure and in which pairwise combinations the marker is not informative, preventing such analysis. In the four crosses in which the marker is informative, two will yield positive cosegregation of the marker with blood pressure, and two will not, depending on whether different QTL alleles are present in the cross. See text for detailed explanation.

Consider crossing strains 1 and 3 of Fig 2. No information about the existence of the QTL is obtained by genotyping at the M locus in segregating populations derived from this cross because both strains 1 and 3 carry the same marker allele and segregation of the chromosomal region cannot be followed (ie, the marker is not informative). This is despite the fact that a QTL with contrasting alleles exists and is in fact segregating and altering BP in the experimental population. Similarly, crossing strains 2 and 4 gives no information about the QTL because the marker is not informative.

In segregating populations derived from strains 1 and 2, it will be possible to follow the chromosomal region of interest because the marker alleles are variant. However, the same QTL allele is present in both strains, and the QTL will not contribute to genetic variation in BP in the experiment. Thus, the marker does not cosegregate with BP despite the fact that it is closely linked to the QTL. Similarly, a cross of strains 3 and 4 does not detect the presence of the QTL.

Finally, segregating populations derived from a cross of strains 1 and 4 detects the presence of the QTL because (1) the marker locus is informative because contrasting marker alleles are present, and (2) contrasting QTL alleles that alter BP are coupled to the marker alleles. Thus, the marker locus will cosegregate with BP. Note that if QTL1 is a minus allele (ie, it reduces BP) and QTL2 is a plus allele (ie, it increases BP), then M1 will cosegregate with lower BP and M2 will cosegregate with increased BP because M1 is coupled on the same chromosome to QTL1, and M2 is coupled to QTL2.

A segregating population derived from strains 2 and 3 will similarly yield a positive cosegregation of the marker locus with BP because here also the marker and the QTL are both informative. Note, however, that in this cross the M1 allele is coupled to QTL2, and M2 is coupled to QTL1; thus, M1 will cosegregate with increased BP and M2 with lower BP. This is just the opposite of the case in which strains 1 and 4 were crossed.

An example of these complexities is provided by the renin locus that cosegregates with BP in an F2 population derived from either SS/Jr and SR/Jr rats, ie, F2(SS/JrxSR/Jr),19 20 21 or in an F2 population derived from Lyon hypertensive (LH) and Lyon normotensive (LN) rats, ie, F2(LHxLN).39 Evidence suggests that one renin allele (r allele) is present in both the SR/Jr and LH strains and that a different renin allele (s allele) is present in both the SS/Jr and LN strains.21 In the F2(SS/JrxSR/Jr) population the r allele cosegregates with reduced and the s allele with increased BP,19 20 21 whereas in the F2(LHxLN) population the r allele cosegregates with increased and the s allele with reduced BP.21 39 This is comprehensible if the renin locus is acting as a marker for an unknown linked QTL and the coupling between renin alleles and QTL alleles is reversed in the two pairs of strains.

The actual situation is more complex than that shown in Fig 2. There can be multiple alleles (not just two) at the marker locus, and multiple functionally different alleles (not just two) at the QTL. Also the genetic background is different in each cross involving two inbred strains. There is evidence for a strong effect of genetic background on the expression of the BP effect of the 11ß-hydroxylase locus,26 the chromosome 3 region marked by endothelin-3,38 and the chromosome 13 region marked by renin20 in crosses of SS/Jr and SR/Jr rats. There is also evidence for an interaction between the QTLs on chromosomes 2 and 10 in an F2 population derived from SS/Jr and the Milan normotensive strain.14

Of course another reason two populations may yield different results for BP linkage to genetic markers is simply that one population may be a false positive (or false negative). Further data in the form of congenic strain development (see below) can help establish or refute the existence of a QTL.


*    Fine Mapping of BP QTL
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowLinkage Analysis: Candidate...
 up arrowPopulation Specificity in QTL...
 *Fine Mapping of BP...
down arrowPositional Cloning
 down arrowConclusions
 down arrowReferences
 
For identification of the DNA structure of an unknown disease-causing locus through linkage and ultimately DNA cloning, so-called positional cloning,40 41 the locus has to be localized to within a 1- to 2-centimorgan (cM) region (1 cM equals approximately 106 bp). This is a major challenge for QTL identification. Using simulated data for QTL of modest effect, in experiments with large numbers, and using an infinite number of markers, Darvasi et al42 have shown that confidence intervals for QTL map location are approximately 10 cM. It does, however, still seem possible that for QTL with favorably large effect, better localization will be obtained. In practice, existing data for BP QTL have so far provided localization only in the range of 20 to 30 cM. It is this difficulty of QTL localization that drives the following paradigm.

A complementary approach to interval mapping for QTL localization is fine genetic mapping by the use of congenic strains. This approach requires a dense (1 to 2 cM) genetic map of the chromosomal region of interest and requires that the markers in the map be variant between the parental donor and recipient strains used to produce the congenic strains.

The use of congenic strains was originally conceived by Snell43 in studying histocompatibility phenomena and has been widely used in the study of genetic problems.44 45 In the usual procedure, one allele at a locus is moved from one inbred strain (donor) to the genetic background of another inbred strain (recipient) by a series of (at least eight) backcrosses. For example, consider a marker locus M with two alleles M1 and M2, where the donor rat strain is homozygous M1M1 and the recipient strain is homozygous M2M2. Fig 3 illustrates the procedure for producing a congenic strain in which the M1 allele of the donor strain replaces the M2 allele of the recipient strain. Donor and recipient strains are crossed to produce F1 heterozygotes, genotype M1M2. F1 are crossed with the recipient strain to produce the first backcross (BC1) population in which the marker locus will be segregating one homozygote M2M2 to one heterozygote M1M2. Heterozygotes at the marker locus are selected and backcrossed again to the recipient strain to produce the second backcross (BC2). After eight or more backcrosses with selection of heterozygotes at the marker locus as breeders, the genetic background of the new line is greater than 99% that of the recipient strain, but the selected locus, M, is still segregating. Note in Fig 3 that the percentage of the recipient rat genome increases with each backcross. This is because at each backcross, loci not linked to M have a 50% chance of fixing the recipient allele in the homozygous state. After the eighth backcross it is then a simple matter to breed two heterozygotes, M1M2, to each other. The offspring will segregate 1 M1M1: 2 M1M2: 1 M2M2. Two homozygotes, M1M1, for the donor M1 allele are selected and bred to fix the M1 allele in the homozygous M1M1 state now on the recipient genetic background.



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  		Figure 3. Diagram shows breeding scheme for production of a congenic strain from two inbred strains. The M1 allele of the donor strain replaces the M2 allele of the recipient strain at locus M. The genetic background of the donor strain is represented by a clear symbol; the genetic background of the recipient strain by a black symbol. Increasing shades of gray represent the progressive increase in genetic background attributable to the recipient strain. See text for detailed explanation. BC indicates backcross. The figure is derived from Klein.45

It is theoretically possible to expedite the production of a congenic strain by selecting for the recipient alleles at many marker loci scattered throughout the genome concomitant with selection of the donor allele at the locus of interest. This procedure is most effective at the BC1 and BC2 generations.46 The recent publication47 of an essentially complete, first-generation map of the rat genome expedites this procedure for the rat. According to Lander and Schork32 it should be possible to produce a congenic strain in three or four generations with this approach.

In producing a congenic strain by selecting on a single marker locus, what is really transferred to the recipient is the donor allele plus flanking DNA at the locus under selection. This flanking DNA is on average 100/N cM on either side of the locus,48 where N is the number of backcrosses. If the marker locus selected is acting as a surrogate for an unknown QTL, then the congenic strain may or may not contain the desired QTL allele, depending on whether a crossover had occurred between the marker and the QTL during the production of the congenic strain (see Fig 4). If the BP of the congenic strain (carrying the donor marker allele) differs (in the expected direction based on cosegregation data) from that of the parental recipient strain, then the desired QTL allele was transferred along with the marker allele. If the two strains have the same BP, then the desired QTL allele was not transferred with the marker allele.



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  		Figure 4. Diagram shows two possible genetic configurations obtainable in constructing a congenic strain when selecting at a single marker locus as a surrogate for a quantitative trait locus (QTL). The M1 allele at the marker locus M is coupled to the QTL1 allele at the QTL. Selection is made only at the M locus. If no crossover occurs between M and QTL, the donor QTL1 allele is introgressed into the recipient strain along with the donor M1 allele (top). If a crossover between M and QTL occurs in a backcross animal selected for breeding in production of the congenic strain, then the M1 allele is transferred, but the QTL1 allele is lost and the allele at the QTL will be that of the recipient strain, denoted QTL2 (bottom).

The probability of successfully transferring the desired QTL allele at each generation of backcrossing, given selection for a linked marker locus, is (1-r), where r is the recombination fraction between the QTL and the marker locus. The probability of successfully selecting for the QTL for N backcrosses is (1-r)N. Thus, if r=0.1 (or approximately 10 cM between the marker and the QTL), the probability of capturing the desired QTL allele in a congenic strain constructed with eight backcrosses is .43.

The ambiguities involved in creating congenic strains for a QTL with the use of a single linked marker locus render the procedure rather unsatisfactory. If an adequate map is available, it is possible to type and simultaneously select for markers over a region larger than the confidence interval for the QTL at each backcross and thus avoid using as breeders those rats with crossovers in the region of interest. This will markedly improve the chances of obtaining the desired QTL allele in the congenic strain.

The successful production of a congenic strain is required to prove that a BP QTL actually does exist in a specific region of a chromosome. This will remove the inherent ambiguity involving the statistical nature of the QTL localization. It is noted that congenic strains can be constructed in two directions: (1) transferring a plus allele (which increases BP) from a hypertensive to a normotensive strain and looking for an increment of pressure, and (2) transferring a minus allele (which decreases BP) from a normotensive to a hypertensive strain and looking for a decrement in BP. Apparent examples of congenic strains in which a BP effect was seen are (1) the transfer of a region of chromosome 20 containing the RT1 (major histocompatibility) complex and heat-shock protein 70 alleles from Brown Norway rats to SHR,49 (2) transfer of the SHR RT1 allele to Lewis rats,50 and (3) transfer of the SHR Y chromosome onto the WKY background.51

Fig 5 shows the hypothetical results for a congenic strain and the concept of fine genetic substitution mapping for the QTL localization that can proceed from such a strain. In the example, it is assumed that a relatively large chromosomal region from a normotensive strain defined by markers A through H was substituted into the genetic background of SS/Jr rats (congenic strain 1 of Fig 5) and that the substituted QTL minus allele decreased BP 20 mm Hg in the homozygous state. Once it is established that congenic strain 1 of Fig 5 has a lower BP than SS/Jr and therefore has actually trapped a QTL allele, it is then possible for congenic substrains to be made. Strain 1 is crossed to SS/Jr, and the offspring are backcrossed to SS/Jr, producing a large population, genotyping at markers A through H and selecting rats with crossovers in various places throughout the region. Congenic substrains 2 through 7 are then produced from such crossover rats. Of course, each selected crossover chromosome has to be fixed by again backcrossing the rat carrying this chromosome to SS/Jr (to replicate the chromosome) and selecting and crossing males and females heterozygous for the crossover chromosome. Fewer than one of four of the offspring will be homozygous for the desired crossover chromosome (depending on the size of the desired region and additional unwanted crossing over), and these homozygotes are bred to fix the desired crossover chromosome in the congenic substrain.



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  		Figure 5. Diagram shows substitution mapping of a quantitative trait locus (QTL) for blood pressure with the use of a set of congenic strains. The thick line represents a chromosome from the donor normotensive strain, and the thin line, a chromosome from the recipient hypertensive strain (assumed to be Dahl salt-sensitive rats [SS/Jr]). A map of informative markers (A to H) is assumed to be known along a chromosomal region thought to contain a blood pressure QTL. In the example, the QTL is located between markers D and E as indicated by the arrow. Strain 1 is a congenic strain in which the entire chromosomal region (A to H) has been substituted into the recipient SS/Jr strain from the donor strain. This substituted region contains a minus QTL allele different from the plus QTL allele present in the recipient SS/Jr strain, and thus the substitution lowers blood pressure of strain 1 relative to SS/Jr. Strains 2 through 6 are congenic substrains carrying various donor fragments as indicated. If the donor fragment contains the QTL, then the congenic substrain carrying it will have its blood pressure lowered. If the QTL is absent in the donor fragment, the congenic strain will have the same blood pressure level as the parental recipient SS/Jr strain. This information on the presence or absence of a blood pressure effect, combined with the location of the substituted chromosomal fragment, can be used to map the blood pressure QTL to a smaller region. See text for detailed explanation.

In Fig 5 the hypothetical QTL is between markers D and E. As the substituted chromosome in congenic substrains 2 through 7 crosses the QTL, there is a step change in BP among strains as shown at the right of Fig 5. This information allows one to considerably narrow down the region containing the QTL. The resolution will depend on the density of the markers on the map and the number of rats used to search for crossovers in appropriate regions. It should be possible to localize the QTL to a region 1 to 2 cM in size and to produce a congenic substrain containing just this small region. Obviously, the method depends on accurate comparison of BP among the recipient strain, the congenic strain, and the congenic substrains of Fig 5. Power calculations52 suggest that the smallest QTL effect (difference between homozygotes) that we can detect in our own work is approximately 15 mm Hg. In most cases, the QTL effect detected (in F2 or on backcross to SS/Jr genetic backgrounds) was at least 20 mm Hg.14 18 19 20 21 25 26 31 38 Thus, we expect that in most cases we need only detect a difference of 20 mm Hg among the strains in Fig 5.

The basic concept of fine genetic mapping is analogous to that of deletion mapping pioneered by Bridges53 and perfected by Benzer54 and used in mapping human genes for muscular dystrophy55 and male-determining function of the Y chromosome.56 In deletion mapping the loss of a chromosomal region is associated with a change in phenotype. The present proposal, however, is more related to substitution mapping, which is useful in agriculture for fine mapping of QTLs known to be in specific chromosomal regions.57

Although the genetic models for hypertension are highly developed in the rat, the genetic map of the rat47 58 59 60 is far behind that of the mouse.61 A map such as shown in Fig 1 is adequate for defining the approximate location of a QTL and for use in construction of an initial congenic strain containing a large chromosomal region to include the QTL, but it is clearly not dense enough for fine mapping of a QTL by subsequent construction of congenic substrains, which require a map with markers 1 to 2 cM apart. It is, however, just a matter of time until the rat genetic map can be developed adequately.

There are methods for obtaining genetic markers in a chromosomal region that differs between two strains, eg, a congenic strain and the parental recipient strain. One method62 63 involves subtractive and kinetic enrichment with the use of the polymerase chain reaction to obtain restriction endonuclease fragments present in one source of DNA but not present in the other. The method was used successfully to obtain probes in an interval of less than 1 cM of mouse chromosome 11 near the nude locus.63 This technique could be used to improve the density of markers in a given QTL-containing region following the construction of an initial congenic strain. Congenic substrains with smaller and smaller QTL-containing regions could then be bred based on the improved map.


*    Positional Cloning
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowLinkage Analysis: Candidate...
 up arrowPopulation Specificity in QTL...
 up arrowFine Mapping of BP...
 *Positional Cloning
down arrowConclusions
 down arrowReferences
 
BP QTL are being found by linkage analysis and can presumably be mapped to a chromosomal region of 1 to 2 cM as suggested above. As outlined by Collins,40 a physical map of such a region is then obtained by a series of overlapping yeast artificial chromosomes (YACs) from a YAC library.64 YAC libraries exist for humans and mice but not for rats. Development of a rat YAC genomic library or a rat bacterial artificial chromosome (BAC) library,65 66 67 like a denser rat linkage map, is expected in the near future. Thus, there is a reason to expect that a 1- to 2-cM region containing a BP QTL identified by fine mapping in congenic strains could subsequently be available in the form of overlapping YACs or BACs.

The next step in positional cloning is to identify all the genes in the YACs or BACs covering the region of interest by identifying regions that are transcribed to produce mRNAs. Methods for doing this are varied and challenging and are listed by Collins40 including such methods as exon amplification.68 One can expect that a region of 1 to 2 cM will yield roughly 50 mRNAs.

At this point a polygenic trait such as BP presents formidable obstacles to definitive QTL identification. In the case of positional cloning of monogenic disease traits, there is usually good reason to expect the disease-causing locus to be expressed in a certain tissue. This narrows down considerably the number of candidate mRNAs to those expressed in the tissue of interest. In BP regulation an unknown QTL could be expressed in any of several obvious organ systems (cardiovascular, renal, nervous, and several endocrine systems) but could also be more obscure (eg, angiotensinogen is produced mainly in the liver). A priori there is no reason to choose one candidate tissue over another.

Once a congenic substrain with a small region known to contain a QTL is obtained, it may be useful to compare this strain with the parental recipient strain at the physiological/biochemical level to get some idea as to which organ system may be different. Such information could guide the screening of candidate mRNAs in appropriate tissues.

For more than two decades research in genetic hypertension using rats focused on comparing hypertensive and control strains at the physiological/biochemical level with little inclination toward further genetic analysis. This approach had no chance of identifying the loci influencing BP because it did not separate strain differences caused by selection for the BP QTLs from strain differences caused by genetic drift (chance selection and fixation of alternate alleles at a locus) or from strain differences arising as a consequence of BP differences. In the case of a congenic substrain compared with the parental recipient strain, the genetic differences between strains are likely to be more easily interpreted because they are limited by the small region of chromosome transferred to the congenic strain from the donor.

The real impediment to identification of BP QTL by positional cloning is likely to be the inability to readily recognize as important a change in a mRNA or regulatory sequence by sequence analysis alone. In many monogenic diseases the gene function is drastically compromised or eliminated because of deletions, frame shifts, addition of stop codons, etc. We do not necessarily expect this to be the case for BP QTL. Rather it is more likely that gene/protein function will be altered in such a way as to create more subtle quantitative differences in function.

If one has identified a candidate QTL and a candidate "lesion" in the QTL, the ultimate test would be to create the lesion in a transgenic rat by homologous recombination and demonstrate a BP effect. This also is a major barrier because at present, methodology for homologous recombination exists for the mouse but not for the rat.


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowLinkage Analysis: Candidate...
 up arrowPopulation Specificity in QTL...
 up arrowFine Mapping of BP...
 up arrowPositional Cloning
 *Conclusions
down arrowReferences
 
There is no doubt that the major BP QTL in the rat can be detected and localized on rat chromosomes to within a few centimorgans with the use of existing methodology. Markers in such a region may prove useful in at least identifying homologous chromosomal regions harboring BP QTL in humans, and thus such localization in rats appears worth considerable research effort. The actual identification of the function of each QTL is highly desirable in order to understand the disease mechanisms in hypertension. The main barrier to achieving this goal is that in general very little information will be available to help direct identification of DNA variants that result in changes in the amount of protein product produced, or in protein products with subtle quantitative functional alterations, as opposed to blatant aberrations or complete loss of function.


*    Acknowledgments
 
This work was supported by research grants from the National Institutes of Health and from Helen and Harold McMaster.


*    Footnotes
 
Reprint requests to John P. Rapp, DVM, PhD, Department of Physiology and Molecular Medicine, Medical College of Ohio, PO Box 10008, Toledo, OH 43699.

Received November 25, 1994; first decision December 16, 1994; accepted February 16, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowLinkage Analysis: Candidate...
 up arrowPopulation Specificity in QTL...
 up arrowFine Mapping of BP...
 up arrowPositional Cloning
 up arrowConclusions
 *References
 

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