Recently, a multiplex ligation-dependent probe amplification (MLPA®) has emerged as a method that is reported to be fast, sensitive, and cost-effective that can detect copy number variation. In the present study, MLPA analysis was compared with chromosome banding analysis (CBA) data to determine the efficiency of MLPA in patients with chronic lymphocytic leukemia (CLL).

CBA karyotyped 65 patients. Chromosomal analysis was performed on G-banded metaphase cells from cultured lymphocytes in a culture medium at 37°C for 72 hours. Numerical, as well as structural abnormalities were reported on at least 20 well-spread and well-banded metaphases. Genomic DNA was extracted from peripheral blood samples and subjected to MLPA through the probemix, kit SALSA®MLPA®P037 containing 54 MLPA probes, including 41 probes for 2p, 6q 8p/q, 9p21, 11q, 12p/q, 13q, and 17p chromosomal regions. The PCR products were separated by capillary electrophoresis using an ABI 3500 DNA analyzer.

Cytogenetics resulted in 41 normal karyotypes, and 25 karyotype aberrations (13 tris12; 3 del13; 2 del11; 4 del17; others 3). Genetic abnormalities observed by MLPA were 45 (14 tris12; 26 del13q; 2 del11q; 3 TP53 deletion), and 22 were normal by MLPA. Of the 41 normal cytogenetics, MLPA was concordant in 20 patients and discordant in 21 patients. Of those 21, MLPA identified 19 del13q, 1 delq11, and 1 tris12. Regarding abnormalities, cytogenetics and MLPA were concordant in 12 tris12, 2 del13, 3 del17, and 1 del11. Statistical difference was seen when comparing both methodologies in favor of MLPA (p= 0.003; chi-square test).

In this study, we aimed to determine the usefulness of the MLPA probemix P037-B1 as a routine for detecting clinically relevant chromosome abnormalities in CLL in laboratories without cytogenetic routine. MLPA identified more abnormalities than cytogenetic (44%). We observed that MLPA was consistent with the cytogenetic results in 20 of 41 normal patients (48%). The main discordant results identified by MLPA were 13q deletion, the most common cytogenetic change in chronic lymphoblastic leukemia (CLL). In one patient, results of the MLPA did not show deletion in the TP53 gene, while isochromosome 17 was observed by cytogenetic. A low proportion of malignant cells with the relevant abnormality could be responsible for the discrepancies in the MLPA technique. Besides that pitfall, our findings highlight the advantage of MLPA compared with conventional cytogenetics. In conclusion, MLPA is a reliable high-throughput technique to detect CNVs and a cost-effective one that can be included in routine diagnostic protocols in laboratories that lack conventional cytogenetics.