Mol. Hum. Reprod. Advance Access originally published online on December 9, 2005
Molecular Human Reproduction 2005 11(11):809-815; doi:10.1093/molehr/gah244
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Variant progesterone receptor mRNAs are co-expressed with the wild-type progesterone receptor mRNA in human endometrium during all phases of the menstrual cycle
1Division of Reproductive Endocrinology and Infertility, 2Cannon Research Center and 3Dickson Institute for Health Studies, Carolinas Medical Center, Charlotte, NC, USA
4 To whom correspondence should be addressed at: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Carolinas Medical Center, 1000 Blythe Boulevard, Charlotte, NC 28232, USA. E-mail: paul.marshburn{at}carolinashealthcare.org
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Abstract |
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Progesterone receptor (PR) variant mRNAs in human endometrium could encode proteins with the potential to alter progesterone action in states of normal and abnormal endometrial development. We have assessed the expression levels of mRNA for the wild-type PR and splice variants of PR mRNA lacking exon 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4&6 PR), and part of exon 4 (del-p4 PR) or part of exon 6 (del-p6 PR) in the human endometrium throughout menstrual cycle development. Eighty-eight endometrial specimens (47 proliferative, 41 secretory) were collected from patients undergoing hysterectomy for benign gynaecologic causes. Measurements by RT–PCR indicated that mRNAs for wild-type PR, and splice variants del-4 PR, del-6 PR, del-4&6 PR, del-p6 PR, and a novel del-p4 PR were detected in all endometrial specimens throughout the menstrual cycle. Higher levels of wild-type PR and all PR variant mRNAs were found in the early and mid-proliferative endometrial phases than in secretory endometrium. The relative expression of mRNA for all PR variants compared to wild-type PR mRNA, however, did not change through all stages of endometrial development. We, therefore, found no evidence of differential co-expression of the PR variants compared with wild-type PR during normal menstrual development. Future studies will determine if the expression profile of PR variant mRNAs will be different in the endometrium of patients with infertility, recurrent pregnancy loss, or endometrial adenocarcinoma.
Key words: endometrium/menstrual cycle/progesterone receptor/progesterone receptor splice variants
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Introduction |
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Estrogen promotes the proliferation of cells in the human endometrium while progesterone counteracts the growth-stimulatory effects of estrogen and induces glandular and stromal differentiation suitable for blastocyst implantation (Levy et al., 1980
; Clarke and Sutherland, 1990; Ace and Okulicz, 1995). The autocrine and paracrine effects of progesterone are mediated by the human progesterone receptor (PR) which exists in two primary isoforms, termed A and B (PR-A and PR-B) (Horwitz and Alexander, 1983; Conneely et al., 1987) and a third less well-characterized isoform, PR-C (Wei and Miner, 1994). The relative levels of PR-A and PR-B are developmentally and hormonally regulated in normal target tissues (Spelsberg and Halberg, 1980; Boyd-Leinen et al., 1982; Kato et al., 1993).
The human PR gene consists of eight exons separated by seven small introns (Kastner et al., 1990). Recently, variant PR mRNAs have been described in endometrial tissue with deletions of one or more exons, generated by the alternative splicing of native (wild type) PR mRNA (Misao et al., 1998, 2000; Yamanaka et al., 2002). These variant PR mRNAs, if translated, would encode PR-like proteins which would lack functional domains for normal progesterone binding and/or DNA binding of the receptor/ligand complex. Richer et al. (1998) described PR-like proteins in breast cells, with sizes similar to predicted sizes of proteins encoded by variant PR mRNAs with exon 6 and exon 5&6 deletions. They showed that PR variants del-6 and del-5&6 proteins expressed in HeLa cells inhibited transcription by wild-type PR (Richer et al., 1998).
No study has systematically determined the presence and relative proportion of PR-splice variant mRNA transcripts in normal human endometrium throughout all phases of the menstrual cycle. If translated, the PR variant mRNAs could encode proteins lacking some functional domains of wild-type PR, thereby modifying PR-mediated gene expression. Since the expression of wild-type PR varies throughout the menstrual cycle, a detailed study of the relative expression of PR-splice variants compared to wild-type PR mRNA expression is required to yield insight into physiological mechanisms whereby progesterone responsive genes may be affected. Such a detailed study of normal endometrium could help determine whether PR variant mRNA expression is altered in the endometrium of patients with infertility, recurrent pregnancy loss or endometrial adenocarcinoma.
We have used RT–PCR amplification of cDNA to identify the presence of PR variant mRNA variants in human endometrium with whole exon deletions, including exon 4 (del-4 PR), exon 6 (del-6 PR), exon 4 and 6 (del-4&6 PR), a partial deletion of exon 6 (del-p6 PR) and a novel PR variant with a partial deletion of exon 4 (del-p4 PR). We further report the relative expression profile of these PR variants with wild-type PR mRNA expression in all stages of normal human proliferative and secretory endometrium.
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Materials and methods |
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Endometrial tissue specimens
All endometrial tissue specimens were acquired following an Institutional Review Board-approved protocol from the Department of Obstetrics and Gynecology of the Carolinas Medical Center. Endometrial tissue was obtained from women, ages 18–40, undergoing hysterectomy for benign gynaecological causes. Specimens were included if pathology did not involve the endometrium. After removal, the uterus was rapidly placed on ice and immediately taken to surgical pathology for evaluation. Two endometrial tissue strips measuring 1 by 2 cm were excised after careful inspection of the uterine cavity. One specimen was immediately stored in liquid nitrogen and the other was submitted for routine formalin fixation, paraffin embedding and staining with hematoxylin and eosin. The unfixed specimens in liquid nitrogen were transferred for freezer storage at –70°C until the time of RNA extraction by methods discussed below. The fixed and stained specimens were evaluated for endometrial dating by the method of Noyes, Hertig and Rock (1950)
. Endometrium was classified histologically by this method into early, mid- and late groups for the proliferative phase and then the closest post-ovulatory day groups for the secretory phase. These stages are well characterized histologically, allowing precise designation of endometrial development for comparison of levels of mRNA for wild-type and splice variants of PR. Specimens were included for analysis only after agreement on endometrial dating occurred between an experienced gynaecological pathologist and an investigating gynaecological physician (PM).
Isolation of RNA
Total RNA was isolated from 100 to 200 mg of endometrial tissue samples, using Trizol reagent (Gibco/BRL, Gaithersburg, MD, USA) according to manufacturer’s protocol.
RT–PCR
RT–PCR was carried out using the Qiagen OneStep RT–PCR Kit (Qiagen Inc., Valencia, CA, USA), following the conditions recommended by the manufacturer. For a 25-µl reaction, 150 ng of total RNA from each tissue sample was mixed with 5µl of 5x QIAGEN OneStep RT–PCR Buffer, 1 µl of dNTP mix containing 10 mM of each dNTP, 0.5 µM each of PR primers, 0.2 µM each of hypoxanthine-guanine phosphoribosyltransferanse (HPRT) primers, and 1 µl of Qiagen OneStep RT-PCR Enzyme Mix. HPRT was used as an internal control. The primer sequences are indicated in Figure 1. RT–PCR cycling was performed on GeneAmp PCR System 2400 or 9700 (PE Applied Biosystems, Foster City, CA, USA). RT was carried out at 50°C for 30 min, followed by a denaturation step at 95°C for 15 min. PCR was carried out as follows: 30 s of denaturation at 94°C, 30 s of annealing at 62°C and 1 min of extension at 72°C for 26 cycles, followed by a final extension step at 72°C for 10 min.
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Cloning of PR variants
The RT–PCR product of an endometrial RNA sample was resolved on a 1% agarose gel. The bands of PR variants were cut out and purified with QIAquick Gel Extraction Kit (Qiagen Inc.). The purified variants were ligated into pCR 2.1 vector and transformed into One Shot Competent cells with an Original TA Cloning Kit (Invitrogen, San Diego, CA, USA). The transformed cells were plated onto LB agar plates containing 50 µg/ml ampicillin and incubated overnight at 37°C in an incubator. The colonies were checked for sizes of the inserts by EcoRI restriction digestion. The colonies with correct insert sizes were cultured in LB broth with ampicillin, and the plasmid DNA was extracted with Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA).
Sequencing of PR variants
The extracted plasmids were cycle-sequenced with ABI BigDye sequencing kit (Applied Biosystems, Foster City, CA, USA). The cycling conditions were as follows: 94°C for 2 min, then 25 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. The reaction products were precipitated by mixing each 20-µl reaction mixture with 2 µl of sodium acetate (3 M, pH 4.6) and 50 µl of 95% ethanol and incubated at –20°C for 15 min. The pellets were spun down, washed with 70% ethanol, then dried with a Speedvac (Savant Instrument Inc., Farmingdale, NY, USA) for 20 min. Each product was resuspended in 8 µl of deionized formamide and 3 µl of EDTA (50 mM) and denatured at 95°C for 3 min. The sequencing was performed on an ABI 310 sequencer (Applied Biosystems).
Southern blot analysis
RT–PCR products (10 µl) were separated on a 1% agarose gel. The gel was soaked in denaturation solution (1.16 M NaCl and 0.5 M NaOH) for 15 min, then in neutralization solution (1.16 M NaCl and 1 M Tris) twice for 30 min. Capillary blotting was performed to transfer DNA from the gel to a piece of nitrocellulose membrane. The membrane was hybridized with Rapid Hyb (Amersham, Piscataway, NJ, USA) following the manufacturer’s instruction. The probe sequences are indicated in Figure 1. Oligonucleotide probe was labelled in a 25-µl reaction including 30 pmol probe, 2.5 µl kinase buffer, 5.0 µl Redivue adenosine 5'-[
-32P]-triphosphate (Amersham) and 1.0 µl T4-kinase on a thermocycler with a program of 30 min at 37°C followed by 10 min at 70°C. The labelled probe was purified with an Amersham MicroSpin G-25 column (Amersham) before adding to Rapid Hyb. After washing, the membrane was wrapped in saran wrap and exposed to a Fuji Imaging Plate and analysed with a Fuji Film BAS-1500 Image Reader (Fuji Photo Film Co., Miyanodai, Japan).
Radioactive RT–PCR and polyacrylamide gel electrophoresis
Radioactive RT–PCR was performed by adding 17.5 µCi redivue deoxyadenosine 5'-[
-32P]-triphosphate (Amersham) to each 25-µl reaction. Other conditions were the same as regular RT–PCR described above. Each sample was prepared by mixing 2.0 µl of radiolabelled product, 2.5 µl of deionized formamide and 1 µl of xylene cyanol dye and denaturing at 95°C for 3 min. The radiolabelled samples were resolved on denaturing polyacylamide gels containing 4.5% acrylamide (BIO-RAD, Hercules, CA, USA) and 50% urea (BIO-RAD, Hercules, CA, USA) in 1 x TBE (BIO-RAD). The gels were then transferred to filter paper and dried at 80°C for 1 h on a SpeedGel SG210D gel dryer (Savant Instrument Inc.). Then the gels were exposed to a Fuji Imaging Plate and analysed with a Fuji Film BAS-1500 Image Reader (Fuji Photo Film Co.).
Statistical analysis
The density of the wild-type PR and variant PR signals was normalized with the HPRT signal intensity. Different sets of experiments were adjusted with a reference sample which was included in all sets of experiments. The ratios of each PR band to HPRT and each PR variant to PR wild-type signal intensity were calculated and compared during phases of the menstrual cycle. The data for each sample was the average of two or three separate RT–PCR experiments. The samples were grouped as early, mid- and late proliferative stages, and PODs 2–4, PODs 5–8, and PODs 9–13 secretory stages and averaged. Extreme values were identified and excluded from the data set with the r ratio outlier test based on the 95th percentile. The exact sample number for each category after removal of outliers is indicated in Table I. As the variables in this data set were not distributed normally, it was necessary to perform non-parametric tests (Kruskal–Wallis) to assess the overall difference for each of the variants across the six stages. Signed rank tests were then used to show differences among the variants within the same sample and were compared at each of the six stages. Multiple comparisons using the Wilcoxon test (significance level: P < 0.0033) were run to determine differences between any two phases for each variant. Differences were also tested with a Bonferroni approach (significance level: P < 0.0033) to control for Type I error (overestimation of the treatment effect) thus ensuring that significant differences were not due to chance. Signed rank tests were then used to analyse differences among the variants within the same sample and were compared at each of the six stages. Again, a Bonferroni approach (significance level: P > 0.0008) was necessary to ensure that significant differences were not due to chance.
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Results |
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From an initial group of 221 human endometrial specimens, 88 samples demonstrated intact mRNA and normal histology for accurate menstrual dating (47 proliferative, 41 secretory). Samples were excluded if patients had been treated with any hormonal medications or had significant medical illnesses with the potential to effect endometrial integrity.
By RT–PCR using primers from exons 2 and 8 (which are common to PR-A and PR-B), five distinct bands representing wild-type PR and PR variants were detected on agarose gel in normal endometrial tissues (data not shown). These bands were extracted from the agarose gel, cloned and sequenced. Figure 1 shows the mRNA structural organization of these PR variants. The largest RT–PCR band represents the wild-type PR. The other four bands contain five PR splice variants with different exon deletions. The two PR variants with an entire deletion of exon 4 (del-4 PR) or deletion of exon 6 (del-6 PR) were detected in human endometrium in agreement with prior reports (Misao et al., 1998; Yamanaka et al., 2002). We confirmed the presence of the PR variants with a partial deletion of exon 6 (del-p6 PR) or deletion of both exon 4 and 6 (del-4&6 PR) recently reported by Hisatomi et al. (2003) and Nagao et al. (2003). Our study documents the first report of a novel PR variant mRNA with a partial deletion of exon 4 (del-p4 PR) in human endometrium. The mRNA for the del-p4 PR would encode a protein with an in-frame deletion, deleting part of the nuclear localization signal from the corresponding protein. The variants of del-p4 PR (967 bp) and del-6 PR (962 bp) comigrated on an agarose gel. To resolve the presence of both PR variant species in that band, two probes were designed so that only one of the two PR variants could be detected but not the other. Southern blot analysis was then performed, and the results indicate that the del-p4 PR and del-6 PR mRNA are both real and contribute similar proportions to the band intensity (Figure 2).
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For the purpose of quantification, RT–PCR conditions for cycle number and initial total RNA input were optimized in the exponential phase of amplification (data not shown). Under the optimized conditions (26 PCR cycles and 150 ng RNA), semi-quantitative analysis by radioactive RT–PCR was carried out to measure the mRNA expression of wild-type PR and PR variants. The transcripts for wild-type PR and all PR variants were co-expressed in all proliferative and secretory endometrial samples. The mRNA expression of wild-type PR and all PR variants was higher in the proliferative phase compared to the secretory phase (Figure 3; Table I). Statistical analysis demonstrated the above mentioned significant differences in mRNA expression of wild-type PR and all PR variants throughout the menstrual cycle, when normalized to HPRT mRNA (P < 0.0001). The ratio of mRNA expression for each PR variant compared to wild-type PR was constant throughout the menstrual cycle (Figure 4). Statistical analysis showed no significant differences in the ratio for each PR variant to wild-type PR mRNA throughout the menstrual cycle and between any two phases (Table I). Analysis of the relative hybridization intensity indicated del-4 PR, del-p6 PR. and del-4&6 PR were approximately 2, 2 and 1%, respectively, of wild-type PR mRNA expression. The combined hybridization intensity of del-6 PR and del-p4 PR together was approximately 10% of wild-type PR (Figure 4). Within each phase of the menstrual cycle except the late proliferative phase (sample size too small), the ratio of del-4&6 PR to wild-type PR mRNA expression was significantly lower than the ratios of del-4 PR or del-p6 PR to wild-type PR mRNA expression (P < 0.0008) (Table I). Southern blot analysis was used to confirm the above results.
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Discussion |
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We present the largest and most systematic study of the expression of mRNA for wild-type PR and PR variants in the human endometrium throughout its development. Semi-quantitative analysis indicated that the expression of mRNA for wild-type PR was higher in the proliferative phase compared to the secretory phase, a finding consistent with the known profile of protein for wild-type PR during endometrial development (Lessey et al., 1988
; Mylonas et al., 2004). It further parallels the expression profile seen by wild-type estrogen receptor (ER) alpha that we reported previously (Marshburn et al., 2004). This data is consistent with the findings that ER induces PR expression and that antagonizing estrogen action promotes PR down-regulation.
In the human endometrium, progesterone induces cellular differentiation and is primarily antagonistic to estrogen-mediated cell proliferation (Clarke and Sutherland, 1990). Estradiol appears to up-regulate ER and PR expression, whereas progesterone down-regulates expression of both receptors (Lessey et al., 1988). Endogenous influences that alter progesterone-mediated gene expression could play a physiological role in modulating the action of estrogen and progesterone in human endometrial development. Furthermore, understanding factors that may induce dysregulation of progesterone-mediated gene expression could elucidate mechanisms promoting unopposed estrogen stimulation, a common factor underlying the development of endometrial carcinoma. Indeed, expression of high levels of PR is associated with improved outcome with endometrial carcinoma (Fukuda et al., 1998).
Findings from in vitro studies suggest that PR variants could impair PR-mediated gene expression in breast cancer tissues, an effect that theoretically could lessen the hormone responsiveness of breast cancer and worsen prognosis. The PR variant with deletion of all of exon 6 (del-6 PR) could bind to DNA but be transcriptionally inactive, thereby inhibiting PR mediated gene expression by blocking DNA binding with activated wild-type PR. Richer et al. (1998) documented that transfection with the del-6 PR variant inhibits the activity of the wild-type PR in breast cancer cell lines. Leygue et al. (1999) showed that the relative mRNA expression of del-6 PR compared to wild-type PR mRNA was higher in breast cancer than normal breast tissue specimens. This finding was in concert with another investigation reporting that mRNA for a 52 base pair, partial deletion of exon 6 (del-p6 PR), was expressed more frequently in breast cancer tissue than in non-cancerous breast tissue (Hisatomi et al., 2003). The significance of these findings in breast cancer pathogenesis is presently unknown because of the low relative expression of PR variant mRNA, the co-existence of PR variant mRNA in both normal and breast cancer tissue and because variant PR proteins encoded by PR variant mRNA have not yet been conclusively demonstrated (Balleine et al., 1999; Nagao et al., 2003).
PR variants with whole or partial exon deletions have been identified in endometrium and in endometrial cancer (Misao et al., 1998, 2000; Yamanaka et al., 2002). No systematic study, however, has been accomplished to assess the relative expression of mRNA for PR and PR variants in the human endometrium throughout its development during the menstrual cycle. Differential co-expression of mRNA for PR variants compared to wild-type PR could modulate regulation of progesterone action during endometrial development if the mRNA for these PR variants were translated into functional proteins.
The expression of mRNA for all of the PR variants with deletion of whole exons [exons 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4 & 6 PR)] and partial exon deletions [partial 6 (del-p 6 PR), partial 4 (del-p4 PR)] was detected at low levels during all stages of endometrial development. We demonstrate that the relative expression of mRNA for these PR variants compared to wild-type PR did not vary through early, mid- and late proliferative and secretory endometrial development. Thus, we find no evidence of differential expression of PR variant mRNA during proliferative and secretory endometrial development and conclude that PR variant expression is affected predominately by the expression of the wild-type PR. Therefore, even if PR variant mRNA is translated into protein, we would expect no impact on wild-type PR function during normal endometrial development.
Our data agree with prior investigations indicating the presence of the del-4 PR and del-6 PR mRNAs in normal endometrium (Misao et al., 1998; Yamanaka et al., 2002). Our findings contrast with other studies on expression of mRNA for PR variants in endometrium in the following ways. Misao et al. (1998) found that wild-type PR, del-6 PR and del-4 PR variant mRNAs were observed in all samples of proliferative and secretory phase of the endometrium. This group, however, did not detect mRNA for del-4&6 PR in normal endometrium but only in ovarian endometriosis. Yamanaka et al. (2002) also examined proliferative phase endometrium and found the del-4 PR and del-6 PR mRNAs but could not clone the del-4&6 PR variant by RT–PCR. We did detect the del-4&6 PR variant in all phases of endometrium, but its ratio to wild-type PR was the lowest of all PR variant mRNAs investigated. Putative proteins encoded by mRNA for del-4 PR and del-4&6 PR lack the DNA binding domain in addition to the steroid-binding domain. Therefore, it is doubtful that these putative proteins are functional as a PR.
The potential exists for alternatively spliced variants of PR to play a role in the pathogenesis of hormone dependent cancers (e.g. endometrial and breast). Expression of mRNA for the del-p6 PR (Hisatomi et al., 2003) and del-4&6 (Nagao et al., 2003) variants was detected more frequently in breast cancer tissues than in non-cancer tissues. Misao et al. (2000) demonstrated the presence of mRNA for the del-4 PR, del-6 PR and del-3&4 PR variants in all specimens of well, moderately and poorly differentiated endometrial cancers. This group, however, did not detect the expression of del-5,6 PR, del-4,5,6 PR, and/or del-3,4,5,6 PR mRNA in some cases of endometrial cancer, especially poorly differentiated adenocarcinoma compared to well and moderately differentiated adenocarcinoma.
Reports to date on endometrial and breast cancer have focused on the presence or absence of mRNA for PR variants in cancer compared to normal tissue without providing information about their relative expression with wild-type PR (Misao et al., 2000; Nagao et al., 2003). Data on the relative expression of mRNA for PR variants and wild-type PR are important because the absolute density of wild-type PR varies during endometrial development and is altered in endometrial adenocarcinoma (Fukuda et al., 1998; Arnett-Mansfield et al., 2001, 2004). We report that mRNA for PR variants is not differentially co-expressed with wild-type PR during endometrial development. As disruption of PR expression has been observed in complex, atypical endometrial hyperplasia and endometrial adenocarcinoma (Arnett-Mansfield et al., 2001), our data can serve as a basis for systematic evaluation of the presence of protein for these specimens during endometrial development and in abnormal endometrial changes such as hyperplasia and adenocarcinoma.
It is not known if abnormal PR variant expression may be an underlying factor in some women who experience unexplained infertility, recurrent pregnancy losses, abnormal uterine bleeding endometrial and hyperplasia. We hope that the data presented in this study will promote further investigation and provide insight into endometrial causes for these conditions. Studies are underway from our group to determine whether a different profile of PR variant expression, compared to the profile during normal endometrial development, is detected in the endometrium of patients with these clinical disorders.
In conclusion, we are the first to report and characterize the novel PR variant mRNA with a partial deletion of exon 4. Furthermore, the co-expression of the variant PR mRNA is constant relative to the wild-type PR throughout the menstrual cycle. We would not expect that these variant PR mRNAs would play a prominent role in normal endometrial physiology because their relatively constant and low level of expression would be masked by the progesterone responsive gene expression mediated by its action upon the wild-type PR. The data presented in this study will allow our group and other investigators the opportunity to determine if these PR variant mRNAs are translated into functional proteins and if they are, examine whether these proteins influence progesterone-mediated gene expression in the human endometrium.
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Acknowledgements |
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The study and manuscript could not have been accomplished without the expert scientific advice of Joseph Wagstaff, MD, PhD, Department of Pediatrics, assistance of Virginia K. Bond, Research Technician from Cannon Research and Lillian Lawrence from the Carolinas Medical Center. This study was support by the Health Services Foundation, Carolinas Medical Center, Charlotte, NC.
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Submitted on September 28, 2005; accepted on October 26, 2005.
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