A novel, essential control for clonality analysis with human androgen receptor gene polymerase chain reaction

Abstract

The most widely used technique for determining clonality based on X-chromosome inactivation is the human androgen receptor gene polymerase chain reaction (PCR). The reliability of this assay depends critically on the digestion of DNA before PCR with the methylation-sensitive restriction enzyme HpaII. We have developed a novel method for quantitatively monitoring the HpaII digestion in individual samples. Using real-time quantitative PCR we measured the efficiency of HpaII digestion by measuring the amplification of a gene that escapes X-chromosome inactivation (XE169) before and after digestion. This method was tested in blood samples from 30 individuals: 2 healthy donors and 28 patients with myelodysplastic syndrome. We found a lack of XE169 DNA reduction after digestion in the granulocytes of two myelodysplastic syndrome patients leading to a false polyclonal X-chromosome inactivation pattern. In all other samples a significant reduction of XE169 DNA was observed after HpaII digestion. The median reduction was 220-fold, ranging from a 9.0-fold to a 57,000-fold reduction. Also paraffin-embedded malignant tissue was investigated from two samples of patients with mantle cell lymphoma and two samples of patients with colon carcinoma. In three of these cases inefficient HpaII digestion led to inaccurate X-chromosome inactivation pattern ratios. We conclude that monitoring the efficiency of the HpaII digestion in a human androgen receptor gene PCR setting is both necessary and feasible.

Figure 1.

HpaII digestion time course of DNA from a healthy female donor. The digestion time in hours is plotted against the fold reduction of XE169 DNA after HpaII digestion. For each time point the amount of XE169 DNA was compared with real-time quantitative PCR between a reaction mixture with or without HpaII enzyme. The fold reduction after digestion reached a plateau between 4 and 24 hours. The DNA was isolated from the mononuclear cells of the peripheral blood.

Figure 2.

False polyclonality in the granulocytes of two MDS patients. The XCIP for four DNA samples of two MDS patients is represented by the relative intensity of the two alleles after HUMARA PCR. The two alleles were visible as peaks in the electropherogram of the PCR products, and are indicated with A and B. The relative intensities of the peaks are given as percentages. In both patients a polyclonal XCIP in the granulocytes coincided with a failure of HpaII digestion, whereas a clonal XCIP in the monocytes coincided with successful HpaII digestion. The success of HpaII digestion is expressed as the fold reduction of XE169 DNA measured with real-time quantitative PCR after digestion.

Figure 3.

HpaII digestion is successful in the majority of samples. The quantity of XE169 in a reaction mixture without HpaII (in arbitrary units) is plotted against the fold reduction after HpaII digestion for 70 different digestions of the DNA of 28 female MDS patients and 1 male healthy donor. The difference before and after digestion was not significant in four digestions of the DNA of granulocytes of two patients. In the other 66 cases a significant reduction (more than fivefold) of XE169 DNA was observed after HpaII digestion.