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DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations
YBCMD-01827; No. of pages: 5; 4C: Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
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DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations Jin Wang a, Qi-Zhi Xiao b, You-Ming Chen c, Sheng Yi a, Dun Liu a, Yan-Hui Liu d, Cui-Mei Zhang e, Xiao-Feng Wei a, Yu-Qiu Zhou b, Xing-Ming Zhong f, Cun-You Zhao a, Fu Xiong a, Xiang-Cai Wei a,f, Xiang-Min Xu a,⁎ a
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China Department of Birth Health and Heredity, Zhuhai Women and Children Care Hospital, Zhuhai, Guangdong, China c Department of Clinical Laboratory, the Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China d Prenatal Diagnosis Center, Dongguan Maternal and Child Health Hospital, Dongguan, Guangdong, China e Department of Child Health, Boai Hospital of Zhongshan, Zhongshan, Guangdong, China f Department of Reproductive Immunology and Endocrinology, Family Planning Research Institute of Guangdong, Guangzhou, Guangdong, China b
a r t i c l e
i n f o
Article history: Submitted 19 May 2014 Accepted 1 June 2014 Available online xxxx Communicated by M. Narla, DSc, 01 June 2014 Keywords: G6PD Phenotypic variation Women heterozygous Methylation X chromosome inactivation
a b s t r a c t Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely dominant enzyme deficiency that results from G6PD gene mutations. Women heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity but the cause of this phenotypic variation is unclear. We determined DNA methylation and X-inactivation patterns in 71 G6PD-deficient female heterozygotes and 68 G6PD non-deficient controls with the same missense mutations (G6PD Canton c.1376GNT or Kaiping c.1388GNA) to correlate determinants with variable phenotypes. Specific CpG methylations within the G6PD promoter were significantly higher in G6PDdeficient heterozygotes than in controls. Preferential X-inactivation of the G6PD wild-type allele was determined in heterozygotes. The incidence of preferential X-inactivation was 86.2% in the deficient heterozygote group and 31.7% in the non-deficient heterozygote group. A significant negative correlation was observed between X-inactivation ratios of the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in heterozygous G6PD Canton (r = −0.657, p b 0.001) or Kaiping (r = −0.668, p b 0.001). Multivariate logistic regression indicated that heterozygotes with hypermethylation of specific CpG sites in the G6PD promoter and preferential X-inactivation of the wild-type allele were at risk of enzyme deficiency. © 2014 Elsevier Inc. All rights reserved.
Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common inherited enzymopathies, affecting approximately 400 million people worldwide, predominantly in Africa, the Middle East and Southeast Asia [1]. G6PD deficiency is caused by mutations in the G6PD gene and more than 160 different mutations associated with G6PD have been described [2]. The inheritance of G6PD deficiency shows a typical X-linked incomplete dominant pattern and most G6PD deficient patients are men. The majority of heterozygous women have been reported with normal G6PD activity, and variable loss of enzyme activity has been observed in some heterozygous females [1,3], suggesting
⁎ Corresponding author at: Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, 1838 Guangzhou Dadao Rd., Guangzhou 510515, China. Fax: +86 20 87278766. E-mail address: [email protected] (X.-M. Xu).
that stratification according to G6PD mutation does not fully explain the clinical heterogeneity of heterozygotes for G6PD deficiency. G6PD expression is regulated by many factors such as mutations in G6PD, exonic splicing silencer, and transcription factors as well as epigenetic states [1,4–7]. For female heterozygotes, DNA methylation and X chromosome inactivation (XCI) might be major factors in the variation in loss of enzyme activity [1,6,7]. DNA methylation is a type of epigenetic silencing and hypermethylation of CpG islands near transcription start sites (TSSs) is crucial to gene silencing [8]. Toniolo et al. reported that in mice, G6PD expression is associated with the methylation status of CpG islands in the 5′-promoter region of G6PD [6]. However, little is known about the impact of specific gene methylations on G6PD enzyme activity in humans. Women heterozygous for G6PD mutations are genetic mosaics because of XCI. XCI is regarded as an essentially stochastic process. However, in certain situations, nonrandom XCI might be under the control of genetic determinants [9]. In a heterozygote, skewed inactivation of the X chromosome with normal allele results in expression of the mutant allele [10]. Consequently, these women can be as susceptible
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J. Wang et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
to a G6PD deficiency as a G6PD-deficient male. Prior reports indicated that skewed XCI influences G6PD activity in elderly women heterozygous for G6PD mutations [7,11]. However, they didn't exclude the variable effect of different G6PD mutations and the X chromosome with the G6PD mutant allele was not determined, so the relationship between XCI and phenotype might partly be explained by lack of persuasive power. Further research is necessary to investigate the causes of phenotypic variation in women heterozygous for G6PD mutations. In this study, we investigated the impact of DNA methylation and XCI on G6PD activity using 71 G6PD-deficient heterozygous women and 68 non-deficient controls with either G6PD Canton (c.1376GNT) or Kaiping (c.1388GNA) mutation. The results showed that G6PD promoter hypermethylation and preferential XCI of the wild-type allele reduced G6PD activity, suggesting that these factors contributed to phenotypic variation in women heterozygous for G6PD mutations. Materials and methods Subjects Recruited subjects were 21- to 43-year-old women and their sons: 71 women heterozygous with G6PD-deficiency and 68 non-deficient controls, all with either G6PD Canton or Kaiping mutation. Characteristics of the individuals in this study are in Supplemental Table S1. The clinical diagnosis of G6PD deficiency in the Chinese population was carried out through the G6PD/6-phosphogluconate dehydrogenase (6PGD) ratio method [12]. G6PD/6PGD positively correlated with the G6PD-enzyme activity: G6PD-deficiency, G6PD/6PGD b 1.3; G6PD nondeficiency, G6PD/6PGD ≥ 1.3. Genomic DNA was extracted from leukocytes in peripheral blood samples by standard phenol/chloroform extraction method. Genotypes for G6PD were determined by multiplex primer extension/denaturing high performance liquid chromatography (PE/DHPLC) [13]. The local medical research ethics committee reviewed the study, and informed consent was obtained from all participants. Examination of CpG methylation within the G6PD promoter Bisulfite modification of DNA was performed as described [14] and treated DNA was amplified using PCR primers G6PD-F and G6PD-R (Supplemental Table S2). Amplification efficiency was verified by gel electrophoresis and sequencing (Invitrogen Life Technologies) using primer G6PD-F. Unmethylated C was converted to U by modification with sodium bisulfite and subsequently replaced by T after PCR. The presence of C at CpG sites indicated a methylated C allele in bisulfiteconverted DNA. The methylation status of CpG sites was calculated from C and T peaks using the BioEdit Sequence alignment Editor v7090 (Carlsbad, CA, USA) [14]. Determination of the X chromosome inactivation pattern The XCI patterns (XCIPs) in heterozygous women were evaluated by androgen receptor assay [15]. The CAG polymorphism in the androgen receptor (AR) gene enables the maternal and paternal alleles to be discriminated. The HpaII site is methylated on the inactivated X chromosome, which blocks the restriction site, while the site is cut by HpaII on the active X chromosome. The HpaII tiny fragment island, which is associated with the promoter region of the MIC2 gene, is unmethylated on the active and inactive X and Y chromosomes [16], and was used as a reference gene as it can also be amplified and digested by HpaII. A Beckman GeXP Genome Lab Genetic Analyzer was used to study the CAG repeat on the AR gene. In brief, 1 μg of heterozygous female genomic DNA was treated overnight with and without HpaII. Reactions were stopped by incubation at 70 °C for 20 min, and processed DNA (200 ng) was used in duplex PCR. Untreated genomic DNA samples from the sons were amplified with primers HUMARA-F and HUMARAR. Primers are in Supplemental Table S2. All amplification products
were analyzed with Beckman GeXP Genome Lab Genetic Analyzer and Genome-Lab software (Beckman Coulter, USA). For families, the allele genotypes of the heterozygous mother were determined from the fragment length and G6PD genotype of the son's gene. Assuming that the wild-type allele is I, the mutant allele is II. The degree of XCI was calculated as area I / (area I + area II × E), where areas I and II were the peak areas in an electropherogram with HpaII digestion, and E was the ratio of amplification of allele I and allele II without digestion [11]. Statistical analysis Multivariate logistic regression was performed for specific CpG methylations in the G6PD promoter and XCIPs of the wild-type allele as independent variables and G6PD/6PGD ratios as the dependent variable. Statistical analysis was done using the Mann–Whitney test, Spearman's correlation test, Fisher's exact test, chi-square test and multivariate logistic regression. p b 0.05 was regarded as statistically significant. All statistical analyses were conducted using SPSS 13.0 for Windows (SPSS Version 13, SPSS Inc., 2003). Results Hypermethylation of specific CpG sites in the G6PD promoter diminishes G6PD activity To ascertain whether specific CpG methylations in the G6PD promoter were involved in reducing G6PD activity similar to genome-wide methylation, we evaluated the DNA methylation patterns of the G6PD promoter. The region in G6PD promoter containing 15 CpG sites located −15 to −200 bp upstream of the transcription start site was mapped (Fig. 1A). The methylation levels of the CpG sites were investigated for women heterozygous for G6PD with enzyme deficiency and non-deficient controls, and data were analyzed by the Mann–Whitney U-test. In women with G6PD Canton mutation (Fig. 1B), deficient heterozygous women had a higher degree of methylation than controls at CpG sites 6, 12, 13 and 14 (p = 0.046, 0.022, 0.042 and 0.040, respectively). Similar results were obtained in women with Kaiping mutation (Fig. 1C), with deficient heterozygous women showing greater methylation than controls at CpG sites 1, 8, 10, 11, 12, 13, 14 and 15 (p = 0.049, 0.028, 0.038, 0.005, 0.041, 0.018, 0.013 and 0.002, respectively). Significant CpG methylations (p b 0.05) were included in subsequent statistical analyses. Correlation between the selected CpG methylations and G6PD activity was analyzed by Spearman's rank correlation test. Women with G6PD Canton mutation had significantly negative correlations between G6PD activity and methylation levels of CpG 12 and CpG 14 (r = −0.298 and − 0.266) (Table 1). Women with G6PD Kaiping mutation had significantly negative correlations between G6PD activity and methylation levels of 7 CpG sites (1, 10, 11, 12, 13, 14 and 15) (r = −0.347, −0.311, −0.291, −0.307, − 0.322, − 0.266 and − 0.373, respectively). The results indicated that hypermethylation of specific CpG sites within the G6PD promoter reduced enzyme activity in female heterozygotes with the same G6PD mutations. Preferential X-inactivation of the wild-type allele reduces G6PD activity To verify the impact of X-inactivation on enzyme activity in the G6PD heterozygotes, 71 G6PD-deficient heterozygous families and 68 G6PD non-deficient heterozygous families were examined for X-inactivation status of the G6PD wild-type allele using the polymorphic trinucleotide repeat sequence CAG in the androgen receptor (AR) gene. Effective tests were conducted on 65 deficient heterozygous families and 63 nondeficient heterozygous families; 11 were excluded because of homozygous polymorphic alleles (Supplemental Table S3A). Varying levels of XCIPs were observed for the cohort of heterozygous women (Fig. 2) ranging from 4.01% to 97.28% (mean ± SD = 54.66 ± 21.95%). Skewed
Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001
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(Supplemental Table S3B). The incidence of preferential X-inactivation of the wild allele in heterozygous women with deficiency (86.2%) was much higher than in controls (31.7%; p b 0.001) (Supplemental Table S3C). A negative correlation between the XCIPs of the wild-type allele and G6PD/6PGD ratios was seen for identical genotypes (r = − 0.657, p b 0.001 for Canton; r = − 0.668, p b 0.001 for Kaiping) (Spearman's correlation Fig. 3), suggesting that abnormal XCIPs affected the variation in G6PD activity. We used multivariate logistic regression analysis to evaluate the effects of specific CpG methylation levels and XCI on the relative risk of G6PD deficiency in heterozygotes. In a logistic regression model of the group with the G6PD Canton mutation (Table 2), methylation of CpG 12 in the G6PD promoter (odds ratio (OR), 1.10; 95% confidence interval (CI), 1.03–1.17; p = 0.004) and XCI of the wild-type allele (OR, 1.11; 95% CI, 1.05–1.17; p b 0.001) were significantly associated with G6PD deficiency. In the group with Kaiping mutation, logistic regression analysis revealed that hypermethylation of CpG 15 (OR, 1.05; 95% CI, 1.01– 1.09; p = 0.023) and preferential XCI of the wild-type allele (OR, 1.06; 95% CI, 1.02–1.10; p = 0.001) remained significantly correlated with G6PD enzyme deficiency. Thus, hypermethylation of specific CpG sites within the G6PD promoter and preferential XCI of the wild-type allele were identified as risk factors for G6PD deficiency. Discussion
Fig. 1. CpG methylation status in the G6PD promoter region. (A) Nucleotide sequence of the CpG islands in the G6PD promoter. (B) In women with the G6PD Canton mutation, methylation status in deficient heterozygotes was higher than that in non-deficient heterozygotes at CpG sites 6, 12, 13 and 14. (C) For the G6PD Kaiping mutation, deficient heterozygous women showed greater methylation than non-deficient heterozygous women at CpG sites 1, 8, 10, 11, 12, 13, 14 and 15. *p b 0.05 and **p b 0.01; bars, standard error. TSS, transcription start site; ND, G6PD non-deficient heterozygous women; D, G6PD-deficient heterozygous women.
X-inactivation, defined as more than 80% or less than 20% activity from the G6PD wild-type allele, was found more frequently in deficient heterozygous women (30.8%) than in controls (15.9%; p = 0.047)
Table 1 Spearman's correlation tests between G6PD activity and CpG methylation levels. Groups
Variables
r
p
Canton
M-CpG 6 M-CpG 12 M-CpG 13 M-CpG 14 M-CpG 1 M-CpG 8 M-CpG 10 M-CpG 11 M-CpG 12 M-CpG 13 M-CpG 14 M-CpG 15
−0.236 −0.298 −0.185 −0.266 −0.347 −0.214 −0.311 −0.291 −0.307 −0.322 −0.266 −0.373
0.053 0.014⁎ 0.132 0.028⁎ 0.003⁎
Kaiping
0.073 0.008⁎ 0.014⁎ 0.009⁎ 0.006⁎ 0.013⁎ 0.001⁎
Abbreviations: r, Spearman's correlation coefficient; M-CpG, methylation levels of CpG site. ⁎ Correlation is significant at the 0.05 level (two-tailed).
G6PD activity was expected to correlate with different mutations in the G6PD gene [7]. Women heterozygous for identical G6PD mutations have recognized phenotypic differences ranging from asymptomatic to severe deficiency [1,7]. This variable phenotype might be closely related to DNA methylation or XCI [1,6,7]. The relationship between G6PD promoter methylation and G6PD expression has been demonstrated in mice [6], however in humans, little research has been done on G6PD-specific methylation. The methylation of CpG sites close to the TSS was also found to be important in gene expression regulation [8]. Therefore, we chose a region containing 15 CpG sites surrounding the G6PD gene TSS, and the region confirmed the promoter activity through dual luciferase reporter assays (data not shown). To exclude possible genotypic effects, heterozygous women with G6PD Canton or Kaiping mutation were investigated separately. Our results indicated that G6PD promoter hypermethylation aggravated the degree of G6PD deficiency in women heterozygous for the same G6PD mutation. Furthermore, multivariate logistic regression analysis showed that the methylation level of CpG 12 was a risk factor for G6PD deficiency in women with Canton mutation. Hypermethylation of CpG 15 was identified as a risk factor for women with G6PD Kaiping mutation, suggesting that genetic variation affected DNA methylation. This finding was consistent with previous studies showing genetic influences on methylation [8,17]. In addition, DNA methylation status is also regulated by DNA methyltransferase (DNMT) family, histone modifications, noncoding RNAs, genomic imprinting and so on [18]. In this study, hypermethylation of G6PD promoter could diminish G6PD activity. However, few studies have provided clear mechanism for DNA methylationmediated gene repression, and one potential mechanism may be by altering protein binding [8,18]. Future work is necessary to investigate the functional relevance of methylation to explain the phenotypic variation in women heterozygous for G6PD. Finally, methylation at CpG dinucleotides is biallelic and no current method is suitable for detecting monoallelic methylation of the G6PD promoter. Additionally, no or very low methylation in the promoter region of G6PD was detected in 67 male individuals (data not shown). Previous reports indicated that 5′-CpG island methylation of most X-linked genes subject to XCI are methylated in females and unmethylated in males [19,20], suggesting that XCI should be investigated. XCI is generally assumed to be essentially random and extremely skewed inactivation patterns are rare in the general population. XCI is tissue specific and dependent on many factors [19]. The frequency of
Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001
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J. Wang et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
Fig. 2. Genetic analysis of X chromosome inactivation using the androgen receptor assay in family systems. Electropherograms: upper panel, G6PD heterozygous women without HpaII digestion; middle panel, heterozygous women with HpaII digestion; lower panel, sons. HpaII digestion was complete by total abrogation of MIC2 amplification. Genotypes corresponding to the alleles of heterozygous mothers were determined using allele gene fragment length and G6PD genotypes of sons. AI, wild-type allele; AII, mutant allele, HUMARA, human androgen receptor gene. (A) Non-skewed family containing a G6PD heterozygous female, whose X-inactivation is random (XCIP = 47.03%). (B) Skewed family containing a G6PD heterozygous female, whose X-inactivation is skewed (XCIP = 84.96%).
skewed XCI increases with age. In neonates, the incidence is low and is abundant in women over the age of 60 [21]. To eliminate the impact of age in this study, heterozygous women in family cohorts were selected to be 21–43 years old. XCI patterns can also be affected by mutations in some X-linked genes: there is skewing in favor of the wild-type allele or mutation allele in different X-linked disorders [22,23]. In this study, skewed XCI in deficient female heterozygotes was much higher than that in non-deficient controls (30.8% vs. 15.9%). In contrast, only a small subset (8.1%) of women with the wild-type alleles had skewing of XCI (data not shown). Furthermore, we observed that the inactivation of the wild-type allele was significantly negatively correlated with G6PD activity. Skewed selective inactivation of the X chromosome with the wild-type allele would be expected to favor the expression of the mutant allele, resulting in enzyme deficiency. XCI skewing might lead to potential clinical manifestations of X-linked recessive genetic diseases [22–24]. With these reports, Au et al. found that skewed XCI gave rise to G6PD deficiency in elderly women in a study of 18
heterozygotes for prevalent G6PD mutations [11]. However, this study did not determine if the inactivated X chromosome carried the mutant allele and thus could not correlate XCI with G6PD activity very well. In this study, investigating the son's allele gene fragment length and G6PD genotype allowed us to ascertain the chromosome in heterozygous females with the mutant G6PD gene. Furthermore, XCIPs and specific CpG methylations were found in multivariate logistic models to distinguish between enzyme deficiency states, indicating that XCI of the wild-type allele and DNA hypermethylation contributed to reducing enzyme activity. Finally, it should be noted that although methylation and XCI are related to the variability of G6PD expression, other mechanisms may also be involved, such as exonic splicing silencer, transcription factors and nutritional status [4,5,25]. In conclusion, our results indicated that specific CpG hypermethylation within the G6PD promoter and preferential XCI of the wild-type allele decreased G6PD activity in women heterozygous for G6PD. Specific CpG methylations and XCI of the G6PD wild-type allele were identified
Fig. 3. Correlation between X chromosome inactivation and G6PD activity. Scatter graphs of Spearman's correlation show significant negative correlation between X-chromosome inactivation pattern ratios and G6PD/6PGD ratios in heterozygous women with (A) G6PD Canton mutation (r = −0.657, p b 0.001) and (B) G6PD Kaiping mutation (r = −0.668, p b 0.001). XCIP, X chromosome inactivation pattern of the wild-type allele.
Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001
J. Wang et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx Table 2 Logistic regression analysis of variables predictive for G6PD deficiency. Groups
Variables
B
Odds ratio
95% CI
p
Canton
M-CpG 12 XCIPs M-CpG 15 XCIPs
0.095 0.101 0.047 0.058
1.100 1.107 1.048 1.059
1.03–1.17 1.05–1.17 1.01–1.09 1.02–1.10
0.004⁎ b0.001⁎ 0.023⁎ 0.001⁎
Kaiping
Abbreviations: M-CpG, methylation level of CpG site; XCIPs, X-inactivation of the wild allele. ⁎ Correlation is significant at the 0.05 level (two-tailed).
as risk factors associated with G6PD deficiency through multivariate logistic regression analyses. A similar approach could be employed in the future to investigate possible associations among methylation, XCI and other X-linked inherited disorders. Acknowledgments The authors would like to thank all the participants for providing their samples in this project. This work was supported by grants from National Public Health Grand Research Foundation (No. 201202017 and No. 201302001) and National Natural Science Foundation of China (No. 30900806). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bcmd.2014.06.001. References [1] M.D. Cappellini, G. Fiorelli, Glucose-6-phosphate dehydrogenase deficiency, Lancet 371 (2008) 64–74. [2] B. Mizukawa, A. George, S. Pushkaran, L. Weckbach, K. Kalinyak, J.E. Heubi, et al., Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis, Pediatr. Blood Cancer 56 (2011) 840–842. [3] R.E. Howes, F.B. Piel, A.P. Patil, O.A. Nyangiri, P.W. Gething, M. Dewi, et al., G6PD deficiency prevalence and estimates of affected populations in malaria endemic countries: a geostatistical model-based map, PLoS Med. 9 (2012) e1001339. [4] T.J. Cyphert, A.L. Suchanek, B.N. Griffith, L.M. Salati, Starvation actively inhibits splicing of glucose-6-phosphate dehydrogenase mRNA via a bifunctional ESE/ESS element bound by hnRNP K, BBA-Gene. Regul. Mech. 1829 (2013) 905–915. [5] C.M. Walsh, A.L. Suchanek, T.J. Cyphert, A.B. Kohan, F.W. Szeszel, L.M. Salati, Serine arginine splicing factor 3 is involved in enhanced splicing of glucose-6-phosphate dehydrogenase RNA in response to nutrients and hormones in liver, J. Biol. Chem. 288 (2013) 2816–2828.
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DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations Jin Wang a, Qi-Zhi Xiao b, You-Ming Chen c, Sheng Yi a, Dun Liu a, Yan-Hui Liu d, Cui-Mei Zhang e, Xiao-Feng Wei a, Yu-Qiu Zhou b, Xing-Ming Zhong f, Cun-You Zhao a, Fu Xiong a, Xiang-Cai Wei a,f, Xiang-Min Xu a,⁎ a
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China Department of Birth Health and Heredity, Zhuhai Women and Children Care Hospital, Zhuhai, Guangdong, China c Department of Clinical Laboratory, the Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China d Prenatal Diagnosis Center, Dongguan Maternal and Child Health Hospital, Dongguan, Guangdong, China e Department of Child Health, Boai Hospital of Zhongshan, Zhongshan, Guangdong, China f Department of Reproductive Immunology and Endocrinology, Family Planning Research Institute of Guangdong, Guangzhou, Guangdong, China b
a r t i c l e
i n f o
Article history: Submitted 19 May 2014 Accepted 1 June 2014 Available online xxxx Communicated by M. Narla, DSc, 01 June 2014 Keywords: G6PD Phenotypic variation Women heterozygous Methylation X chromosome inactivation
a b s t r a c t Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely dominant enzyme deficiency that results from G6PD gene mutations. Women heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity but the cause of this phenotypic variation is unclear. We determined DNA methylation and X-inactivation patterns in 71 G6PD-deficient female heterozygotes and 68 G6PD non-deficient controls with the same missense mutations (G6PD Canton c.1376GNT or Kaiping c.1388GNA) to correlate determinants with variable phenotypes. Specific CpG methylations within the G6PD promoter were significantly higher in G6PDdeficient heterozygotes than in controls. Preferential X-inactivation of the G6PD wild-type allele was determined in heterozygotes. The incidence of preferential X-inactivation was 86.2% in the deficient heterozygote group and 31.7% in the non-deficient heterozygote group. A significant negative correlation was observed between X-inactivation ratios of the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in heterozygous G6PD Canton (r = −0.657, p b 0.001) or Kaiping (r = −0.668, p b 0.001). Multivariate logistic regression indicated that heterozygotes with hypermethylation of specific CpG sites in the G6PD promoter and preferential X-inactivation of the wild-type allele were at risk of enzyme deficiency. © 2014 Elsevier Inc. All rights reserved.
Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common inherited enzymopathies, affecting approximately 400 million people worldwide, predominantly in Africa, the Middle East and Southeast Asia [1]. G6PD deficiency is caused by mutations in the G6PD gene and more than 160 different mutations associated with G6PD have been described [2]. The inheritance of G6PD deficiency shows a typical X-linked incomplete dominant pattern and most G6PD deficient patients are men. The majority of heterozygous women have been reported with normal G6PD activity, and variable loss of enzyme activity has been observed in some heterozygous females [1,3], suggesting
⁎ Corresponding author at: Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, 1838 Guangzhou Dadao Rd., Guangzhou 510515, China. Fax: +86 20 87278766. E-mail address: [email protected] (X.-M. Xu).
that stratification according to G6PD mutation does not fully explain the clinical heterogeneity of heterozygotes for G6PD deficiency. G6PD expression is regulated by many factors such as mutations in G6PD, exonic splicing silencer, and transcription factors as well as epigenetic states [1,4–7]. For female heterozygotes, DNA methylation and X chromosome inactivation (XCI) might be major factors in the variation in loss of enzyme activity [1,6,7]. DNA methylation is a type of epigenetic silencing and hypermethylation of CpG islands near transcription start sites (TSSs) is crucial to gene silencing [8]. Toniolo et al. reported that in mice, G6PD expression is associated with the methylation status of CpG islands in the 5′-promoter region of G6PD [6]. However, little is known about the impact of specific gene methylations on G6PD enzyme activity in humans. Women heterozygous for G6PD mutations are genetic mosaics because of XCI. XCI is regarded as an essentially stochastic process. However, in certain situations, nonrandom XCI might be under the control of genetic determinants [9]. In a heterozygote, skewed inactivation of the X chromosome with normal allele results in expression of the mutant allele [10]. Consequently, these women can be as susceptible
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J. Wang et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx
to a G6PD deficiency as a G6PD-deficient male. Prior reports indicated that skewed XCI influences G6PD activity in elderly women heterozygous for G6PD mutations [7,11]. However, they didn't exclude the variable effect of different G6PD mutations and the X chromosome with the G6PD mutant allele was not determined, so the relationship between XCI and phenotype might partly be explained by lack of persuasive power. Further research is necessary to investigate the causes of phenotypic variation in women heterozygous for G6PD mutations. In this study, we investigated the impact of DNA methylation and XCI on G6PD activity using 71 G6PD-deficient heterozygous women and 68 non-deficient controls with either G6PD Canton (c.1376GNT) or Kaiping (c.1388GNA) mutation. The results showed that G6PD promoter hypermethylation and preferential XCI of the wild-type allele reduced G6PD activity, suggesting that these factors contributed to phenotypic variation in women heterozygous for G6PD mutations. Materials and methods Subjects Recruited subjects were 21- to 43-year-old women and their sons: 71 women heterozygous with G6PD-deficiency and 68 non-deficient controls, all with either G6PD Canton or Kaiping mutation. Characteristics of the individuals in this study are in Supplemental Table S1. The clinical diagnosis of G6PD deficiency in the Chinese population was carried out through the G6PD/6-phosphogluconate dehydrogenase (6PGD) ratio method [12]. G6PD/6PGD positively correlated with the G6PD-enzyme activity: G6PD-deficiency, G6PD/6PGD b 1.3; G6PD nondeficiency, G6PD/6PGD ≥ 1.3. Genomic DNA was extracted from leukocytes in peripheral blood samples by standard phenol/chloroform extraction method. Genotypes for G6PD were determined by multiplex primer extension/denaturing high performance liquid chromatography (PE/DHPLC) [13]. The local medical research ethics committee reviewed the study, and informed consent was obtained from all participants. Examination of CpG methylation within the G6PD promoter Bisulfite modification of DNA was performed as described [14] and treated DNA was amplified using PCR primers G6PD-F and G6PD-R (Supplemental Table S2). Amplification efficiency was verified by gel electrophoresis and sequencing (Invitrogen Life Technologies) using primer G6PD-F. Unmethylated C was converted to U by modification with sodium bisulfite and subsequently replaced by T after PCR. The presence of C at CpG sites indicated a methylated C allele in bisulfiteconverted DNA. The methylation status of CpG sites was calculated from C and T peaks using the BioEdit Sequence alignment Editor v7090 (Carlsbad, CA, USA) [14]. Determination of the X chromosome inactivation pattern The XCI patterns (XCIPs) in heterozygous women were evaluated by androgen receptor assay [15]. The CAG polymorphism in the androgen receptor (AR) gene enables the maternal and paternal alleles to be discriminated. The HpaII site is methylated on the inactivated X chromosome, which blocks the restriction site, while the site is cut by HpaII on the active X chromosome. The HpaII tiny fragment island, which is associated with the promoter region of the MIC2 gene, is unmethylated on the active and inactive X and Y chromosomes [16], and was used as a reference gene as it can also be amplified and digested by HpaII. A Beckman GeXP Genome Lab Genetic Analyzer was used to study the CAG repeat on the AR gene. In brief, 1 μg of heterozygous female genomic DNA was treated overnight with and without HpaII. Reactions were stopped by incubation at 70 °C for 20 min, and processed DNA (200 ng) was used in duplex PCR. Untreated genomic DNA samples from the sons were amplified with primers HUMARA-F and HUMARAR. Primers are in Supplemental Table S2. All amplification products
were analyzed with Beckman GeXP Genome Lab Genetic Analyzer and Genome-Lab software (Beckman Coulter, USA). For families, the allele genotypes of the heterozygous mother were determined from the fragment length and G6PD genotype of the son's gene. Assuming that the wild-type allele is I, the mutant allele is II. The degree of XCI was calculated as area I / (area I + area II × E), where areas I and II were the peak areas in an electropherogram with HpaII digestion, and E was the ratio of amplification of allele I and allele II without digestion [11]. Statistical analysis Multivariate logistic regression was performed for specific CpG methylations in the G6PD promoter and XCIPs of the wild-type allele as independent variables and G6PD/6PGD ratios as the dependent variable. Statistical analysis was done using the Mann–Whitney test, Spearman's correlation test, Fisher's exact test, chi-square test and multivariate logistic regression. p b 0.05 was regarded as statistically significant. All statistical analyses were conducted using SPSS 13.0 for Windows (SPSS Version 13, SPSS Inc., 2003). Results Hypermethylation of specific CpG sites in the G6PD promoter diminishes G6PD activity To ascertain whether specific CpG methylations in the G6PD promoter were involved in reducing G6PD activity similar to genome-wide methylation, we evaluated the DNA methylation patterns of the G6PD promoter. The region in G6PD promoter containing 15 CpG sites located −15 to −200 bp upstream of the transcription start site was mapped (Fig. 1A). The methylation levels of the CpG sites were investigated for women heterozygous for G6PD with enzyme deficiency and non-deficient controls, and data were analyzed by the Mann–Whitney U-test. In women with G6PD Canton mutation (Fig. 1B), deficient heterozygous women had a higher degree of methylation than controls at CpG sites 6, 12, 13 and 14 (p = 0.046, 0.022, 0.042 and 0.040, respectively). Similar results were obtained in women with Kaiping mutation (Fig. 1C), with deficient heterozygous women showing greater methylation than controls at CpG sites 1, 8, 10, 11, 12, 13, 14 and 15 (p = 0.049, 0.028, 0.038, 0.005, 0.041, 0.018, 0.013 and 0.002, respectively). Significant CpG methylations (p b 0.05) were included in subsequent statistical analyses. Correlation between the selected CpG methylations and G6PD activity was analyzed by Spearman's rank correlation test. Women with G6PD Canton mutation had significantly negative correlations between G6PD activity and methylation levels of CpG 12 and CpG 14 (r = −0.298 and − 0.266) (Table 1). Women with G6PD Kaiping mutation had significantly negative correlations between G6PD activity and methylation levels of 7 CpG sites (1, 10, 11, 12, 13, 14 and 15) (r = −0.347, −0.311, −0.291, −0.307, − 0.322, − 0.266 and − 0.373, respectively). The results indicated that hypermethylation of specific CpG sites within the G6PD promoter reduced enzyme activity in female heterozygotes with the same G6PD mutations. Preferential X-inactivation of the wild-type allele reduces G6PD activity To verify the impact of X-inactivation on enzyme activity in the G6PD heterozygotes, 71 G6PD-deficient heterozygous families and 68 G6PD non-deficient heterozygous families were examined for X-inactivation status of the G6PD wild-type allele using the polymorphic trinucleotide repeat sequence CAG in the androgen receptor (AR) gene. Effective tests were conducted on 65 deficient heterozygous families and 63 nondeficient heterozygous families; 11 were excluded because of homozygous polymorphic alleles (Supplemental Table S3A). Varying levels of XCIPs were observed for the cohort of heterozygous women (Fig. 2) ranging from 4.01% to 97.28% (mean ± SD = 54.66 ± 21.95%). Skewed
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(Supplemental Table S3B). The incidence of preferential X-inactivation of the wild allele in heterozygous women with deficiency (86.2%) was much higher than in controls (31.7%; p b 0.001) (Supplemental Table S3C). A negative correlation between the XCIPs of the wild-type allele and G6PD/6PGD ratios was seen for identical genotypes (r = − 0.657, p b 0.001 for Canton; r = − 0.668, p b 0.001 for Kaiping) (Spearman's correlation Fig. 3), suggesting that abnormal XCIPs affected the variation in G6PD activity. We used multivariate logistic regression analysis to evaluate the effects of specific CpG methylation levels and XCI on the relative risk of G6PD deficiency in heterozygotes. In a logistic regression model of the group with the G6PD Canton mutation (Table 2), methylation of CpG 12 in the G6PD promoter (odds ratio (OR), 1.10; 95% confidence interval (CI), 1.03–1.17; p = 0.004) and XCI of the wild-type allele (OR, 1.11; 95% CI, 1.05–1.17; p b 0.001) were significantly associated with G6PD deficiency. In the group with Kaiping mutation, logistic regression analysis revealed that hypermethylation of CpG 15 (OR, 1.05; 95% CI, 1.01– 1.09; p = 0.023) and preferential XCI of the wild-type allele (OR, 1.06; 95% CI, 1.02–1.10; p = 0.001) remained significantly correlated with G6PD enzyme deficiency. Thus, hypermethylation of specific CpG sites within the G6PD promoter and preferential XCI of the wild-type allele were identified as risk factors for G6PD deficiency. Discussion
Fig. 1. CpG methylation status in the G6PD promoter region. (A) Nucleotide sequence of the CpG islands in the G6PD promoter. (B) In women with the G6PD Canton mutation, methylation status in deficient heterozygotes was higher than that in non-deficient heterozygotes at CpG sites 6, 12, 13 and 14. (C) For the G6PD Kaiping mutation, deficient heterozygous women showed greater methylation than non-deficient heterozygous women at CpG sites 1, 8, 10, 11, 12, 13, 14 and 15. *p b 0.05 and **p b 0.01; bars, standard error. TSS, transcription start site; ND, G6PD non-deficient heterozygous women; D, G6PD-deficient heterozygous women.
X-inactivation, defined as more than 80% or less than 20% activity from the G6PD wild-type allele, was found more frequently in deficient heterozygous women (30.8%) than in controls (15.9%; p = 0.047)
Table 1 Spearman's correlation tests between G6PD activity and CpG methylation levels. Groups
Variables
r
p
Canton
M-CpG 6 M-CpG 12 M-CpG 13 M-CpG 14 M-CpG 1 M-CpG 8 M-CpG 10 M-CpG 11 M-CpG 12 M-CpG 13 M-CpG 14 M-CpG 15
−0.236 −0.298 −0.185 −0.266 −0.347 −0.214 −0.311 −0.291 −0.307 −0.322 −0.266 −0.373
0.053 0.014⁎ 0.132 0.028⁎ 0.003⁎
Kaiping
0.073 0.008⁎ 0.014⁎ 0.009⁎ 0.006⁎ 0.013⁎ 0.001⁎
Abbreviations: r, Spearman's correlation coefficient; M-CpG, methylation levels of CpG site. ⁎ Correlation is significant at the 0.05 level (two-tailed).
G6PD activity was expected to correlate with different mutations in the G6PD gene [7]. Women heterozygous for identical G6PD mutations have recognized phenotypic differences ranging from asymptomatic to severe deficiency [1,7]. This variable phenotype might be closely related to DNA methylation or XCI [1,6,7]. The relationship between G6PD promoter methylation and G6PD expression has been demonstrated in mice [6], however in humans, little research has been done on G6PD-specific methylation. The methylation of CpG sites close to the TSS was also found to be important in gene expression regulation [8]. Therefore, we chose a region containing 15 CpG sites surrounding the G6PD gene TSS, and the region confirmed the promoter activity through dual luciferase reporter assays (data not shown). To exclude possible genotypic effects, heterozygous women with G6PD Canton or Kaiping mutation were investigated separately. Our results indicated that G6PD promoter hypermethylation aggravated the degree of G6PD deficiency in women heterozygous for the same G6PD mutation. Furthermore, multivariate logistic regression analysis showed that the methylation level of CpG 12 was a risk factor for G6PD deficiency in women with Canton mutation. Hypermethylation of CpG 15 was identified as a risk factor for women with G6PD Kaiping mutation, suggesting that genetic variation affected DNA methylation. This finding was consistent with previous studies showing genetic influences on methylation [8,17]. In addition, DNA methylation status is also regulated by DNA methyltransferase (DNMT) family, histone modifications, noncoding RNAs, genomic imprinting and so on [18]. In this study, hypermethylation of G6PD promoter could diminish G6PD activity. However, few studies have provided clear mechanism for DNA methylationmediated gene repression, and one potential mechanism may be by altering protein binding [8,18]. Future work is necessary to investigate the functional relevance of methylation to explain the phenotypic variation in women heterozygous for G6PD. Finally, methylation at CpG dinucleotides is biallelic and no current method is suitable for detecting monoallelic methylation of the G6PD promoter. Additionally, no or very low methylation in the promoter region of G6PD was detected in 67 male individuals (data not shown). Previous reports indicated that 5′-CpG island methylation of most X-linked genes subject to XCI are methylated in females and unmethylated in males [19,20], suggesting that XCI should be investigated. XCI is generally assumed to be essentially random and extremely skewed inactivation patterns are rare in the general population. XCI is tissue specific and dependent on many factors [19]. The frequency of
Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001
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Fig. 2. Genetic analysis of X chromosome inactivation using the androgen receptor assay in family systems. Electropherograms: upper panel, G6PD heterozygous women without HpaII digestion; middle panel, heterozygous women with HpaII digestion; lower panel, sons. HpaII digestion was complete by total abrogation of MIC2 amplification. Genotypes corresponding to the alleles of heterozygous mothers were determined using allele gene fragment length and G6PD genotypes of sons. AI, wild-type allele; AII, mutant allele, HUMARA, human androgen receptor gene. (A) Non-skewed family containing a G6PD heterozygous female, whose X-inactivation is random (XCIP = 47.03%). (B) Skewed family containing a G6PD heterozygous female, whose X-inactivation is skewed (XCIP = 84.96%).
skewed XCI increases with age. In neonates, the incidence is low and is abundant in women over the age of 60 [21]. To eliminate the impact of age in this study, heterozygous women in family cohorts were selected to be 21–43 years old. XCI patterns can also be affected by mutations in some X-linked genes: there is skewing in favor of the wild-type allele or mutation allele in different X-linked disorders [22,23]. In this study, skewed XCI in deficient female heterozygotes was much higher than that in non-deficient controls (30.8% vs. 15.9%). In contrast, only a small subset (8.1%) of women with the wild-type alleles had skewing of XCI (data not shown). Furthermore, we observed that the inactivation of the wild-type allele was significantly negatively correlated with G6PD activity. Skewed selective inactivation of the X chromosome with the wild-type allele would be expected to favor the expression of the mutant allele, resulting in enzyme deficiency. XCI skewing might lead to potential clinical manifestations of X-linked recessive genetic diseases [22–24]. With these reports, Au et al. found that skewed XCI gave rise to G6PD deficiency in elderly women in a study of 18
heterozygotes for prevalent G6PD mutations [11]. However, this study did not determine if the inactivated X chromosome carried the mutant allele and thus could not correlate XCI with G6PD activity very well. In this study, investigating the son's allele gene fragment length and G6PD genotype allowed us to ascertain the chromosome in heterozygous females with the mutant G6PD gene. Furthermore, XCIPs and specific CpG methylations were found in multivariate logistic models to distinguish between enzyme deficiency states, indicating that XCI of the wild-type allele and DNA hypermethylation contributed to reducing enzyme activity. Finally, it should be noted that although methylation and XCI are related to the variability of G6PD expression, other mechanisms may also be involved, such as exonic splicing silencer, transcription factors and nutritional status [4,5,25]. In conclusion, our results indicated that specific CpG hypermethylation within the G6PD promoter and preferential XCI of the wild-type allele decreased G6PD activity in women heterozygous for G6PD. Specific CpG methylations and XCI of the G6PD wild-type allele were identified
Fig. 3. Correlation between X chromosome inactivation and G6PD activity. Scatter graphs of Spearman's correlation show significant negative correlation between X-chromosome inactivation pattern ratios and G6PD/6PGD ratios in heterozygous women with (A) G6PD Canton mutation (r = −0.657, p b 0.001) and (B) G6PD Kaiping mutation (r = −0.668, p b 0.001). XCIP, X chromosome inactivation pattern of the wild-type allele.
Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001
J. Wang et al. / Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx Table 2 Logistic regression analysis of variables predictive for G6PD deficiency. Groups
Variables
B
Odds ratio
95% CI
p
Canton
M-CpG 12 XCIPs M-CpG 15 XCIPs
0.095 0.101 0.047 0.058
1.100 1.107 1.048 1.059
1.03–1.17 1.05–1.17 1.01–1.09 1.02–1.10
0.004⁎ b0.001⁎ 0.023⁎ 0.001⁎
Kaiping
Abbreviations: M-CpG, methylation level of CpG site; XCIPs, X-inactivation of the wild allele. ⁎ Correlation is significant at the 0.05 level (two-tailed).
as risk factors associated with G6PD deficiency through multivariate logistic regression analyses. A similar approach could be employed in the future to investigate possible associations among methylation, XCI and other X-linked inherited disorders. Acknowledgments The authors would like to thank all the participants for providing their samples in this project. This work was supported by grants from National Public Health Grand Research Foundation (No. 201202017 and No. 201302001) and National Natural Science Foundation of China (No. 30900806). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bcmd.2014.06.001. References [1] M.D. Cappellini, G. Fiorelli, Glucose-6-phosphate dehydrogenase deficiency, Lancet 371 (2008) 64–74. [2] B. Mizukawa, A. George, S. Pushkaran, L. Weckbach, K. Kalinyak, J.E. Heubi, et al., Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis, Pediatr. Blood Cancer 56 (2011) 840–842. [3] R.E. Howes, F.B. Piel, A.P. Patil, O.A. Nyangiri, P.W. Gething, M. Dewi, et al., G6PD deficiency prevalence and estimates of affected populations in malaria endemic countries: a geostatistical model-based map, PLoS Med. 9 (2012) e1001339. [4] T.J. Cyphert, A.L. Suchanek, B.N. Griffith, L.M. Salati, Starvation actively inhibits splicing of glucose-6-phosphate dehydrogenase mRNA via a bifunctional ESE/ESS element bound by hnRNP K, BBA-Gene. Regul. Mech. 1829 (2013) 905–915. [5] C.M. Walsh, A.L. Suchanek, T.J. Cyphert, A.B. Kohan, F.W. Szeszel, L.M. Salati, Serine arginine splicing factor 3 is involved in enhanced splicing of glucose-6-phosphate dehydrogenase RNA in response to nutrients and hormones in liver, J. Biol. Chem. 288 (2013) 2816–2828.
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Please cite this article as: J. Wang, et al., Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/j.bcmd.2014.06.001