Evaluation of Microsatellite Instability and Loss of Heterozygosity in Defining the Instability Phenotype of Myelodysplastic Syndromes.
Authors: Vazquez Maria Lorena , Arbelbide Jorge , Fantl Dorotea Beatriz , Nucifora Elsa , Larripa Irene , Fundia Ariela 
Published: October 2019

Abstract
It has long been proposed that Myelodysplastic syndromes (MDS) arise into a context of genomic instability leading to accumulation of multiple mutations. However, the underlying mechanism remains elusive. Two different genetic instability pathways, chromosome instability (CIN) and microsatellite instability (MSI) can be study using repetitive polymorphic markers (STR). Mutations of CIN genes increase the rate of gross chromosomal changes and loss of heterozygosity (LOH), which is an important mechanism of tumor suppressor gene inactivation. MSI is manifested by alterations of the length of STR due to germline mutations or methylation-induced silencing of key DNA mismatch repair genes (MMR). To verify whether both mechanisms might be involved in MDS, microsatellite instability and loss of heterozygosity (LOH) were analyzed with 10 specific STR markers. Bone marrow samples from 21 de novo MDS patients (12 females/9 males) with a mean age of 70.7 years (range 38–93), including 13 RA, 4 RAEB, 1 RAEB-T, 2 RARS and 1 CMML, were analyzed. All patients gave informed consent and the study was approved by our Ethics Committee. Mononuclear cells were separated by density gradient centrifugation through Ficoll Hypaque and depleted of adherent cells by 1 h incubation at 37°C. Myelodysplastic DNA was obtained from non-adherent mononuclear cells, while normal DNA was extracted from polymorphonuclear cells. Ten STR were amplified at different annealing temperatures (55–58°C), depending on the average size of each primer. Four mono- or dinucleotide markers (BAT 25, BAT 26, D2S123 and D18S58) were chosen from the Bethesda reference panel for MSI studies. Six additional loci at CRTL (5q13-14), IRF (5q31), D5S209 (5q31-33), CSF1RT (5q33-35), D7S525 (7q21-31) and TP53 (17p13.1), were selected because of their location on specific genomic points involved in MDS and acute leukemia. PCR products were analyzed by medium size non-denaturing polyacrylamide (15%) gel electrophoresis and silver staining (0.1%). Allele variations between normal and myelodysplastic DNA were scored according international criteria. Six out of 21 cases (28.6%) presented an unstable phenotype with band shifts or losses. No STR alterations were observed with markers BAT 25, BAT 26, IRF and D2S123. Two cases (9.6%) presented LOH at TP53, D5S209 and D7S525. Bethesda panel revealed MSI only in one patient (4.8%) at D18S58. STR at hematological loci allowed the identification of 5 cases (23.8%) with amplifications or deletions of the repeat sequences at CSF1RT, CRTL and TP53. MSI was detected only at 1/9 or 2/9 STR per patient and therefore these cases were classified to exhibit low grade of MSI (MSI-L). The mean frequency of mutated STR/individual showed a significant difference between hematological markers (0.08±0.03) respect to Bethesda STR (0.01±0.03) (Student test, p=0.007), suggesting that colon markers are not useful to evaluate MSI/LOH in MDS. MSI-L was more frequent than LOH in patients with unstable phenotype, suggesting that putative tumor suppressor genes at the loci studied are not involved in MDS genomic instability. Our data suggest that a subset of patients with MSI-L, are not related to MMR mutations but are probably associated to other genetic alterations, critical in MDS development. Moreover, a STR panel specific for MDS should be defined in order to deep evaluate genomic instability in this disease.