Single nucleotide polymorphism associated with nonsyndromic cleft palate influences the processing of miR-140.код для вставкиСкачать
RESEARCH ARTICLE Single Nucleotide Polymorphism Associated With Nonsyndromic Cleft Palate Influences the Processing of miR-140 Ling Li,1 Tian Meng,1 Zhonglin Jia,1 Guiquan Zhu,1 and Bing Shi1,2* 1 State Key Laboratory of Oral Disease, West China College of Stomatology, Sichuan University, Chengdu, PR China 2 Department of Cleft Lip and Palate Surgery, West China College of Stomatology, Sichuan University, Chengdu, PR China Received 10 June 2009; Accepted 13 November 2009 Nonsyndromic oral cleft is a common developmental malformation of humans. Embryonic development is regulated by microRNAs. MicroRNA-140-5p (miR-140-5p) was found to regulate palatal development. As sequence variants in miRNA genes are likely to affect miRNA expression and/or maturation, we investigated the miRNA-140 gene and identified a SNP (rs7205289: C>A) located in precursor miRNA-140. We carried out a case–control analysis in 557 patients with nonsyndromic oral clefts and 306 unaffected controls from west China and found that the frequency of minor allele (A allele) was significantly increased (P ¼ 0.003 after Bonferroni correction) in nonsyndromic cleft palate (NSCP) patients in comparison with that in controls. We constructed expression vectors of primary miRNA-140 (pri-miR140) with the major and minor alleles of rs7205289. The vectors were transfected into HEK293 cells, and the mature forms of miR -140 were detected by Northern blot. Compared to the vector with the C allele, the vector with the A allele was found to influence the miR-140 processing, resulting in a significant decrease of miR-140-5p and an increase of miR-140-3p. These results suggest that the SNP located in pre-miR-140 contributes to NSCP susceptibility by influencing the processing of miR-140. 2010 Wiley-Liss, Inc. Key words: microRNA-140; cleft palate; SNP INTRODUCTION Nonsyndromic oral clefts (NSOC) have a prevalence of approximately 1/700 worldwide [Bender, 2000]. There are three main types of NSOC: nonsyndromic cleft lip (NSCL), nonsyndromic cleft lip and palate (NSCLP), and nonsyndromic cleft palate (NSCP). The genesis of NSOC involves numerous cellular and genetic factors during embryonic development. Vertebrate embryonic development is extensively regulated by microRNAs [Wienholds and Plasterk, 2005; Wienholds et al., 2005; Darnell et al., 2006; Kloosterman et al., 2006], including craniofacial development [Darnell et al., 2006]. MicroRNAs (miRNAs) are 22 nucleotide RNAs that regulate post-transcriptional eukaryotic gene expression. Recently, Eberhart 2010 Wiley-Liss, Inc. How to Cite this Article: Li L, Meng T, Jia Z, Zhu G, Shi B. 2010. Single nucleotide polymorphism associated with nonsyndromic cleft palate influences the processing of miR-140. Am J Med Genet Part A 152A:856–862. et al.  found that microRNA-140-5p (miR-140-5p) regulated craniofacial development, because it targeted PDGFRA, and thus regulated PDGF signaling during neural crest cell migration. In zebrafish, the reduction of miR-140-5p increased the level of PDGFRA protein and altered palatal shape and an increased level of miR-140-5p reduced the PDGFRA protein and caused cleft palate [Eberhart et al., 2008]. As the interaction of miR-140-5p and PDGFRA is conserved across species [Eberhart et al., 2008], human craniofacial development could also be regulated by miR140-5p. Generally, miRNAs are initially transcribed as primary miRNAs (pri-miRNA), which are then processed into a 60 nt hairpin shaped precursor miRNAs (pre-miRNA) by the Drosha–DGCR8 complex [Lee et al., 2003; Han et al., 2004]. Precursor miRNAs are cleaved by RNase III, Dicer, to produce a 22 base-pair duplex RNA [Kim, 2005]. The duplex generates mature miRNAs from each of the strands with different levels, called miRNA-5p and miRNA-3p [Landgraf et al., 2007]. Subsequently, miRNA guides RNA-induced silencing complex (RISC) to specific target sites within mRNAs to induce immediate cleavage or post-translational repression [Song Grant sponsor: State Key Laboratory of Oral Diseases; Grant Number: 2008.1–2009.12; Grant sponsor: National Science Funds of China; Grant Number: 30530730. *Correspondence to: Bing Shi, Department of Cleft Lip and Palate Surgery, West China College of Stomatology, Sichuan University, No. 14, Section 3, RenMinNan Road, Chengdu 610041, PR China. E-mail: email@example.com Published online 23 March 2010 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.a.33236 856 LI ET AL. et al., 2004]. However, the normal miRNA processing can be influenced by sequence variants in miRNA genes, because processing requires precise structure of the transcripts [Lee et al., 2003; Han et al., 2004, 2006; Kim, 2005; Winter et al., 2009]. As it has been shown that sequence variants in miRNA genes could potentially alter miRNA processing and contribute to human diseases [Gottwein et al., 2006; Duan et al., 2007; Sethupathy and Collins, 2008], the aim of our study was to investigate if a SNP located in miR-140 gene could change the processing of that miRNA and contribute to NSOC phenotype. MATERIALS AND METHODS Subjects of Case–Control Analysis Our sample consisted of 557 patients with NSOC and 306 unaffected controls. The patients were ascertained from the Department of Cleft Lip and Palate Surgery, West China College of Stomatology, Sichuan University, and the unaffected controls were ascertained from the West China Women’s and Children’s Hospital, Sichuan University. The study was prospectively approved by the Ethics Committee of Sichuan University. The subjects were recruited between 2005 and 2008, and self-identified as western Han Chinese. To assess the nonsyndromic status of the affected patients, all were screened for the presence of associated anomalies [Calzolari et al., 2007] or syndromes [Cohen, 1978] by a physician, and only those determined to have an isolated cleft were included in this study. None of the unaffected controls had signs of oral clefting or associated anomalies [Calzolari et al., 2007]. Each participant was interviewed in person by trained interviewers (if the participant was a child, his/her parents were interviewed), and the participants with a family history of oral cleft or associated anomalies in first or second degree relatives were excluded from this study. Cell Line and Reagents 857 ABI PRISM 7900HT Sequence Detection System and SDS 2.2 software (Applied Biosystems). The reaction employed the TaqMan Universal PCR Master Mix, TaqMan primers and probes, water and 5 ng of DNA. The thermal cycle program included one cycle at 95 C for 10 min to activate the AmpliTaq Gold enzyme activation, 40 cycles at 92 C for 15 sec to denature the DNA and at 60 C for 1 min for the stage of annealing/extension. The sequences of primers and probes for this polymorphism were 50 -GCTTGGTGGGCTTCTGGT (forward primer), 50 -CCCGGTATCCTGTCCGTGGT (reverse primer), VIC-50 -TAAAACCACTGGCAGGACAC (reporter 1) and FAM-50 -TAAAACCACTGTCAGGACAC (reporter 2). Construction of a Recombinant Expression Vector We used pSUPER RNAi System. Vectors were constructed in pSUPER containing the H1 RNA polymerase III promoter according to the manufacturer’s instructions. As Drosha function requires single-stranded RNA extensions outside the pri-miRNA hairpin [Zeng and Cullen, 2005], the sequence of the mature miR-140 and the region including 18 nt upstream of the 50 cleavage site of Drosha and 20 nt downstream of the 30 cleavage site of Drosha were used as the minimal pri-miRNA [Han et al., 2004] (Fig. 1). The 100 nt minimal pri-miRNA-140 linked 30 to the sequence 50 -TTTTT-30 . The oligos were self-annealed in 6 SSC using standard protocols and ligated on to the BglII and HindIII sites of the vector pSUPER. The pri-miR-140-C and pri-miR-140-A was generated by introducing a C-A substitution (tctctctctgtgtcctgC/Acagtggttttaccctatggtaggttacgtcatgctgttctaccacagggtagaaccacggacaggataccggggcaccct). The two kinds of vectors were referred to as pSUPER-miR-140-C and pSUPER-miR-140-A. We further constructed pSUPER-miR125a which served as a positive control for the miRNA biogenesis machinery (catgttgccagtctctaggtccctgagaccctttaacctgtgaggacatccagggtcacaggtgaggttcttgggagcctggcgtctggcccaac). The primers and probes for polymorphism genotyping were purchased from Shanghai Genecore BioTechnologies Co., Ltd (Shanghai, China). Human HEK293 cells were obtained from State Key Laboratory of Oral Disease, Sichuan University. The TaqMan Universal PCR Master Mix was purchased from Applied Biosystems (Foster City, CA). The pSUPER Vector was purchased from OligoEngine (Seattle, WA). The oligonucleotides were synthesized by Takara (Dalian, China). Lipofectamine2000 Reagent was purchased from Invitrogen Corporation (Rockville, MD). DNA probes complementary to miR-140-5p, miR-140-3p, and miR-125a were synthesized by Takara Biotechnology Co., Ltd (Dalian, China). The MEGALABEL Kit (Takara) was used for probe labeling. RNA size marker was from Ambion (Austin, TX), and positive charged nylon membrane was from Bio-Rad Laboratories (Hercules, CA). SNP Genotyping We drew venous blood samples from participants, and extracted DNA by a phenol chloroform extraction protocol. The DNA samples were sequenced by Takara. Genotyping for SNPs was carried out with 50 nuclease assay (Taqman; Applied Biosystems) as described [Landi et al., 2008]. Analysis was performed using the FIG. 1. The SNP is located in pre-miR-140. The stem–loop structure of pre-miR-140 with major (A) and minor (B) alleles, as predicted by Mfold [Zuker, 2003], are shown. Mature miRNA nucleotides are bold highlighted, and the polymorphic nucleotide is underlined. The free energy calculated by Mfold is indicated on the right. The arrow indicates the normal cleavage site of Drosha. 858 AMERICAN JOURNAL OF MEDICAL GENETICS PART A Cell Culture and Transfection Human HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Plasmids were transfected into human HEK293 cells by Lipofectamine 2000 (Invitrogen Corporation) following the manufacturer’s protocol. After 6 h of transfection, the medium was replaced by serum-containing medium and incubated for a further 48 hr. Case–Control Analysis of SNP Northern Blot Total RNA was isolated from the nuclear and cytoplasmic fractions of cells with Trizol Reagent (Invitrogen Corporation), and separated on 15% TBE urea gel, then transferred at 250 mA. The transferred RNA was cross-linked to nylon membrane (Bio-Rad Laboratories) by UV irradiation. Oligonucleotide probes used for detecting miR-140-5p (50 -CCTACCATAGGGTAAAACCACT), miR-140-3p (50 -TCCGAGGTTCTACCCTGTGGTA), and miR125a (50 -TCACAGGTTAAAGGGTCTCAC) were labeled according to the manufacturer’s protocol. The membranes were pre-hybridized at 42 C for 1 hr and hybridized for 12–16 hr at room temperature (RT). Then, the membranes were washed three times at RT and once at 42 C with 2 SSC and 0.2 SSC, respectively, and exposed to X-ray film (Kodak) for 19 hr and analyzed by densitometer (GS-700; Bio-Rad Laboratories). Statistical Analysis Hardy–Weinberg equilibrium was assessed with alpha threshold of 0.05 among the normal controls as samples of the western Han Chinese population (Table I). Chi-square statistics were used to assess significance in case–control comparisons. Two-tailed P value, odds ratio, and 95% confidence intervals (95% CI) were calculated with the SAS 8.1 statistical software package (SAS Institute). Bonferroni correction was used to adjust P values. RESULTS Identification of SNP Associated With MiR-140 To identify the SNP associated with miR-140, we began with a bioinformatic search in a miRNA-SNP database (http://www. patrocles.org/), and verified it on dbSNP (http://www.ncbi.nlm. nih.gov/SNP/). As a result, we identified that one SNP (rs7205289) was located in pre-miR-140 (Fig. 1A). Using Mfold [Zuker, 2003], TABLE I. Cleft Type and Sex of Controls and Patients NSCL NSCLP NSCP Control Total 105 283 169 306 we further found that rs7205289 could introduce a base-pairing mismatch, alter the free energy value, and create an RNA bulge around the Drosha cleavage site (Fig. 1B). As the precise structure of the stem outside pre-miRNA was found to be critical for miRNA processing and could slightly modify the precise cleavage site of Drosha [Zeng et al., 2005], we hypothesized that the processing of miR-140 could be influenced by rs7205289. Male 60 159 78 150 Female 45 124 91 156 x2 2.064 3.026 0.358 P-value 0.151 0.082 0.549 NSCL, nonsyndromic cleft lip; NSCLP, nonsyndromic cleft lip and palate; NSCP, nonsyndromic cleft palate; c2, Chi-square. To confirm the presence of this SNP in the general population, 20 samples [Zhang et al., 2002] were randomly selected from 306 unaffected controls. Eleven samples were sequenced initially and a single AA genotype was found. The other nine samples were not sequenced because the minor allele frequency would be 5% (1/20) [Unneberg et al., 2005] even if we could not find any more minor alleles in the other nine samples. We hypothesized that miR140 could modulate craniofacial development [Darnell et al., 2006; Eberhart et al., 2008] and that rs7205289 might have a different allelic or/and genotypic frequency in NSOC patients. To test our hypothesis, a case–control study of rs7205289 was undertaken in 557 patients with NSOC and 306 unaffected controls. The patients were divided by sex and cleft types (Table I). There was no significant difference in the sex distribution between the patients and controls, suggesting that our frequency-matching on sex was satisfactory. According to Hardy–Weinberg equilibrium, the observed genotypic distribution in controls was not different from the expected distribution using an alpha level of 0.05 as significant threshold (Table II). Genotypic and allelic frequencies were shown in Table III. The CC genotype was compared with a group that contained the other two genotypes (CA þ AA). The genotypic frequency comparison showed that the frequency of CC genotype was significantly lower in NSCP patients than that in controls (P ¼ 0.027 after Bonferroni correction), with an odds ratio of 0.54 (95% CI, 0.36–0.83). However, the genotypic frequency of NSCL and NSCLP groups were not different from that of the control group. The allelic frequency comparison showed that the C allele frequency was significantly decreased in NSCP patients than that in controls (P ¼ 0.003 after Bonferroni correction), with an odds ratio of 0.55 (95% CI, 0.38–0.79). However, the C frequency in patients with NSCL and NSCLP was not significantly different from that of controls. These results indicated that, compared with the controls, patients with NSCP had significantly different genotypic and allelic frequencies of rs7205289. Construction of MiR-140 Expression Vector To explore the function of rs7205289, we generated expression vectors using the pSUPER system. As Drosha function requires single-strand RNA extensions outside the pre-miRNA hairpin [Zeng and Cullen, 2005], the minimal pri-miRNA [Han et al., 2004] of miR-140 was used (Fig. 1A,B). Human HEK293 cells were transfected with pSUPER-miR-140-C and empty pSUPER vectors. The expression of miR-140 was assessed by Northern blot (Fig. 2). Both miR-140-5p and miR-140-3p were increased in the cells transfected with pSUPER-miR-140-C, compared with that in the LI ET AL. 859 TABLE II. Genotype Frequencies of Controls and Patients followed Expected Hardy–Weinberg Equilibrium Distribution Genotype CC CA AA Total x 2 (P, df ¼ 2) P-value NSCL 90 (85.7%) 14 (13.3%) 1 (0.95%) 105 0.293 0.588 NSCLP 234 (82.7%) 45 (15.9%) 4 (1.41%) 283 1.131 0.288 NSCP 113 (66.9%) 47 (27.8%) 9 (5.33%) 169 1.855 0.173 Control 241 (78.8%) 59 (19.3%) 6 (1.96%) 306 1.100 0.294 NSCL, nonsyndromic cleft lip; NSCLP, nonsyndromic cleft lip and palate; NSCP, nonsyndromic cleft palate; c2, Chi-square. The percentages of genotype frequency are in parentheses. cells transfected with empty vector (Fig. 2). This result suggests that pSUPER-miR-140 was sufficient for expression. Pre-miR-140 SNP Influenced the Processing of MiR-140 In order to test the function of the minor allele, the C allele was substituted for the A allele in the expression vector. Human HEK293 cells were transfected with pSUPER-miR-140-C and pSUPER-miR-140-A, respectively. The empty pSUPER vector was used as a negative control. Upon pSUPER-miR-140-C transfection, we detected the mature form of miR-140-5p and miR-140-3p by Northern blot, and the quantity of miR-140-5p was higher than that of miR-140-3p (Fig. 3). However, in the cells transfected with pSUPER-miR-140-A, we detected a reduction of miR-140-5p and increase of miR-140-3p, compared with that in cells transfected with pSUPER-miR-140-C (Fig. 3). This phenomenon could be due to either altered miRNA processing or a defect in miRNA biogenesis machinery in the transfected cells [Duan et al., 2007]. To exclude the possibility that the expression of pSUPER-miR-140-A interfered with the miRNA biogenesis machinery, we used pSUPER-miR125a as a positive control, because human HEK293 cells do not express miR-125a endogenously [Landgraf et al., 2007]. After simultaneously transfecting human HEK293 cells with pSUPERmiR-125a and miR-140 expression vectors, we observed comparable miR-125a expression in the cells with either miR-140 expression vector (Fig. 3). These results suggest that the miRNA-140 SNP did not alter the biogenesis machinery of miRNA in human HEK293 cells, and that the vector with the A allele generated less miR-140-5p and more miR-140-3p than that of the vector with the C allele. DISCUSSION Embryonic development can be regulated by miRNAs at the posttranscriptional level. MicroRNA-140 was found to have a conserved regulatory role in craniofacial development [Eberhart et al., 2008]. TABLE III. Rs7205289 Genotype and Allele Distributions in Controls and Patients Genotype CC CA AA Total x 2 (P, df ¼ 2) P-value P-value* NSCL 90 (85.7%) 14 (13.3%) 1 (0.95%) 105 2.493 0.287 NS OR 95% CI 1.62 0.878–2.982 C A x 2 (P, df ¼ 2) P-value P-value* OR 95% CI 194 (92.4%) 16 (7.6%) 2.62 0.106 NS 1.59 0.903–2.80 NSCLP 234 (82.7%) 45 (15.9%) 4 (1.41%) 283 1.429 0.474 NS CC vs. CA þ AA 1.29 0.853–1.945 Alleles 513 (90.6%) 53 (9.4%) 1.563 0.211 NS 1.27 0.872–1.85 NSCP 113 (66.9%) 47 (27.8%) 9 (5.33%) 169 9.519 0.009 0.027 Control 241 (78.8%) 59 (19.3%) 6 (1.96%) 306 0.544 0.357–0.830 273 (80.8%) 65 (19.2%) 10.33 0.001 0.003 0.55 0.382–0.795 541 (88.4%) 71 (11.6%) NSCL, nonsyndromic cleft lip; NSCLP, nonsyndromic cleft lip and palate; NSCP, nonsyndromic cleft palate; c2, Chi-square; OR, odds ratio; NS, not significant after Bonferroni correction. *P values after Bonferroni correction. The percentages of genotype and allele frequency are in parentheses. The P values less than 0.05 are in bold. 860 AMERICAN JOURNAL OF MEDICAL GENETICS PART A FIG. 2. Human HEK293 cells transfected with pSUPER-miR-140-C expressed more miR-140-5p and miR-140-3p than those transfected with pSUPER empty vector. In the present study, we performed a case–control study and found that rs7205289, located in pre-miR-140, was associated with an increased risk of NSCP. In subsequent in vitro analysis, pSUPERmiR-140-A was found to increase miR-140-3p expression and decrease miR-140-5p expression, compared to pSUPER-miR-140-C. These data suggest that rs7205289 may contribute to NSCP susceptibility and the A allele at rs7205289 could influence the processing of miR-140. In this study, we found that rs7205289 was associated with NSCP. After Bonferroni correction, the CC genotypic frequency of patients with NSCP was significantly lower than that of the unaffected controls, indicating that, compared with CA and AA genotypes, the CC genotype has a protective effect on NSCP. There was no difference in the allelic and genotypic frequencies between patients with NSCL or NSCLP and controls, indicating that NSCL and NSCLP may not share a common causal variant with NSCP on miR140 gene. This result is in accordance with previous studies that NSCL and NSCLP are considered etiologically distinct from NSCP [Harville et al., 2007; Weinberg et al., 2008]. However, the modest sample size and Chinese Han ethnic are limitations of this study. Alarger sample size of patients with NSOC from other ethnic origins should be performed to confirm the allelic and genotypic distribution. Eberhart et al.  found in zebrafish that an increase of miR140-5p caused cleft palate. On the contrary, we found that the A allele generated less miR-140-5p, and that the A allele frequency was increased in patients with NSCP. This discrepancy could be understood when we take the etiology of NSCP into consideration. FIG. 3. Human HEK293 cells were transfected with miR-140 expression vector and pSUPER-miR-125a (as a positive control). Total RNAs isolated from transfected cells was used for Northern blots as shown. The expression vector of pri-miR-140-A (pSUPERmiR-140-A) produced more miR-140-3p, but less miR-140-5p than that of pri-miR-140-C (pSUPER-miR-140-C). We used 5S RNA as a loading control. NSCP is a multifactorial disease with strong genetic background [Murray, 2002; Weinberg et al., 2008]. It has been demonstrated that reduced PDGF signaling during neural crest migration could lead to the failure of palatal precursors to reach the oral ectoderm, whereas elevated PDGF signaling could reduce the number of rostrally migrating crest cells that reach the oral ectoderm and alter palatal shape [Eberhart et al., 2008]. Both of the cases result in abnormal development of the palate. Therefore, the A allele probably contributes to NSCP susceptibility as one of its genetic factors, which could regulate PDGF signaling and may interact with other factors. In addition, we found that miR-140-3p is increased by the A allele. However, as the function of miR-140-3p remains unclear, further studies of miR-140-3p may provide new insights into the function of rs7205289. As the amount of common SNPs in miRNA genes is less than that in miRNA target sites [Saunders et al., 2007], most recent studies focused on the SNPs in miRNA target sites. However, polymorphisms in miRNA genes, including pri-miRNA, pre-miRNA and mature miRNA, could impact various biological processing [Gottwein et al., 2006; Duan et al., 2007; Georges et al., 2007; Saunders et al., 2007; Yu et al., 2007; Sethupathy and Collins, 2008]. In this study, we found that the A allele generated a bulge on the stem, altered the free energy value at the cleavage site of Drosha, and LI ET AL. thus probably influenced miRNA processing. This is consistent with previous studies suggesting that variations around the Drosha cleavage site could fine-tune the actual cleavage sites [Zeng and Cullen, 2003; Zeng et al., 2005]. Therefore, our result demonstrates the function of SNPs, especially those located at the Drosha cleavage site in pre-miRNA genes, is important for miRNA processing. Our results suggest that the A allele might induce cleavage site shift and thereby influence the processing of miR-140. There are two possible explanations for this phenomenon. First, as both primiR-140-C and pri-miR-140-A could effectively generate miR-140 -5p and miR-140-3p, the SNP may not disturb the biogenesis machinery of miR-140. Second, although miRNA processing includes cleavage by Drosha and Dicer, Drosha actually selects its cleavage site, dictates where Dicer will cleave and hence, which strand of the miRNA duplex enters RISC [Lee et al., 2003; Bartel, 2004]. In our study, RISC favored miR-140-5p from pri-miR-140C and miR-140-3p from pri-miR-140-A, respectively. Given the fact that RISC favors the strand with relatively unstable 50 -end in the duplex [Schwarz et al., 2003], we suggest that miRNA duplexes generated by pri-miR-140-C and pri-miR-140-A may have different 50 -ends with different stabilities. This difference might come from the shift of Drosha cleavage site because of rs7205289. However, further studies should be conducted to confirm our hypothesis. In summary, our findings show that rs7205289 in pre-miR-140 may contribute to NSCP susceptibility by influencing the processing of miR-140. We suggest a pathogenic correlation among the SNP, miRNA, and NSCP. Further exploration of these miRNA-associated SNPs would improve our understanding of the potential contribution of these SNPs to the pathogenesis of human disease. ACKNOWLEDGMENTS This study was supported by the State Key Laboratory of Oral Diseases (2008.1–2009.12) and the National Science Funds of China (30530730). We thank the participants who donated samples to this study. 861 Eberhart JK, He XJ, Swartz ME, Yan YL, Song H, Boling TC, Kunerth AK, Walker MB, Kimmel CB, Postlethwait JH. 2008. MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis. Nat Genet 40:290–298. Georges M, Coppieters W, Charlier C. 2007. Polymorphic miRNA-mediated gene regulation: Contribution to phenotypic variation and disease. Curr Opin Genet Dev 17:166–176. Gottwein E, Cai XZ, Cullen BR. 2006. 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