The human androgen-receptor gene (HUMARA) has been used for analysis of X chromosome inactivation (XCI) pattern because of a polymorphic short tandem repeat (STR) near the 5′-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzes methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. Because DNA modified by sodium bisulfite is also available for assessment of methylation status of other genes by setting specific primers for them, we performed the simultaneous assessment of clonality and aberrant hypermethylation of p15INK4B gene in myelodysplastic syndromes. These simultaneous assessments were easily possible and provided much information despite requiring only a small volume of DNA. The HUMARA-MSP assay may facilitate the analyses for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability.
The human androgen-receptor gene (HUMARA) is located at Xcen-q13 and contains a polymorphic short tandem repeat (STR) in the first exon.12 This STR resides near the 5′-promoter region of the HUMARA gene which contains frequent CpG dinucleotides, ie CpG islands.3 A previous study demonstrated that methylation of HpaII and HhaI sites near this polymorphic STR is correlated with X chromosome inactivation (XCI).4 This unique feature allowed us to identify the methylation patterns of the maternally and paternally derived X chromosomes by a simple and sensitive PCR assay (HUMARA assay).4 Although the HUMARA assay uses a methylation-sensitive restriction enzyme to distinguish between the inactive (methylated) and active (unmethylated) alleles, bisulfite modification of cytosine is also available to distinguish methylated from unmethylated alleles. Unmethylated cytosines are modified by sodium bisulfite and changed to uracil, whereas methylated cytosines remain unaffected,56 producing sequence differences between unmethylated and methylated alleles. PCR amplification with primers specific for unmethylated and methylated alleles allow identification of methylation status of maternally and paternally derived alleles, which are polymorphic for STR numbers, and show the XCI pattern. In this study, we describe a novel method to analyze XCI pattern using bisulfite modification. Since our method is based on methylation-specific PCR (MSP) developed recently to detect aberrant methylation of various tumor-related gene promoters,7 we named it the HUMARA-MSP assay. Because modified DNA is available for MSP of other genes by setting specific primers for them, it is easily possible to assess clonality and methylation status of various genes simultaneously. We also demonstrate simultaneous assessment of clonality and methylation of p15INK4B gene promoter of which the importance during the development of myelodysplastic syndromes (MDS) was reported in our previous study.8
Materials and methods
Preparation of DNA
Clinical samples were obtained with informed consent from 11 healthy women and five female patients with MDS. A bone marrow (BM) sample from a female patient with aplastic anemia was also obtained for validation of semi-quantitative nature of HUMARA-MSP assay. Polymorphonuclear cells (PMNs), mononuclear cells (MNCs) and T-lymphocytes were separated from BM or peripheral blood (PB) and their DNAs were extracted as described previously.9
Conventional HUMARA assay
Conventional HUMARA assay was performed essentially as described by Allen et al.4 Briefly, aliquots of 25 ng of DNA digested by HhaI and the same volume of undigested DNA were amplified with primers surrounding polymorphic CAG repeats in each PCR tube after denaturation at 95°C for 10 min (Table 1). PCR products were electrophoresed on 11% polyacrylamide gels and visualized on a UV transilluminator after staining with ethidium bromide.
The HUMARA-MSP assay consists of three steps: chemical modification by sodium bisulfite, PCR amplification and gel electrophoresis. We modified aliquots of 500 ng of DNA with sodium bisulfite using a DNA modification kit (CpGenome DNA Modification Kit; Intergen, Purchase, NY, USA). Aliquots of 25 ng of modified DNA were added to 20 μl of PCR solution containing 1 × PCR buffer (TAKARA, Kyoto, Japan), dNTP mixture (0.25 mM of each), 200 pmole of primer sets specific for unmethylated (U)- or methylated (M)-DNA. Sense primers (Us2 and Ms2) corresponded to a region containing two HhaI sites and one HpaII site at which DNA methylation was correlated with XCI. The antisense primer (UMa1) was designed corresponding to a sequence in a region containing no CpG dinucleotides (Figure 1a, Table 1). After denaturation at 95°C for 5 min, PCR amplification was ‘hot started’ with 1 unit of Taq DNA polymerase (TAKARA). PCR conditions were shown in Table 1. PCR products were confirmed in 11% polyacrylamide gels as the conventional HUMARA assay.
To validate the semi-quantitative nature of the HUMARA-MSP assay, we prepared DNA from two lymphoblastoid cell lines (LCL) immortalized by Epstein–Barr virus and made a series of mixed samples. These LCLs were established in separate dishes from PB cells and showed two different monoclonal patterns of XCI by the conventional HUMARA assay (pattern A, LCL-A and pattern B, LCL-B, Figure 2a). LCL-A and LCL-B were mixed at the ratios of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100, respectively. DNA series of mixed samples were modified and subsequently amplified under the conditions described above.
We also examined the possibility of false results due to mispriming of the U- and M-specific primers to incompletely modified DNA. DNAs before and after modification from the individual normal female were amplified with U- and M-specific primers, respectively, under the same conditions as described above.
For hematopoietic colonies, we performed semi-nested HUMARA-MSP. BM-MNCs from a patient with MDS were cultured in methylcellulose medium with supplemented cytokines, and subsequently individual hematopoietic colonies were picked up by hand with a micropipette as described previously.10 We used one tenth of the DNA obtained from a single colony (approximately 50 cells) in first PCR. In nested PCR, an aliquot of one quarter of the first PCR product was amplified with the inner primers (Table 1, Figure 3a).
MSP assay for p15INK4B gene
The same modified DNAs used in the HUMARA-MSP assay were analyzed for methylation of p15INK4B gene. Aliquots of 25 ng of modified DNA were mixed with the PCR buffer (TAKARA), dNTP mixture (0.25 mM of each), 5% dimethylsulfoxide and 200 pmole of primer sets specific for U- and M-DNAs described by Herman et al.7 For the primer set specific U-DNA of p15INK4B gene, we used the PCR buffer described by Quesnel et al.11 PCR amplification was ‘hot started’ with 1 unit of Taq DNA polymerase (TAKARA) under the conditions as shown in Table 1. PCR products were confirmed in 11% polyacrylamide gel after staining with ethidium bromide.
Results and discussion
XCI pattern by the conventional HUMARA assay is based on identification of methylated (inactive) allele by the use of methylation-sensitive restriction enzyme. In HUMARA-MSP assay, since unmethylated (active) allele may also be identified by another PCR in addition to methylated allele, we can assess XCI pattern independently twice by the use of the same modified DNA. As shown in Figure 1b, the expected band patterns of PCR products amplified with M primers (M products) were similar to those observed by conventional HUMARA assay. The band patterns of PCR products amplified with U primers (U products) should be opposite to those of M products. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands.
Although the MSP method is not intended to provide quantitative information on methylation, it was able to assess the methylation status semi-quantitatively as well as Southern blotting.7 We validated the semi-quantitative nature of HUMARA-MSP assay by mixing experiments of LCLs showing different monoclonal XCI patterns. As shown in Figure 2b, the known proportion of each LCL prior to PCR correlated well with the band patterns of both U and M products.
XCI patterns of PB-PMN from healthy women are shown in Figure 4a. The HUMARA-MSP assay shows normal polyclonal patterns such as the results obtained by conventional HUMARA assay in all 11 healthy women.
As the HUMARA-MSP assay is based on bisulfite modification, a method that avoids the use of restriction enzymes,12 false results by incomplete enzyme digestion can be avoided similarly to the expression-based HUMARA assay which uses RT-PCR without relying on differential methylation of X chromosomes.13 Incomplete bisulfite modification will not affect the results because of the use of specific primers in the MSP method.7 The possibilities of false results due to mispriming of the U- and M-specific primers to incompletely modified DNA were denied by no amplification of unmodified DNA with these primers (Figure 4b).
Since the MSP method is very sensitive, we attempted the assessment of XCI in small cell populations, ie hematopoietic colonies, using semi-nested HUMARA-MSP assay. A hematopoietic colony, considered to be derived from one progenitor cell and thus to be monoclonal, should show one or the other monoclonal patterns of methylation. As shown in Figure 3b, this assay identified two different patterns consistent with the pattern of the PB-PMN in hematopoietic colonies derived from a female patient with MDS although the conventional HUMARA assay failed despite using half of the DNA obtained from a single colony (approximately 250 cells, data not shown). Decreased loss of DNA by the omission of restriction enzyme digestion may allow us to analyze very small quantities of DNA. This result is consistent with our previous demonstration of the evidence for nonclonal hematopoietic progenitor cells in BM of patients with MDS by the restriction fragment length polymorphisms of the X-linked phosphoglycerate kinase gene.10 Although expression-based HUMARA assay is a direct assay to assess XCI and is also sensitive, requirement of RNA may limit the number of available samples. Thus, the HUMARA-MSP assay can target various samples for XCI pattern because of its requirement of a very small volume of DNA.
This bisulfite modification-based method, which is theoretically applicable to any gene regardless of its location in the genome, may enable simultaneous assessment of methylation status of various genes by setting specific primers for them, while restriction enzyme digestion-based methods are limited by the presence of specific digestion sites. We performed the simultaneous assessment of XCI pattern, ie clonality and aberrant hypermethylation of p15INK4B gene in five female patients with myelodysplastic syndromes (Figure 5). We also performed the conventional HUMARA assay in patients 4 and 5 for comparison between these two methods.
Patient 1 showed a skewed XCI pattern not only in BM-MNC but also in PB-PMN, but a polyclonal pattern in the T-lymphocyte population, suggesting that the MDS clone was involved in PMN population but not in the T-lymphocyte population. Patient 2 showed a skewed XCI pattern and aberrant hypermethylation of p15INK4B gene promoter except for T-lymphocyte population, suggesting that p15INK4B gene hypermethylation is limited in monoclonal MDS clone. Both patients 3 and 4 were diagnosed with refractory anemia at first presentation. Patient 3 showed slight methylation of p15INK4B gene but her XCI was not so skewed. She has remained with refractory anemia for a long time. In contrast, patient 4 showing a skewed XCI pattern and methylated p15INK4B gene developed overt leukemia after 5 months. It was demonstrated that monoclonal MDS clone with p15INK4B gene hypermethylation disappeared after complete remission achieved by chemotherapy in patient 5. The T-lymphocyte population again showed polyclonal pattern even in overt leukemia.
These simultaneous assessments of clonality and aberrant methylation of p15INK4B gene were easily possible and provided much information as described above. Since promoter hypermethylation has been proposed as a mechanism to remove gene function as an alternative to loss-of-function type mutations and deletions in various tumor-related genes,1415 such simultaneous assessments may be a useful means to investigate the pathogenesis of hematological disorders.
In conclusion, many researchers have used the HUMARA assay to analyze X chromosome inactivation1617 or clonality.1819 The HUMARA-MSP assay may also be useful for such studies because of its simplicity, sensitivity and wide applicability.
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We thank Sonoko Hatano and Kimie Kondo for their excellent technical assistance. This study was supported in part by Grants-in-Aid from the Ministry of Health and Welfare of Japan.
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Cite this article
Uchida, T., Ohashi, H., Aoki, E. et al. Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP). Leukemia 14, 207–212 (2000) doi:10.1038/sj.leu.2401631
- human androgen-receptor (HUMARA) gene
- X chromosome inactivation (XCI) pattern
- bisulfite modification
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