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Determination of Sequence Variation and Haplotype Structure for the Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Genes: Investigation of Role in Pubertal Timing

Divisions of Endocrinology and Genetics (I.L.S., J.L.B., T.B., J.N.H.), Department of Medicine, Children’s Hospital, and Departments of Genetics and Pediatrics (I.L.S., J.L.B., T.B., J.N.H.), Harvard Medical School, Boston, Massachusetts 02115; Department of Preventive Medicine (C.L.P., B.E.H.), University of Southern California, Keck School of Medicine, Los Angeles, California 90089; Endocrine Science Research Group (J.A.T., P.E.C.), University of Manchester, Manchester M13 9PT, United Kingdom; Academic Unit of Medical Genetics (A.P.R.), St. Mary’s Hospital, Manchester M13 0JH, United Kingdom; Cancer Research Center (L.N.K.), University of Hawaii, Honolulu, Hawaii 96813; Program in Medical and Population Genetics (J.N.H.), Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02139; and Division of Pediatric Endocrinology and Metabolism (M.R.P.), Rainbow Babies and Children’s Hospital, University Hospitals of Cleveland, Departments of Pediatrics and Genetics, Case School of Medicine, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Mark R. Palmert, M.D., Ph.D., Division of Pediatric Endocrinology and Metabolism, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: mark.palmert{at}cwru.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Because GnRH and its receptor (GnRHR) are pivotal regulators of the reproductive endocrine axis and mutations in GNRHR lead to hypogonadotropic hypogonadism, we investigated whether genetic variation in GNRHR or GNRH1 affects pubertal timing in the general population.

To screen for missense mutations in these genes that might affect pubertal timing, we resequenced the coding regions of these genes in 48 probands with late but otherwise normal pubertal development. No missense variants were found in either gene, except for a previously identified single nucleotide polymorphism (SNP) in GNRH1 that was not associated with late pubertal development. To search for common variants that might affect pubertal timing, we took a haplotype-based association approach. To identify common haplotypes in these genes, we genotyped 41 SNPs in DNA from commercially available European-derived multigenerational pedigrees and participants in a multiethnic cohort (MEC). Two blocks of strong linkage disequilibrium were identified that spanned GNRHR and one was identified spanning GNRH1; within each block, more than 80% of chromosomes carried one of a few common haplotypes. A set of haplotype-tagging SNPs that mark these common haplotypes in all five ethnic groups within the MEC were defined and used to perform association studies among 125 trios (probands with late pubertal development and their parents) and 506 women from the MEC who had early (menarche < 11 yr of age, n = 216) or late (menarche 15 yr of age, n = 290) pubertal development. Three SNPs in GNRHR showed modest association with late pubertal development in the trios; among the 506 women, a different SNP was associated with late menarche, and one rare haplotype was associated with early age of menarche. All of the observed associations were relatively modest and only nominally statistically significant; replication is needed to determine their validity.

We conclude that genetic variation in GNRH1 and GNRHR is not likely to be a substantial modulator of pubertal timing in the general population.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GENETIC FACTORS MODULATE pubertal timing, as shown by the correlation in the timing of puberty among parents and children, among racial groups, and between monozygotic twins (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Constitutional delay of growth and maturation (CD), which likely represents the extreme end of normal pubertal timing, clusters in families and has a strong genetic component, as indicated by case series (11, 12, 13, 14) and pedigree analysis (15). Through the analysis of 53 CD pedigrees, we recently demonstrated that CD is often transmitted through autosomal dominant inheritance with variable penetrance (15). Thus, although the timing of puberty is likely to be a multigenic trait (16), as has been suggested for other complex traits (17, 18, 19), the data from CD families indicate that there may also be single genes that exert major effects on the timing of puberty within the general population.

GnRH and its receptor (GnRHR) are pivotal regulators of the reproductive endocrine axis, and abnormalities in their function have significant impacts on human physiology and pubertal development (20, 21). Several mutations in GNRHR have been identified (22, 23, 24, 25, 26, 27, 28, 29, 30, 31). When assessed, these mutations (in either homozygous or compound heterozygous states) represent the most commonly identified genetic cause of idiopathic hypogonadotropic hypogonadism (IHH) (29). Approximately 10–15% of IHH probands have a family history of delayed puberty (32, 33), suggesting that carriers of IHH mutations might have a tendency toward late pubertal development. Furthermore, because severe genetic disruption of the GnRH pathway leads to the phenotype of IHH, it is possible that more mild genetic variation in this pathway may lead to the milder phenotype of late pubertal development. Such mild variation could be either a heterozygous severe mutation or less severe polymorphisms.

Here we report results from sequence analysis and haplotype-based association studies performed among individuals with later-than-average pubertal development to determine whether genetic variation within either GNRHR or GNRH1 contributes to the regulation of pubertal timing in the general population.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject population

The study was approved by the Institutional Review Boards at Children’s Hospital in Boston, University Hospitals of Cleveland, the Salford and Trafford Local Research Ethics Committee (Manchester, UK), the University of Hawaii, and the University of Southern California School of Medicine. Written informed consent and, when appropriate, assent was obtained from all study participants. The trio samples were derived from U.S. and U.K. collections. For the U.S.-based trios, endocrine division and hospital-wide databases (34, 35) were used to identify adolescents seen for delayed puberty in the endocrine clinic at Children’s Hospital in Boston from approximately January 1995 to June 2000 and at Rainbow Babies and Children’s Hospital (University Hospitals of Cleveland) between February 2000 and June 2002. Individual records were then reviewed, and eligible families were contacted regarding participation. Eighty-one subjects (72 from Boston and nine from Cleveland), and their parents agreed to participate and met the inclusion criteria [no underlying medical conditions that affect pubertal development; boys with testicular enlargement (testis size 2.5 cm in length or 4 cc in volume) after 13 yr of age (pubertal onset 1 SD beyond the mean) or after 14 yr of age (pubertal onset 2 SD beyond the mean); girls with breast development after 12 yr of age (pubertal onset 1 SD beyond the mean) or after 13 yr of age (pubertal onset 2 SD beyond the mean)]. The U.S. study group consisted of 39 boys and 10 girls with pubertal onset 2 SD or more beyond the mean and 24 boys and 8 girls who met the 1 SD criteria. All subjects had documented spontaneous pubertal development. No family had a history of consanguinity. Ethnic distribution among the 81 subjects was as follows: 76 (94%) declared themselves as white or non-Hispanic; two (3%) as Cape Verdian; one (1%) as Hispanic; one (1%) as mixed CapeVerdian/white; and one (1%) as Asian.

The second set of trios was recruited from the endocrine outpatient clinics in Greater Manchester, UK. This group consisted of subjects diagnosed with constitutional delay of growth and pubertal development and their parents. In all cases, the probands were seen by a pediatric endocrinologist who established the diagnosis, and case notes were reviewed to establish eligibility. The included subjects met the following criteria: 1) delayed puberty and/or height below that predicted by their genetic potential with reduced growth velocity; 2) significant delay in bone maturation; and 3) no underlying medical conditions that would account for the pubertal delay. This group consisted of 37 boys (21 with pubertal onset 2 SD, 16 with pubertal onset 1 SD) and seven girls (four with pubertal onset 2 SD, three with pubertal onset 1 SD). Forty-two of the subjects were whites of European origin and two were Pakistani. It is important to note that in both the U.S. and U.K. study groups, we likely underestimated the proportion of individuals with pubertal onset 2 SD or more beyond the mean. This is because we classified individuals as 1 SD or more unless we had documentation of pubertal timing that met the criteria of 2 SD or more. See Table 1A for phenotypic and demographic information regarding the U.S. and U.K. trio population.

Genetic analysis

Genomic DNA was isolated from peripheral blood cells (U.S. trios and MEC subjects) or cheek swabs (U.K. trios) using conventional procedures before DNA sequencing and genotyping.

To screen for missense mutations in GNRHR and GNRH1 (accession no. NM_000406 and NM_000825.2), we used a subset of 48 subjects from the U.S. trio population with late pubertal development. See Table 1C for phenotypic and demographic information. For both genes, we directly sequenced the coding regions as well as the intron/exon and untranslated region/coding region borders. For GNRHR, the following primer pairs were used: exon 1, two overlapping primer sets, 5'-CAGGGACAAAATTTGACATACG-3' and 5'-ATGTTCCACATCCCATCCAG-3' along with 5'-TTCTGCTCTCTGCGACCTTT-3' and 5'-CTGACTTCCAGAACCCAAGC-3'; exon 2, 5'-GGCTAGCAGAGTACCAAAGAGAA-3' and 5'-TGCCACTCTGTTTTGAGCAT-3'; exon 3, 5'-TCCTTTTTGTCCACTTTGGTTT-3'and 5'-TCCCAGATGGAGAGATTCA-3'. The same strategy was employed for GNRH1 except that for completeness and because of uncertainty regarding the GNRH1 genomic structure (see NM_000825 and revision NM_000825.2 at http://www.ncbi.nlm.nih.gov for details), we also sequenced the region previously designated as exon 1. The following primer pairs were used: previously designated exon 1, 5'-GCAGGAAAGATTTCAATGTCC-3' and 5'-GATTTAGCCCTTGGGCTGTC-3'; exon 1, 5'-CCATCTTCTGCAGGGTTAGTG-3' and 5'-GCCTTATCTCACCTGGAGCA-3'; exon 2, 5'-CCCCACTCTCCACAATTTTT-3' and 5'-CAGGAATGTAAGCCCCACAG-3'; exon 3, 5'-CAAACCCAATTTATCATGTCTCC-3' and 5'-ACATGGAGGGCTCCCTTTG-3'.

For both genes, bidirectional fluorescent dideoxy sequencing was performed (Megabace 1000, Amersham, Aylesbury, UK) and sequence variants were identified using Polyphred (37). High-quality sequence (phred score > 20 in at least one direction through the entire exon) was obtained for GNRHR in 40 subjects for exon 1B, 46 subjects for exon 2, and 36 subjects for exon 3. For GNRH1 high-quality sequence was obtained in 45 subjects for exon 1, 46 subjects for exon 2, and 46 subjects for exon 3. This degree of coverage yields a power of 77–84% to detect a missense mutation that is present at an allele frequency of 2% or greater in individuals with late pubertal timing.

A panel of single nucleotide polymorphisms (SNPs) from the SNP Data-base (dbSNP) (http://www.ncbi.nlm.nih.gov/SNP/) and Celera (www.celeradiscoverysystem.com) were genotyped for both genes within 12 commercially available European-derived multigenerational pedigrees (CEPHs) [93 CEPH samples described elsewhere (38), representing 96 independent chromosomes] to define the haplotype structure of GNRHR and GNRH1. This haplotype structure was verified for the five ethnic groups represented in the MEC. The three sequence variants in GNRH1 discovered during resequencing were also genotyped within the U.S.-based trios to confirm sequencing results and to determine whether these were common variants. Genotyping was performed as described elsewhere (38) using the MassARRAY platform (Sequenom, Inc., San Diego, CA), which entails primer extension of multiplex products with detection by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (39). The complete list of SNPs, coordinates in the hg16 freeze (July 2003) of the human genome, and flanking sequences are given in Table 2 and are depicted in Figs. 1 and 2.

View larger version (55K): [in this window] [in a new window]   FIG. 1. Haplotype structure of GNRHR. A, Location of genotyped SNPs. See Table 2 for SNP names and coordinates; SNPs are numbered 1–30, from 5' to 3'. Exons are shown as thick bars (untranslated regions as half-height). Haplotype blocks (SNPs in linkage disequilibrium) are represented below by long black bars and were defined using established criteria in Ref. 38 as implemented in the Haploview software. B, Common haplotypes within GnRHR. SNP numbers 1–14 are included in block 1, and SNP numbers 16–30 are included in block 2. The allele for each SNP within the most common haplotypes is also displayed; each row represents a particular haplotype, and the columns represent the SNPs in order of position. The frequencies of the individual haplotypes are displayed at the end of each row. The data displayed are for a CEPH sample. C, Haploview output for GnRHR. The CI output of the Haploview program is displayed, using data from a CEPH sample. Black squares indicate pairs of SNPs for which there is good evidence that there has been little or no recombination, white squares indicate pairs of SNPs for which there is strong evidence of recombination, and gray squares indicate noninformative SNP pairs. Two haplotype blocks appear as outlined triangular regions with mostly black or gray squares.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Haplotype structure of the GnRH gene (GNRH1). Details of SNP locations, haplotypes, haplotype blocks, and Haploview output are displayed for GNRH1 as described in the legend to Fig. 1. See Table 2 for SNP names and coordinates; SNPs are numbered 1–11, from 5' to 3'.

It is now known that much of the genome can be parsed into blocks of linkage disequilibrium within which common genetic variants, such as SNPs, generally fall into a few simple patterns called haplotypes (38). Blocks of linkage disequilibrium within GNRHR and GNRH1 were defined using the normalized measure of allelic association, D' and the previously established criteria (38) implemented in the Haploview software package (Jeffrey Barrett and Mark Daly, Genome Center, Whitehead Institute, Cambridge, MA, http://www.broad.mit.edu/personal/jcbarret/haploview/). Within each block, haplotype frequencies were estimated by an expectation-maximization algorithm (described in Ref. 40) implemented in the Haploview package.

The minimum set of haplotype tagging SNPs (htSNPs) needed to identify confidently the common haplotypes (5% in any one study population and among all five ethnic groups) within a block was selected as described by Stram et al. (41). The degree to which the htSNPs mark the common haplotypes is measured by the square of the correlations (R_h²) between the estimated haplotype frequencies when all SNPs are typed and the estimated frequencies using only the htSNPs. A set of htSNPs within each block was selected to ensure a minimum R_h² of 0.8 or more for all haplotypes observed at a frequency of 5% or more in any study population and among all five ethnic groups (i.e. minimum R_h² 0.8). For the set of htSNPs we selected, the minimum R_h² was more than 0.9 for nearly all haplotypes. Consistent with previous studies (38), the African-American population contained a more diverse set of haplotypes, necessitating the genotyping of additional htSNPs in this population to provide sufficient coverage. Fifteen htSNPs were genotyped in all ethnic groups; six additive SNPs were genotyped in GNRHR and four in GNRH1 to provide adequate coverage in the African-American population (Table 2). See Table 3 for details of minimum R_h² for each block and each ethnic group.

The MEC samples were used for a cohort control analysis in which haplotype frequencies were computed from the htSNP genotypes as described by Stram et al. (44). For each subject an estimate of the number of copies (haplotype dosage) of each predicted haplotype was computed using that individual’s genotype data and the overall haplotype frequency estimates obtained from the E-M algorithm (45). These individual haplotype dosage estimates were then phased by assigning individuals to the most likely haplotype(s) based on the assigned dosage to allow for more flexible modeling of recessive and dominant effects. This method of phasing provided identical results to the method of Stephens and colleagues (43, 46) using the program Phase 2 (http://archimedes.well.ox.ac.uk/pise/). Unconditional logistic regression was used to analyze the resulting data in which individuals with early menarche were assigned to be unaffected, and individuals with late menarche were assigned to be affected in the outcome variable (version 8.0, SAS Institute, Cary, NC). Analyses were also conducted for the single SNP results using the same unconditional logistic regression approach. In instances in which a nominally significant association was observed between the younger than 11 and 15+ age at menarche groups, women with an age at menarche between 11 and 14 yr were compared with the younger than 11 age group to determine whether a trend in risk of late menarche was evident. Analyses were stratified by ethnicity, and a summary odds ratio was estimated controlling for ethnicity.

The P values reported for all tests are nominal values and have not been corrected for multiple testing.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Our analysis of genetic variation in GNRH1 and GNRHR consisted of three components. First, we investigated whether any IHH-causing mutations in GNRHR were present in a group of subjects with late pubertal development. None of these mutations was identified by direct, bidirectional sequencing within our population. We did, however, identify one silent C/T polymorphism in exon1, nucleotide 2204 (Ser151Ser), which has been described previously (47) and referred to as rs4986942.

We also screened GNRH1 for missense mutations that might underlie late pubertal development. We identified three novel sequence variants and one SNP (SNP 6, see Table 2) that has been previously reported (48). The three novel variants included one in the untranslated portion of exon 1 (T/C at nucleotide 171), one silent polymorphism in exon 2 (A/G at nucleotide 1251, Gln59Gln), and one that alters the amino acid sequence in exon 1 [G/C at nucleotide 1215, Glu47Asp, a conservative substitution that is less likely to have functional significance (49)]. The nucleotide variants identified through direct sequencing were confirmed during genotype analysis, but their frequencies (<1%) were too low to permit further analysis.

The second phase of our analysis involved defining the common haplotypes for GNRHR and GNRH1. Each of the SNPs listed in Table 2 and shown in Figs. 1 and 2 were genotyped in 12 CEPH pedigrees. The SNP genotypes were then used to define the patterns of linkage disequilibrium (correlation among markers) and common haplotype structure empirically. For GNRHR, two blocks of strong linkage disequilibrium were identified that spanned SNPs 1 and 14 and SNPs 16 and 30, respectively (Fig. 1). For the first block, two2 common haplotypes were observed that captured more than 80% of the chromosomes studied (Fig. 1B); for the second haplotype block, four common haplotypes captured more than 80% of the chromosomes studied (Fig. 1B). For GNRH1, all SNPs were located within a single block, with four common haplotypes explaining more than 90% of the chromosomes (Fig. 2). These haplotype structures were confirmed in the U.S.-based trios and among the five different ethnic groups from the MEC. The haplotype frequencies were essentially identical among CEPHs, the U.S. trios, and the white population in the MEC. Haplotype frequencies but not block structure did vary slightly among ethnic groups, with increased haplotype diversity in the African-American population (data not shown; detailed results of haplotype analysis in multiple ethnic groups for these and other genes will be reported separately). Within each haplotype block, we identified a subset of SNPs that tag these haplotypes (htSNPs) and therefore capture information about the other variants contained in the haplotype block (41, 50). To ensure that all common haplotypes were well captured by these htSNPs in each of the ethnic groups represented in the MEC, six additional SNPs for GNRHR and four additional SNPs for GNRH1 were selected to capture genetic variation within the African-American population (see Subjects and Methods).

Having selected a set of htSNPs that can serve as proxies for most of the common variation in these genes, we first tested these htSNPs for association with late pubertal development among the trio population (Tables 4 and 5). Only very modest associations between the timing of puberty and particular genetic variants were observed. Using transmission disequilibrium testing (42), three htSNPs in GNRHR (rs3822196, hCV1960913, and rs3796718) showed nominal association with late pubertal development in the sample of 125 parent-offspring trios. It is not surprising that all three SNPs showed association with late pubertal development because, as seen in Fig. 1B, they all are contained in the same haplotypes (numbers 2, 3, 4, and 5 of block 2). We also assessed whether any of the common haplotypes in GNRHR and GNRH1 were preferentially transmitted to offspring with late pubertal timing. No individual haplotype showed significantly preferred transmission, but haplotype 4 of block 2 of GNRHR, a common haplotype marked by the three associated SNPs, trended toward overtransmission (25:16) to the offspring with late pubertal development.

View this table: [in this window] [in a new window]   TABLE 6. Analysis of htSNPs in GNRHR in 506 women with late (15 yr) vs. early (<11 yr) menarche

View this table: [in this window] [in a new window]   TABLE 7. Analysis of htSNPs in GNRH1 in 506 women with late (15 yr) vs. early (<11 yr) menarche

None of the SNPs or haplotypes that showed a nominal association with late puberty in the trios had a similar association in the MEC, and none of the SNPs and haplotypes associated with altered pubertal timing in the MEC was associated with late pubertal development in the trios.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We have examined GNRHR and GNRH1 for genetic variants that might lead to late pubertal development. We found no evidence that heterozygosity for IHH-causing GNRHR mutations or other missense variants in GNRHR or GNRH1 is a common cause of late pubertal development within the general population. This sequence analysis was performed in a predominantly white population; therefore, it remains a formal, but unlikely, possibility that such mutations cause delayed, but otherwise normal, pubertal development in rare instances or certain minority populations. Three htSNPs in GNRHR showed very modest nominally significant associations with late pubertal development in the trios; among the women in the MEC cohort, one htSNP was associated with late menarche and one rare haplotype was associated with early age of menarche. None of these findings was seen in both study populations. This lack of replication could stem from the associations being false positives (none would be significant after correction for multiple hypothesis testing), gender-specific associations (the trios are 80% males, whereas the MEC cohort is all female), lack of power to replicate real associations, or other differences between these populations (51). Additional studies in other populations with early and late pubertal development are needed to determine the validity of the preliminary associations we observed; until they are replicated, these nominally significant findings are best viewed as hypotheses rather than evidence of association between common variation in GNRHR and altered pubertal timing.

Although our results are intriguing, it does not appear that genetic variation in GNRH1 and GNRHR is a major regulator of pubertal timing in the general population. Thus, if major genes exist, they may encode proteins that function downstream of the GnRH receptor, proteins that function as upstream regulators of GnRH secretion, or proteins in completely independent pathways.

We acknowledge that our study has limitations. Although the IHH-causing mutations thus far identified are limited to the coding region sequence and intron/exon boundaries of GNRHR (22, 23, 24, 25, 26, 27, 28, 29, 30, 31), it is possible that cases of pubertal delay could derive from mutations in the unsequenced portions of GNRHR. It also remains possible that rare but functionally important polymorphisms might be present in GNRH1 and GNRHR but not identified yet in the public databases or in our analysis. Our study is underpowered to reproducibly observe associations with polymorphisms that are rare or exert only modest effects and was not designed to examine the possibility of gene-gene interactions between GNRHR and GNRH1. Finally, although our haplotype-based approach likely captured most of the common variation in these genes, it is possible that some common variants were not represented by the htSNPs and haplotypes or that relevant regulatory variation is located at a great distance from the GNRH1 or GNRHR genes.

Although several rare monogenic disorders affect the timing of puberty (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 52, 53, 54, 55), no genetic factors that modulate the timing of puberty within the general population have been identified. Our current study failed to identify substantial modulators of pubertal timing, but we believe that genetic approaches hold great promise for identifying factors that regulate the hypothalamic-pituitary-gonadal axis and modulate the timing of puberty. The structure of the current analysis is a model of an efficient strategy for ongoing investigation of other potential modifiers of the reproductive endocrine axis, such as leptin, -aminobutyric acid, glutamate, neuropeptide Y, fibroblast growth factor receptor 1, and G protein-coupled receptor 54 (53, 54, 56, 57) as well as other yet-to-be-identified genes such as human timing genes that regulate maturational processes (58) or genes derived from animal models (59). Moreover, the definition of the haplotype structure of GNRH1 and GNRHR and the identification of htSNPs will greatly facilitate investigation of these genes in other complex traits and disorders of the reproductive endocrine axis.

	Acknowledgments

 
We thank Drs. Daniel Nigrin and William Dahms for help in identification of research subjects through database queries. We also thank Dr. Matthew Warman for providing support and guidance regarding experimentation and Drs. William F. Crowley, Jr., Paul Boepple, and Stephanie Seminara for providing critical review of the manuscript. Finally, we thank all the subjects who participated and made these analyses possible.

	Footnotes

 
First Published Online November 16, 2004

¹ J.N.H. and M.R.P. codirected this project.

Abbreviations: CD, Constitutional delay of growth and maturation; CEPH, commercially available European-derived multigenerational pedigree; CI, confidence interval; GnRHR, GnRH receptor; ht, haplotype tagging; IHH, idiopathic hypogonadotropic hypogonadism; MEC, multiethnic cohort; OR, odds ratio; R_h², haplotype measured by the square of the correlations; SNP, single nucleotide polymorphism.

This work was supported by Lawson Wilkins Genentech Clinical Scholar Award (to M.R.P.); National Institutes of Health Grants K23 RR15544 (to M.R.P.), RR 002172 (Children’s Hospital GCRC), RR00080 (University Hospitals of Cleveland GCRC), and NCI R01-CA63464 (to B.E.H.); Burroughs Wellcome Career Award in Biomedical Science (to J.H.N.); and California Cancer Research Program Grant 00-01389V-20170 (to C.L.P.).

Received April 5, 2004.

Accepted November 4, 2004.

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