DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background: Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings: To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance: We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.Tina Bianco-Miotto, Damian J. Hussey, Tanya K. Day, Denise S. O'Keefe and Alexander Dobrovi

False negative results from using common PCR reagents

Background\ud The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results.\ud \ud Findings\ud We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA.\ud \ud Conclusions\ud These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is

False negative results from using common PCR reagents

Abstract Background The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results. Findings We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA. Conclusions These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.</p

<i>ABO</i> genotyping, expression and methylation analysis of leukemic cell lines.

<p>‘TYPE’ refers to the cell line type and origin, ‘GENO’ refers to the <i>ABO</i> genotype, ‘EXP’ to <i>ABO</i> mRNA expression and ‘METH’ to the <i>ABO</i> promoter CpG island methylation status as determined by MS-SSCA and COBRA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004788#pone-0004788-g002" target="_blank">Figure 2</a>). ‘5-AZA’ refers to <i>ABO</i> mRNA expression after the cell line was treated with 5-aza-2′-deoxyxytidine as outlined in the ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004788#s2" target="_blank">Materials and Methods</a>’. ‘−’ refers to negative <i>ABO</i> expression, ‘+’ refers to positive <i>ABO</i> expression, ‘U’ refers to unmethylated and ‘M’ to methylated <i>ABO</i> promoter region.</p

<i>ABO</i> methylation analysis in patient specimens with <i>ABO</i> allelic loss of expression.

<p>In the ID column a F prefix denotes patients analyzed by flow cytometry while a S prefix denotes loss of ABH antigen patients as detected by serology. ‘GENO’ refers to <i>ABO</i> genotype, ‘METH’ to <i>ABO</i> promoter CpG island methylation assessed either by MS-SSCA, COBRA and/or melt curve analysis (MCA). ‘U’ unmethylated at the <i>ABO</i> promoter and ‘M’ is methylated at the <i>ABO</i> promoter. For patient F60 two samples were available for analysis; the 1993 ('93) sample was positive for <i>ABO</i> expression whereas the 1996 ('96) sample was negative for <i>ABO</i> expression.</p

Melt curve analysis of the ABO BIS product in patients previously not shown to be methylated by MS-SSCA or COBRA.

<p>The methylated pattern is shown by a dashed line while the unmethylated with a dark black solid line. F9 and F11 both have loss of the <i>A¹</i> allele but are unmethylated. F10 and F60 '96 are patient samples with no <i>ABO</i> allelic expression and show evidence of methylation by melt curve analysis. F27 is an <i>A¹O¹</i> patient with loss of the <i>A¹</i> allele and methylation of 2 different samples, a month apart, which we had previously shown had increasing loss of ABH antigen loss <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004788#pone.0004788-Bianco1" target="_blank">[8]</a> and both showed evidence of methylation.</p

Loss of <i>A</i> expression by RT-PCR and restriction enzyme digestion.

<p>(A) Schematic representation of <i>ABO</i> allelic expression analysis. <i>Kpn</i>I digestion results in a 130 bp band if the O allele is present and no digestion of the A or B allele. <i>BstE</i>II digestion results in a 130 bp band if the A or B allele is present and no digestion of the O allele. (B) Lane M is the pUC19/<i>Hpa</i>II marker while lane 1 is the uncut <i>ABO</i> RT-PCR product. Lanes 2, 4, 6 and 8 are digested with <i>Kpn</i>I while lanes 3, 5, 7 and 9 are digested with <i>BstE</i>II. Lanes 2 and 3 are from cDNA of patient F7, lanes 4 and 5 from F11, lanes 6 and 7 from F15 and lanes 8 and 9 from F17. F7 and F11 are <i>AO</i> patients with loss of the A allele, F17 is an <i>AO</i> patient with no loss of <i>ABO</i> allelic expression. Patient F15 has an <i>A¹A²</i> genotype, hence no cutting with <i>Kpn</i>I was expected. (C) Lanes 1, 3 and 5 are <i>ABO</i> RT-PCR product digested with <i>Kpn</i>I while lanes 2, 4 and 6 are digests with <i>BstE</i>II. Lanes 1 and 2 are from cDNA of patient F23, an <i>A²B</i> genotype, hence no cutting with <i>Kpn</i>I was expected. Lanes 3 and 4 are F53, an <i>A¹O¹</i> patient with loss of <i>A</i> at the mRNA level. Lanes 5 and 6 are S8, which is a patient with an <i>A¹O¹</i> genotype with loss of <i>A</i> allelic expression. (D) The <i>ABO</i> CpG island promoter region assessed for methylation. The methylated and bisulfite modified sequence is shown and the primer sequences are double underlined. The capital Ts identify thymines that are a result of bisulfite modification of cytosines and the CpGs are shown in bold. The start of transcription is marked with +1. The different restriction enzymes used for assessing methylation by digestion are as follows: eight <i>BstU</i>I sites (<u>cg/cg</u>), two <i>Taq</i>I (<u>T/cga</u>) sites (however one is found in the primer and hence will cut regardless of methylation status), one <i>Hinf</i>I (<u>g/aTTc</u>) site. Regions 161–173 and 198–210 harbor Sp1 sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004788#pone.0004788-Hata1" target="_blank">[55]</a>.</p

<i>ABO</i> genotyping, ABH antigen status and <i>ABO</i> allelic expression.

<p>In the ID column a F prefix denotes patients analyzed by flow cytometry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004788#pone.0004788-Bianco1" target="_blank">[8]</a> while a S prefix denotes loss of ABH antigen patients as detected by serology. ‘GENO’ refers to ABO genotype and ‘EXP’ to ABO allelic expression. For SEROLOGY ‘mfr’ refers to a mixed field reaction. In the EXP column, which allele is lost is shown in italics and underline. For patient F60 there were 2 samples analyzed for ABO mRNA expression, one in 1993 and one in 1996. The 1993 sample ('93+) was positive for <i>ABO</i> expression however the 1996 sample ('96–) which was when the flow analysis was performed, was negative for <i>ABO</i> mRNA expression. The bold indicates the samples with loss of ABH antigens. F25 was an <i>A¹A¹</i> sample therefore determining allelic expression was not possible.</p

<i>ABO</i> promoter methylation in leukemic cell lines.

<p>(A) MS-SSCA analysis of the ABO BIS PCR products. PBMNC refers to peripheral blood mononuclear cells and PBSC to peripheral blood stem cells. These were used as unmethylated controls. It is clear from the SSCA gel that only the K-562 leukemic cell line is unmethylated as it has the same banding pattern as the PBMNC and PBSC. The other cell lines all have varying amounts of methylation as seen by the various banding patterns. The JURKAT and RAJI cell lines were hypermethylated, as seen by the dramatic shift of the bottom doublet of bands. (B) Restriction enzyme digests of the ABO BIS PCR products. Digestion with any of the restriction enzymes is indicative of methylation at that CpG site within the restriction enzyme recognition sequence. All the products will cut with <i>Taq</i>I since there is a <i>Taq</i>I site in the reverse primer. (C) <i>ABO</i> re-expression in the JURKAT cell line after 24 h treatment with 5-aza-2′-deoxycytidine treatment. On the gel, the NEGATIVE was an RT control (RNA only), the VEHICLE lane was JURKAT cells treated with ultra pure water, the following lanes are JURKAT cells treated with 1 µM or 2 µM of 5-aza-2′-deoxycytidine respectively showing <i>ABO</i> re-expression. <i>PBGD</i> is the reference gene. (D) The <i>ABO</i> promoter is demethylated in JURKAT cells after 5-aza-2′-deoxycytidine treatment. In the VEHICLE treated JURKAT cells there is no evidence of unmethylated ABO promoter which would be a band at the same size as the UNCUT sample. However, after treatment with 1 or 2 µM of 5-aza-2′-deoxycytidine the <i>ABO</i> promoter is unmethylated as evidenced by a band at the same size as the UNCUT sample.</p