Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review

Abstract

Background. Myelodysplastic syndromes (MDS) represent a heterogeneous group of premalignant hematologic disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of progression to acute leukemia. Cytogenetic analysis still plays a central role in the diagnosis of MDS, as clonal chromosomal abnormalities are observed in 30-50% of MDS patients. Despite their technical limitations, standard karyotyping and fluorescence in situ hybridization (FISH) are routinely used for identifying recurrent chromosomal rearrangements. However, using this approach means that submicroscopic and not targeted chromosomal aberrations, as well as somatic mutations and epigenetic changes remain largely undetected. Methods and Results. Introduction of methods for the analysis of copy-number variations (CNV), including arraybased technologies and Multiplex ligation-dependent probe amplification (MLPA) has provided novel insights into the molecular pathogenesis of MDS and considerably extended possibilities for genetic laboratory testing. Several novel molecular markers have been discovered and used for diagnosis and prognostic evaluation of patients with MDS. At present, mutational analysis is not routinely performed, as the clinical significance of somatic mutations in MDS has only begun to emerge. However, recently introduced Next-generation sequencing (NGS) technologies could help to elucidate the relationship between chromosomal and molecular aberrations in MDS and lead to further improvement in its diagnosis. Conclusion. This review focuses on the advantages, limitations, clinical applications and future perspectives of three molecular methods (array-based analysis, MLPA and NGS) currently used in genetic testing and/ or translational research of MDS. In conclusion, a brief summary for clinicians from the routine diagnostic point of view is given.

Mutations of myelodysplastic syndromes (MDS): An update

“…Besides chromosomal alterations, sequencing of whole genome/exome and targeted deepsequencing have identified a landscape of mutated genes that encode signal transduction proteins (NRAS at 1p13, FLT3-ITD at 13q12, CBL at 11q23, JAK2 at 9p24, KIT at 4q12), transcription factors (RUNX1 at 21q22, TP53 at 17p13, ETV6 at 12p13), tumor suppressors (TP53, WT1), epigenetic modifiers (TET2 at 4q24, ASXL1 at 20q11, IDH1 at 2q34, IDH2 at 15q26, EZH2 at 7q36, DNMT3A at 2p23), RNA splicing machinery (SF3B1 at 2q33, U2AF1 at 21q22, SRSF2 at 17q25, ZRSR2 at Xp22) and components of the cohesin complex (STAG2, RAD21, SMC3, SMC1A) [10][11][12][13][14]. However, no specific mutation has been detected in ~20% of MDS patients.…”

Familiáris myelodysplasiás szindrómában szenvedő család genomikus kópiaszám-változásainak vizsgálata multiplex ligatiofüggő szondaamplifikációval

“…A familiáris MDS genetikai hátterének feltérképezésében az eddig azonosított pontmutációk és más kiegyensúlyozott aberrációk vizsgálatán kívül a DNS-kópiaszámváltozással járó eltérések vizsgálatára is szükség van. A multiplex ligatiofüggő szondaamplifi kációval (MLPA) ilyen, a betegségben jelenlévő kiegyensúlyozatlan genetikai változások, valamint néhány, szintén a familiáris MDSre specifi kus pontmutáció detektálható [15][16][17][18][19][20][21]. A módszer során adott genomikus lókuszokra specifi kus hibridizá ciós próbák (szondák) amplifi kációja történik polimeráz láncreakció (PCR) használatával, majd fl uoreszcens kapilláris elektroforézist alkalmazva méret szerint elkülönítjük a keletkező amplifi kációs termékeket, meghatározzuk azok mennyiségét és az eredmények referencialókuszokról keletkezett termék mennyiségéhez viszonyítva relatív kópiaszámot számolunk.…”

Techniques for detecting chromosomal aberrations in myelodysplastic syndromes

“…So Array-CGH provides a genome-wide analysis of CNV at very high resolution. It has been reported that commercially available Array-CGH platforms have roughly 50-fold higher resolution than traditional cytogenetic methods, and can reveal chromosomal aberrations in 15% to 20% of samples [ 105 ].…”