Simultaneous Detection of Genomic Rearrangements In Myelodysplastic Syndromes (MDS) with the Multiplex Ligation-Dependent Probe Amplification (MLPA) Assay

Clonal chromosome abnormalities are present in the marrow cells in about 50% of patients with myelodysplastic syndromes (MDS) at the time of presentation. Cytogenetic analysis of the bone marrow is not only indicated in MDS for diagnostic purposes, but also to assess the individual prognosis according to IPSS scoring guidelines and plan tailored therapy. Conventional cytogenetics (CC) analysis is performed in clinical practice to detect chromosomal abnormalities. It has been reported that fluorescence in situ hybridization (FISH) is a more sensitive approach, but this analysis is limited to detection of the more frequent abnormalities on chromosomes 5, 7, 8, 11, and 20, and reports from the literature provide contradictory data. A new method has recently been described for the measurement of gene/chromosome copy number using genomic DNA: Multiplex Ligation-dependent Probe Amplification (MLPA).

The purpose of this study was to perform the MLPA assay in a series of 29 MDS patients (M: 21, F: 8, median age 71 years, range 44–84), and to compare the results obtained with CC data. According to the WHO classification, 7 cases were classified as RA, 7 as refractory cytopenia with multilineage dysplasia, 2 as RAEB-1, 8 as RAEB-2, 1 as MDS_U, and 4 as CMML. According to the IPSS score, 8 were considered low risk, 11 intermediate-1 risk, 5 intermediate-2 risk, and 5 high risk.

The MLPA assay was performed for all samples in two independent reactions, one for each probe mix (SALSA Probe-Mix P144 and P145) according to the manufacturer's recommendations (MRC-Holland). This mix contains 61 target sequences specific for different chromosome regions commonly involved in MDS: 5q (9 probes) + 5p (1 probe), 7q (8 probes) + 7p (2 probes), 8q (8 probes) + 8p (2 probes), 11q (8 probes), 12p (6 probes), 17q (2 probes) + 17p (4 probes), 20q (5 probes) + 20p (1 probe) and 21q (5 probes). The Probe mixes also include 21 reference probes selected from chromosomal regions that appear to be “quiet” in MDS. Data were analyzed with Coffalyser Software (MRC-Holland) using DNA from ten healthy donors as controls. The CC study was performed following standard protocols and at least 20 metaphases were analyzed.

Our study showed a good correlation between the MLPA and CC results, as shown in Table I, since most of the alterations were detected by both techniques. Discrepancies were found in 5 (17%) samples. MLPA analysis did not detect: in sample n°5 the presence of a chromosomal (chr.) translocation; in sample n°12 a chr. deletion and a chr. translocation; in sample n°17 a chr. deletion; in sample n°22 several chr. translocations and deletions; in sample n°28 a chr. gain. In fact, MLPA is not able to detect chr. translocations because it can reveal only chr. loss or gain; it can only analyse the chr. regions commonly involved in MDS (5, 7, 8, 11, 12, 17, 20 and 21); it can reveal chr. abnormalities only if the percentage of cells carrying the alterations is about 30–35% and do not show mosaicism. On the other hand, with CC we observed a karyotype failure (no metaphases) in 3 samples. MLPA proved to be rapid, cost effective, relatively easy to perform, had high throughput and enabled simultaneous analysis of many samples by automated data processing. MLPA and CC result complementary techniques, and MLPA is particularly useful in MDS cases with Karyotype failure.

No relevant conflicts of interest to declare.