Comment on “MeDIP real‐time qPCR of maternal peripheral blood reliably identifies trisomy 21”

We read with interest the recent publication in your journal by Tsaliki et al. titled ‘MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21’. In a correspondence published in September issue of Nature Medicine, Tong et al. raised technical concerns about this methodology. Experimentally, Tong et al. failed to reproduce the results using 15 normal and 3 trisomy cases. We wish to communicate our preliminary results with colleagues working in this field. We used cell-free circulating DNA extracted from 1.5 to 2mL ofmaternal plasma fromwomen during the first trimester of pregnancy as the starting material. Following the enrichment of methylated DNA using a methodology to be published later by our group, real time Q-PCR was performed using eight molecular markers, EP4–8 and EP10–12, as shown in experiments by Papageorgious and Tong et al. Thirty normal samples and two trisomy 21 cases were examined. We calculated methylation ratios that showed similar ranges for each marker to those presented by Papageorgious et al. We tentatively used the equation published by Papageorgious to calculateD values.Wewere able to correctly identify both trisomy cases and 29 normal cases, although one normal case had a D value of 0.65. We will be able to test the data more accurately once our own equation is developed. We believe that statistically, the equation obtained from work by Papageorgious et al. may only represent the data obtained from their experimental conditions and reagents used. The coefficients in the equation are not necessarily accurate for the data collected in other laboratories unless these coefficients are determined under a standardized system and from a large enough sample size that is statistically able to represent general population of women with first trimester pregnancy. Therefore, we believe that it would be prudent to be cautious when using the equation to explain another set of data. We are not surprised to see that D values varied for the replicates in the reproducibility experiments of Tong et al. Once the coefficients are determined on the basis of a particular set of data, the methylation ratio is an independent variable that ultimately determines theD value. In this context, themethylation ratio is defined as normalized delta Ct for an individual sample against the median normalized delta Ct of a group of known normal samples. In a quantitative polymerase chain reaction (PCR), variation in Ct values amongmultiple replicates of a sample is generally acceptedwithin the half cycle. This allowable, less than half-cycle, difference can produce a range of variation in the D value for any sample given that eight markers contribute to the formation of the D value in this equation. Existence of this variation is logical as long as it does not change the nature of the tested sample that, in this scenario, is trisomy 21 or euploidy. We also tested the reproducibility of methylation enrichment. Within a run, five technical replicates of a single sample were examined. Ct values for five enriched samples were all centered on a value with less than half-cycle difference. With runs on different days, we were able to reach 99–100% consistency for the readings of Ct values when comparing twomeans from each run. In terms of efficiency of enrichment, reproducibility between runs for the eight markers examined is as follows: EP4, 99.30%; EP5, 93.65%; EP6, 98.90%; EP7, 91.14; EP8, 88.89%, EP10, 100%; EP11, 86.96; and EP12, 88.52%. Furthermore, we examined the reproducibility of methylation enrichment using representative markers EP4 and EP5 within a period of 1month, and we are able to reproduce five weekly results with variations of less than half a PCR cycle. Our preliminary results suggest that it is worthwhile to further validate this methodology in a large sample cohort.