Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP)

Abstract

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 5Ј-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylationspecific 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 p15 INK4B 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. Leukemia (2000) 14, 207-212.

Monoclonality and cytogenetic abnormalities in hyaline vascular Castleman disease

“…19 In addition to the conventional HUMARA gene analysis described by Allen et al, 18 some other methods have also been reported, such as the methylation-specific PCR HUMARA assay. 20 This assay can minimize the false polyclonality resulting from incomplete DNA digestion by methylation-sensitive restriction enzymes. Accordingly, we found that methylation-specific PCR HUMARA assay increased the monoclonal rate by 21%.…”

“…For methylation-specific PCR HUMARA analysis, 20 500 ng of DNA was modified with sodium bisulfate by EpiTect Fast DNA Bisulfide kit (Qiagen) according to the manufacturer's instructions. Aliquots of 25 ng of modified DNA were subjected to PCR in a total of 20 ml mixture with primers specific for unmethylated (U: US2, 5 0 -GGTTGTGAG TGTAGTATTTTTTGGT-3 0 ; and HUMARA 1B) or methylated (M: MS2, 5 0 -CGAGCGTAGTATTTTTCG GC-3 0 ; and HUMARA 1B) DNA.…”

Disease-dependent Differently Methylated Regions (D-DMRs) of DNA are Enriched on the X Chromosome in Uterine Leiomyoma

“…The promoter region of FMR1 and the first exon of AR should be 50% methylated if X chromosome inactivation is normal [27,28]. Indeed, in Case 6, FMR1 and AR were about 50% methylated in the normal myometrium exhibiting the typical pattern of genes regulated by X inactivation.…”

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes

“…17 Briefly, the upstream methylated (M) and unmethylated (U) primers were set on the sequence of the human androgen receptor (HUMARA) gene, described by Allen et al to be differentially methylated on the active and inactive X-chromosomes, and the common downstream primer was located in the area without CpG dinucleotides. Between the upstream primer and the downstream primer lies the (CAG) repeat, the number of which is highly polymorphic.…”