Development of a Multiplex PCR Assay for the Detection of Genomic Copy Number Changes in Myelodysplastic Syndromes

Background: Myelodysplastic syndrome (MDS) is a heterogeneous group of disorders characterized by ineffective hematopoiesis. Clonal cytogenetic aberrations are found in about 50% of primary MDS and 80% of secondary MDS. The International Prognostic Scoring System (IPSS) introduces cytogenetic abnormalities as an independent prognostic factor for both survival and progression to AML. However, conventional cytogenetics or FISH substitutes are not available for all myelodysplastic patients. In order to overcome this problem, we developed a multiplex PCR assay (quantitative multiplex PCR of short fluorescent fragments or QMPSF) measuring the copy number of genes located in regions involved in myelodysplastic syndromes on chromosome 7 and 8 and on the 5q, 12p, 17p and 20q regions.

Material and method: In order to select target region, high resolution CGH (Agilent, 44K) was performed in 12 patients (5 complex karyotypes, 2 isolated monosomy 7, 2 isolated trisomy 8, 1 isolated 17p deletion, 1 isolated 20q deletion and 1 normal karyotype) for which DNA extracted from CD34+ sorted cells was available. Twenty three primer pairs were selected on 6 target chromosomes (5, 7, 8, 12, 17 and 20) in regions either commonly deleted or involved in biallelic deletions. A QMPSF assay was developed and applied on the first 12 patients. This assay was then applied on a series of 60 patients for which DNA extracted from either CD34 + sorted cells or total bone marrow (BM) cells or peripheral mononuclear cells (PB) was available.

Results: As previously reported by several groups, the correlation between cytogenetics and CGH was very good although high resolution CGH was able to detect small cytogenetically not detected defects in most patients. For the 23 QMPSF targeted genes, the correlation between CGH log ratio and QMPSF ratio was excellent. Using QMPSF, genomic copy number changes were detected in 21/27 CD34+ samples, 16/20 BM samples and 15/20 PB samples. However, when different samples were available for the same patient (CD34, BM and/or PB) the correlation between samples was not perfect and mainly depends on the MDS subtype, the % of blasts and the presence of cytogenetics abnormalities.

Conclusion: These results demonstrate that a single multiplex PCR assay targeting 23 genomic regions located on the 6 chromosomes most frequently involved in MDS can be helpful for the prognostic evaluation of MDS patients for whom cytogenetic data are not available, and that DNA extracted from PB or total BM cells can be informative in a substantial number of cases.