(Hypertension. 1997;29:158.)
© 1997 American Heart Association, Inc.
Scientific Contributions |
Linkage Analysis Using Platelet-Activating Factor Ca2+ Response in Transformed Lymphoblasts
From the Hypertension Research Program of the University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark (J.P.G., L.H., E.J., X.Y.Y., Z.F., A.A.); the Center for Molecular and Behavioral Neurosciences, Rutgers University, Newark, NJ, and the Department of Psychiatry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark (L.M.B.); and the Statistical Genetics Laboratory, The Rockefeller University, New York, NY (J.O.).
Reprint requests to Dr Abraham Aviv, Hypertension Research Program, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, MSB-F464, 185 S Orange Ave, Newark, NJ 07103-2714
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Abstract |
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Epstein-Barr virus-transformed lymphoblasts from patients with essential hypertension demonstrate enhanced G protein-mediated cytosolic free calcium ([Ca2+]i) response to platelet-activating factor (PAF). To map genes responsible for variation in G protein-coupled signaling, we used this cellular phenotype for a linkage study of transformed cell lines from the Centre d’Etude du Polymorphisme Humain (CEPH) reference pedigrees. The PAF-evoked change in [Ca2+]i ranged from 20 to 392 mmol/L and was highly reproducible within each cell line. PAF-elicited [Ca2+]i responses were obtained in lymphoblastic cell lines from five densely mapped pedigrees of the CEPH collection. Using PAF-evoked [Ca2+]i responses as a quantitative trait, two-point sibpair linkage analyses were conducted using 5150 markers from the Collaborative Human Linkage Center (CHLC) database. Nine loci, located on chromosomes 1, 4, 10, 11, 13, 16, and 17, were suggestive of linkage, with values of P<7.4x10-4. Multipoint linkage analysis produced a significant linkage finding (P=2.1x10-5) in one family at D16S151, with suggestive linkage results for seven additional markers spanning a 40-cM interval of chromosome 16. Multipoint analysis produced suggestive findings of linkage to eight loci from two distinct regions of chromosome 11 in another family. These results indicate that loci involved in the control of G protein-mediated mechanisms, suggested to be involved in the pathophysiology of essential hypertension, can be identified using cell lines from general pedigrees selected without any knowledge of the blood pressure status of the donors. This strategy represents an approach to rapidly and inexpensively mapping loci related to common, complex disorders, using phenotypes that are stable in immortalized lymphoblasts together with existing reference pedigree cell lines and genotype databases.
Key Words: platelet-activating factor • cytosolic calcium • G protein
Abbreviations: CEPH = Centre d’Etude du Polymorphisme Humain • CHLC = Collaborative Human Linkage Center • cM = centimorgan • cytosolic free Ca2+ = [Ca2+]i • EBV = Epstein-Barr virus • G protein = guanine nucleotide-binding regulatory protein • HBS = HEPES-buffered solution • NHE-1 = Na+/H+ exchanger • PAF = platelet-activating factor • Vmax = maximal reaction velocity
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Introduction |
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Blood pressure is controlled by a network of genes that interact with one another, with their protein products, and with the environment. Unlike monogenic forms of hypertension (see References 1 and 2), essential hypertension is quite common in human beings3 and reflects in part mutations in several and perhaps many genes.4–6 These mutations should be considered as genetic variations that raise the blood pressure in a segment of the population. It is thus unlikely that the genetics of essential hypertension will be readily resolved by linear paradigms, which rely on the concept that a mutation in a given gene would generate a well-defined phenotype. It is reasonable to assume, however, that essential hypertension is an expression of variations of physiological processes in control of blood pressure. The genes responsible for variations in these processes among human beings are likely to harbor susceptibility for essential hypertension. Identifying these genes would therefore be an important step toward dissecting the genetics of essential hypertension. One way to identify these genes is to use phenotypes representing biological processes in control of blood pressure for genetic linkage analysis. Moreover, the use of cellular phenotypes instead of systemic ones positions genetic linkage studies closely to the presumptive genetic variations that underlie elevated blood pressure in a subset of the human population.
If immortalized cell lines express the cellular processes that are involved in blood pressure control, then it is possible to use cell lines from large reference pedigrees for the linkage mapping of genes responsible for variations in these processes. Such cells are available from the CEPH collection.7 The use of these cell lines would accomplish two goals: First, it would drastically reduce the time and expense needed to conduct a genome-wide scan for susceptibility genes for essential hypertension, as genotype data are readily available for thousands of markers for these families. Second, it would eliminate the contribution of environmental factors expressed in vivo to the variations in phenotypic expressions.
To test these ideas, we have examined two cellular phenotypes as candidates for genome-wide linkage mapping in cell lines from the CEPH collection, focusing on the CEPH families that were used to generate the CHLC genetic map.8 The phenotypes we examined were the Vmax of the ubiquitous NHE-1, and a G protein-mediated cellular process,9,10 namely, the [Ca2+]i response to PAF. These choices are based on observations that NHE-111 and G protein-mediated processes10,12–15 are altered in patients with essential hypertension and the recent findings that EBV-transformed lymphoblasts express these phenotypes.10,16,17
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Methods |
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Cell Lines
EBV-transformed cell lines of 15 husband/wife pairs from the CEPH collection(s) were obtained from the Coriell Institute for Medical Research (Camden, NJ) for initial evaluation (Pedigree Reference Numbers 102, 884, 1331, 1332, 1333, 1341, 1344, 1346, 1347, 1349, 1362, 1408, 1413, 1416, and 1423). In further experiments, we obtained all available cell lines from the following CEPH pedigrees: 102, 1331, 1347, 1362, and 1416. Cells were maintained in RPMI 1640 supplemented with 2 mmol/L L-glutamine, plus 100 units/mL penicillin, 100 µg/mL streptomycin, and 15% heat-inactivated fetal bovine serum. Lymphoblasts were passaged twice a week, and stock cultures were frozen once sufficient cell numbers were obtained (approximately 2 to 3 weeks after receiving the cell lines). Mycoplasma contamination was excluded by monitoring cell lines with the Mycotrim TC triphasic culture system (Irvine Scientific). In preparation for the experimental protocols, unless indicated otherwise, cells (2x106/mL) were maintained in serum-free RPMI 1640 plus 1% BSA for 24 hours.
Measurement of the Vmax of NHE-1
Lymphoblasts (4x106/mL) were suspended in HBS comprising (in mmol/L): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 glucose (pH 7.4). Aliquots (106/250 µL) of the cell suspension were incubated with 2 µmol/L BCECF-acetoxymethyl ester (AM) for 30 minutes. Cells were centrifuged, washed gently with 100 µL acidification solution (comprising HBS plus 5 µmol/L each of nigericin and monensin), and resuspended in 200 µL acidification solution for 10 minutes. The pH of the acidification solution was set to 5.9, which is the level of maximum stimulation of the NHE-1.17 At the end of 10 minutes, the ionophores were removed by treatment of the cell suspension with 1% albumin for 1 minute and a brief centrifugation. Cells were resuspended either in Na+-containing or Na+-free buffer (N-methyl-D-glucamine replacing Na+), and cellular fluorescence was monitored in a CM3 spectrophotometer (SPEX Industries) set to 440/503 nm excitation and 530 nm emission. This instrument is equipped with a thermostatically controlled (37°C) cell holder and mechanisms that allow for rapid alterations between excitation wavelengths. The rate of recovery in Na+-free medium was negligible. The product of the initial rate of recovery in Na+-containing medium and the buffer power (measured by adding 15 mmol/L NH4Cl to acidified cells in Na+-free buffer) was expressed as H+ equivalent efflux rate and used as a criterion for the Vmax of the NHE-1. Calibration of pH was performed using the acidification solution with preset pH values between 5.9 and 7.4. The calibration buffer contained 20 µmol/L nigericin.
Measurements of the PAF-Evoked [Ca2+]i Responses
Lymphoblasts were washed at 37°C with HBS containing 0.1% BSA. Cells (2x107/mL) were incubated for 30 minutes at 37°C with 2 µmol/L of fura 2-AM and 0.125 mmol/L sulphinpyrazone (to inhibit dye leakage) as described previously.18 In preparation for experiments, cells (2x106/mL) were washed once with HBS and resuspended in a cuvette containing 3 mL of either Ca2+-free HBS (0.3 mmol/L EGTA substituted for CaCl2) or HBS. [Ca2+]i measurements were performed in a CM3 spectrophotometer. Excitation wavelengths were set at 340/380 nm and emission wavelength at 505 nm. Thirty seconds after resuspension in solution, cells were challenged with 100 nmol/L PAF (Calbiochem) dissolved in ethanol. Similar amounts (0.2% final volume) of vehicle had no effect on basal [Ca2+]i. Experiments were done in duplicate and calibration of [Ca2+]i was performed on a sample aliquot of cells initially suspended in HBS.18 Autofluorescence of unloaded cells in HBS was subtracted from the cellular fluorescence of the dye. Basal [Ca2+]i was determined as the average [Ca2+]i value during the 5 seconds preceding PAF challenge, and peak [Ca2+]i was determined as the maximal [Ca2+]i (8 to 12 seconds after stimulation) with PAF. PAF-evoked [Ca2+]i responses (ie, the difference between basal [Ca2+]i and peak [Ca2+]i in response to PAF) were measured on at least three separate occasions for all cell lines.
Measurements of Subsets of B Cell Lines
B cell subsets in cell lines from pedigree 1347 were monitored by flow cytometry19 with FITC- or phycoerythrin-labeled mouse anti-human antibodies (anti-leu-8[CD62L], -CD20, -CD23, -CD34, and -CD38, from Becton Dickinson Immunocytometry Systems and anti-CD10, -CD21, -CD22, -CD25, -CD30, -CD71, and -Ki67, from DAKO Corp). Cells (1x107) were harvested by centrifugation (180g) for 10 minutes and suspended in PBS, pH 7.6. After a second centrifugation, cells were resuspended in 2.5 mL RPMI 1640 and a 100-µL aliquot was incubated with 20 µL of monoclonal antibody for 30 minutes (4°C). After one wash with PBS, cells were fixed with 1% formaldehyde and analyzed on a FACScan (Becton Dickinson Immunocytometry Systems). Threshold settings for nonspecific fluorescence were obtained by using isotypic anti-IgG2a and anti-IgG1. CD19-positive cells expressed the following markers (percentages±SD are given in parentheses): Leu8(CD62L) (33.7±16.6); CD20 (47.4±14.5); CD23 (86.5 ±15.1); CD34 (0.9±0.7); CD38 (61.7±27.4); CD10 (0.7±0.5); CD21 (44.1±19.9); CD71 (81.6±5.9); CD22 (63.0±12.8); CD25 (29.9±12.9); CD30 (75.5±12.2); Ki67 (1.8±1.0).
Measurements of Telomerase Activity
Telomerase activity (an indicator of immortalization status) was assayed in 14 randomly chosen cell lines. Assays of telomerase activity were performed as previously described.20,21
Genetic Linkage Analysis of PAF-Evoked [Ca2+]i Responses
PAF-evoked [Ca2+]i responses in Ca2+-free and Ca2+-containing medium were analyzed as quantitative traits. The distributions of response values were normalized using the program NO-COM.22 Using the transformation formula x=(ye-1)/e+e, where y is the original value and x the transformed value, optimal transformations were obtained with e=0.5125 for Ca2+-free and e=0.4125 for Ca2+ -containing medium. Genotype data for CEPH families 1331, 1347, 1362, 1416, and 102 were downloaded from the CHLC website (http://www.chlc.org6). Two-point and multipoint autosomal sibpair analyses were conducted using the SIBHE and SIBIHE modules of GAS version 2.0,23 respectively, employing the dfweight option to compensate for the analysis of multiple sibpairs from each sibship. Two-point analyses were conducted with all marker data. Multipoint analyses were conducted using only those markers positioned on the CHLC version 2.0 sex-averaged framework maps. Each entire framework map was analyzed as a single multipoint analysis. Marker data from the X chromosome were analyzed using the SIBMWU module of GAS.
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Results |
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Vmax of the NHE-1 in Transformed Lymphoblasts
In initial experiments we measured the Vmax of NHE-1 in 16 lymphoblastic cell lines from 8 husband/wife pairs from the CEPH collection. The Vmax values for the NHE-1 demonstrated large day-to-day variations for most of the cell lines in quiescent cells deprived of serum for 24 hours (Fig 1) or in growing cells (not shown). Neither the rates of recovery from acidification nor the cellular buffer power were sufficiently stable on different days to serve as reliable cellular phenotypes. Additionally, relatively small differences were observed among cell lines from different individuals in the activity of NHE-1. Thus, the Vmax of NHE-1 was not pursued as a useful cellular phenotype for further genetic linkage studies.
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PAF-Evoked [Ca2+]i Responses in Transformed Lymphoblasts
Initial screening of 15 husband/wife pairs showed that the PAF-evoked [Ca2+]i responses in both 1 mmol/L Ca2+ medium and in Ca2+-free medium were highly reproducible for each cell line with small day-to-day variations (Fig 2). Additionally, substantial differences were observed among lymphoblastic cell lines from different individuals with values ranging from 20 to 392 nmol/L in 1 mmol/L Ca2+ medium and 11 to 304 nmol/L in Ca2+-free medium.
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EBV transformation might yield different subsets of B lymphoblasts or different states of immortalization. For these reasons, we examined the relation between lineage- and proliferation-specific B lymphocyte markers19 and the PAF-evoked [Ca2+]i response. No significant correlations were observed (probability values ranged from 0.15 to 0.94). We also examined the correlation between the PAF-evoked [Ca2+]i responses and the enzymatic manifestations of telomerase, an RNA-dependent DNA polymerase that is frequently activated by immortalization.24,25 No correlation was observed between the expression of the enzyme and the PAF-elicited [Ca2+]i response (data not shown). Thus, variations among cell lines in the PAF-evoked [Ca2+]i responses were not the result of factitious selections of subsets of lymphoblasts, variations in proliferation, or different expressions of immortalization in vitro.
Linkage Results of the PAF-Evoked [Ca2+]i Responses
Five families (102, 1331, 1347, 1362, and 1416) were selected for further study. Selection was made to take advantage of the most densely mapped families. In addition, as the genetic factors underlying the differences in the PAF-evoked [Ca2+]i response are unknown, ideal selection criteria for families to study further were uncertain. For quantitative traits controlled by multiple genetic factors, families with parents with large phenotypic differences are preferable. However, for traits determined by a single gene, crosses of parents with similar, intermediate phenotypes can produce offspring with large phenotypic variation, and the observation of such pedigrees can help elucidate the inheritance of the trait. Therefore, families for further study included both those with a large parental difference in PAF-evoked [Ca2+]i response and others with similar, moderate parental responses. All cell lines available from these families were phenotyped for PAF-evoked [Ca2+]i responses. The PAF-evoked [Ca2+]i responses in family 1347, the family that subsequently showed significant linkage, are presented in Fig 3.
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Genotype data for these five families for 5150 markers were downloaded from the CHLC website.8 Two-point sibpair linkage analyses were conducted with all of the marker data and PAF-evoked [Ca2+]i responses. In addition, multipoint analyses were conducted with 938 markers organized into chromosome framework maps. Analyses were conducted using the PAF-evoked [Ca2+]i response in both Ca2+-free and Ca2+-containing medium; as these analyses generated similar results for the two phenotypes, only the Ca2+-containing medium results are reported here. Table 1
presents a summary of the distribution of results by level of significance and chromosome for the two-point and multipoint analyses of the set of five families. Lander and Kruglyak26 have recently suggested probability values of 7.4x10-4 (equivalent to an LOD score of 2.2) and 2.2x10-5 (equivalent to an LOD score of 3.6) as thresholds for suggestive and significant linkage findings, respectively, for dense, complete genome scans by sibpair analysis. On the basis of these thresholds, nine loci were suggestive of linkage under two-point analysis (Table 2), with no significant two-point and no suggestive or significant multipoint linkages detected. Interestingly, both chromosomes 11 and 16 had two loci each with suggestive two-point linkage findings, as well as the two smallest multipoint probability values, .0019 for chromosome 11 and .0045 for chromosome 16. Three of the nine suggestive loci had a second polymorphism in the CHLC database, but none of these produced even suggestive results (probability values of .7 for PND, .02 for D10S16, and .0027 for D16S151). Inspection of the primary genotype data (not shown) indicated that these differences were probably due to variations in which families were genotyped or informativeness for each of the polymorphisms. This suggests that some families might be contributing to linkage to a certain locus while others were not. On the basis of this observation, we proceeded to analyze our linkage results for each family individually.
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Due to the size of the CEPH families, individual families are capable of producing significant linkage results. As the above findings had suggested that lack of full informativeness could potentially lead to incorrect findings of linkage, we restricted this portion of the study to multipoint analyses of the CLHC framework map markers. Multipoint analysis produced a significant linkage finding (P=2.1x10-5) on chromosome 16 for family 1347 at the location of D16S151, with seven additional markers within a 39.7 cM region producing values in the suggestive range (probability values of 2.5x10-4 to 3.3x10-5; upper panel, Fig 4). In addition, eight markers from chromosome 11 produced probability values in the suggestive range (P= 7.2x10-4 to 2.4x10-4) for family 102. The locus with the lowest probability value in this region was at THY1 (Thy-1 cell surface antigen), with six of the remaining suggestive loci clustering within a 13.9 cM region, and one additional suggestive locus (D11S1385) located approximately 60 cM away from the cluster (lower panel, Fig 4).
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Discussion |
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Essential hypertension reflects the input of several, and perhaps many, independently segregating genes, some of which interact with the environment in a complex fashion (reviewed in references 4 through 6). Blood pressure is a poorly defined phenotype in that it changes with age in industrialized societies and is further modified by gender, menopausal status, race, body mass, dietary intake of salt (NaCl), and a multitude of other factors. In addition, blood pressure is continuously distributed, whereas the definition of essential hypertension is based on arbitrary cutoff points in the blood pressure (eg, systolic/diastolic blood pressure of 145/95 mm Hg). These artificial thresholds have little biological relevance. Given these limitations of defining essential hypertension, stable intermediate phenotypes, reflecting physiological processes that contribute to variations in blood pressure among human beings would be highly desirable for use in linkage studies. Ideally, such phenotypes should represent processes in control of blood pressure, be stable over a long period of time, be resistant to environmental factors, and demonstrate a high degree of heritability. Moreover, shifting the phenotypic assignment from the systemic to the cellular level moves the linkage study one step closer to the genes responsible for variations in blood pressure. This would be expected to lessen problems such as reduced penetrance, phenocopies, and genetic heterogeneity.
On the basis of these general considerations, we have formulated the following hypothesis: What distinguishes patients with essential hypertension from the rest of the population exposed to equivalent environmental risk factors is that they carry a combination of blood pressure regulating genes leading to a rise in blood pressure above an arbitrary cut-off point. Some of these genes exert their blood pressure effect through regulatory systems of cellular Ca2+ and Na+. Thus, identifying the genes responsible for variations in the expression of these processes in the human population would aid in dissecting the genetics of essential hypertension. The evidence that cellular Ca2+ and systemic Na+ regulations are altered in essential hypertension is compelling.27 The effective use of Ca2+ antagonists and diuretics as antihypertensive agents is in line with this concept from the clinical standpoint.28–30 The angiotensin II receptor antagonists used to treat hypertension also function primarily through cellular Ca2+, since angiotensin II acts via the Ca2+ signaling system of target cells (eg, vascular smooth muscle cells).31,32
Accordingly, it is not necessary to study individuals with essential hypertension to identify genes responsible for variations of cellular Ca2+ and Na+ processes involved in blood pressure regulation. The only requisite is that the cellular phenotypes used for the linkage analysis represent biological processes involved in blood pressure control or mechanisms that are altered in patients with essential hypertension. Thus, with relatively small cost, cells from reference pedigrees, such as those in the CEPH collection, can be used to identify linkage of intermediate cellular phenotypes of altered Ca2+ and Na+ metabolism to specific genetic loci. This approach takes advantage of the wealth of genotype data easily available for these families. Screening the cell lines from these families for suitable variations in genetically controlled cellular phenotypes allows for the rapid utilization of this enormous amount of genotype data. The core collection of 40 CEPH families is large enough to expect cellular phenotypes to not only demonstrate variation but also to be present in sufficient, suitable mating combinations to produce an informative linkage study.
To be useful for linkage studies using EBV-transformed lymphoblasts from the CEPH collection, intermediate phenotypes must (1) be expressed in these cells, (2) be stable, and (3) demonstrate variability among cell lines. Although NHE-1 has been excluded as a candidate gene for essential hypertension,16,33,34 the Vmax of the exchanger could have served to trace abnormal genes that may modify the activity of NHE-1 in EBV-transformed lymphoblasts.35 The Vmax for NHE-1 showed little interindividual variations among cell lines from the CEPH collection. One possibility is that increased Vmax of NHE-1 is expressed only in a small segment of EBV-transformed lymphoblasts from the general population. However, this parameter was also unstable, demonstrating substantial day-to-day variations in the majority of cell lines. Therefore, the Vmax of NHE-1 was not pursued for linkage analysis. In contrast, the PAF-evoked [Ca2+]i response met the criteria for stability in a given cell line and variability among cell lines to serve as an intermediate phenotype in EBV immortalized lymphoblasts from the CEPH collection.
Our results show that the PAF-evoked [Ca2+]i response is linked to a locus on chromosome 16 and possibly two other loci on chromosome 11. Since numerous proteins shape the [Ca2+]i signal, it is very likely that a number of interacting genes rather than a single gene are responsible for variations in the PAF-evoked [Ca2+]i response among lymphoblastic cell lines from different pedigrees. The PAF-evoked response is mediated through a family of heterotrimeric G proteins.9,10 G proteins are ubiquitous and they play pivotal roles in a multitude of cellular functions that are initiated through the relay of messages from receptors to effector proteins on the plasma membrane and in the cellular interior.36 Several of these functions are altered in essential hypertension.10,12–15
The locus on chromosome 16 linked to the PAF-evoked [Ca2+]i response is of interest, as this chromosome contains the ß and 
subunits of the epithelial sodium channel (ßENaC and ENaC). Both genes are implicated in the cause of Liddle’s syndrome.1,2 Both ßENaC and ENaC are linked to D16S420 at 
=0.00, but this marker is unfortunately not part of the set of CHLC framework markers used to generate our multipoint linkage results. D16S420 has been assigned to a specific interval on the CHLC framework map, however, between the loci D16S79 and D16S67, the 12th and 17th loci from the left on the upper panel of Fig 3. Examination of the figure reveals that the location of these genes does not overlap the point of our significant multipoint linkage finding in family 1347 but does overlap part of the surrounding interval with suggestive linkage results.
While no single locus produced a significant linkage finding for PAF-evoked [Ca2+]i response when the five families were considered together, significant and suggestive findings were identified when the families were examined individually. Cellular phenotypes that are expressed in cultured lymphoblasts are expected to greatly reduce or even eliminate the role of environmental factors on phenotypic variation. However, deciphering the contribution of all the genes expected to control the normal population variation of a trait such as the PAF-evoked [Ca2+]i response would likely require a sample larger than five families, particularly if the effects of some loci are small. If the genes interact epistatically, then it would be expected that the presence or absence of an effect for a specific locus in a particular family would depend on the genetic background contributed by the other interacting loci.
By using large pedigrees, our strategy allows for the detection of an effect of a particular locus within a subset of families, either those that have an appropriate genetic background to allow for the contribution of a particular locus to be clearly seen, or those that have more extreme mutant alleles that contribute a larger-than-average effect to the variation of the cellular phenotype. Alleles having a larger effect on Ca2+ homeostasis would also likely be of the most clinical interest in predicting an increased risk for the development of hypertension. Accordingly, Fig 4 clearly demonstrates how, as with parametric linkage studies, locus heterogeneity (or apparent locus heterogeneity on the basis of epistasis) can obscure a positive linkage finding in a small sample when all families are considered together. However, analysis of a larger sample of families with the same profile of contributions to PAF-evoked [Ca2+]i response by these various loci would be expected to reach significance even when all families were considered together.
Finally, we would like to caution against putting too much weight on our findings of linkage of the PAF-evoked [Ca2+]i response to a locus on chromosome 16 based on findings from a single family. In fact, a locus on chromosome 11 may be more promising, as markers on this chromosome express a similar trend toward linkage in three of five families. The main purpose of this communication is to illustrate a new approach to the dissection of the genetics of essential hypertension. Loci identified by such an approach can serve as candidates for further linkage studies, using DNA from peripheral blood cells from sibships with family histories of essential hypertension. The paradigm described herein, using lymphoblastic cell lines from pedigrees of the CEPH collection, can serve for linkage studies of any complex trait with pervasive distribution in the general population. The central requirements for such studies are that cellular processes that define such a trait are expressed in EBV-transformed lymphoblasts, they are stable under in vitro conditions, and they demonstrate interindividual variations.
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Acknowledgments |
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This work was supported by grants NH47906 and HL44196 from the National Institutes of Health and HG00008 from the National Center for Human Genome Research. Drs X-Y Yang, E. Jeanclos, and Z. Fekete were postdoctoral fellows of the American Heart Association, New Jersey affiliate. We thank Dana Stein and Hortencia Ong for excellent technical assistance with immunocytometry experiments and Pat Peluso for her secretarial skills.
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Footnotes |
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J.P.G. and L.M.B. contributed equally to this work.
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