Introduction: Myelodysplastic syndromes (MDS) are clonal hematological neoplasms characterized by ineffective hematopoiesis and a risk of progression to acute myeloid leukemia (AML). Epigenetic alterations (including gene silencing through methylation) have been implicated in their pathogenesis and are targeted with hypomethylators.

Disruptions in folate metabolism (with subsequent impairment of nucleotide synthesis, DNA repair and DNA methylation) are also suggested to play a role. MTHFR gene polymorphisms (SNP) such as C677T and A1298C decrease enzyme activity, affecting the folate cycle, with potential epigenetic effects. This study aimed to evaluate the role of MTHFR SNPs and of tumor suppressor gene methylation in MDS.

Methods: 88 MDS patients (pts) were enrolled from Oct-2009 to Mar-2014 and followed up until Apr-2025, and diagnoses (Dx) updated according to ICC and WHO2022 criteria. MTHFR C677T and A1298C SNPs were analyzed at Dx by RFLP, and the methylation status of p15, p16, TP53, DAPK, MGMT, TRAILR1 and TRAILR2 wasdetermined by Methylation-Specific PCR. Prognosis was assessed using the IPSS-R; associations with clinical variables, overall survival (OS) and transformation to AML were analyzed.

Results and discussion: The median age at Dx was 76 years (22-90), with 60.2% females; 55.7% had MDS-Not Otherwise Specified (NOS) with multilineage dysplasia, 20.5% had MDS-NOS with single-lineage dysplasia, 9.1% had MDS with excess blasts, 9.1% had MDS/AML, 4.5% had MDS with del(5q) and 1.1% had MDS-NOS without dysplasia. IPSS-R risk was very low in 15.9%, low in 35.2%, intermediate in 1.3%, high in 12.5% and very high in 3.4%; 13.6% were not classifiable. Median hemoglobin was10.3 g/dL (5.8-15.5), median neutrophil counts were 1.8 G/L (0.1-9.4) and median platelet counts were 91.5 G/L (10-367); 81.8% had <5% bone marrow blasts.

MTHFR was wild-type (WT) in 18.0% of pts, 40.0% had one mutation, 40.0% had two, 2.0% had three, and no pts had simultaneous biallelic mutations for both genotypes; 52.3% were C677T WT, 43.2% were C/T heterozygous (Htz) and 4.5% were T/T homozygous (Hmz); 35.2% were A1298C WT, 55.7% were A/C Htz, and 9.1% were C/C Hmz.

Both p15 and DAPK were methylated in over half of the cohort (57.7% and 52.7% of pts, respectively), TRAILR1 in one-third (32.1%) and p16 (26.9%) and TRAILR2 (25.4%) in one-fourth; no instances of TP53 or MGMT methylation were observed.

At a median follow-up of 152.2 months (mo), the median OS was 53.3mo; 17.1% of pts had transformed to AML, and 13.6% were lost to follow-up. An inverse correlation was observed between the total number of mutations at loci 677 and 1298 of MTHFR and OS, with a median OS of 99.0mo for pts with MTHFR WT, 58.1mo for 1 SNP, 37.3mo for 2 SNPs and 22.2mo for 3 SNPs, with estimated 10-year OS (10yOS) of 46.4%, 24.4%, 6.2% and 0%, respectively (p=0.01), suggesting a cumulative effect on folate metabolism and genomic stability.

Likewise, pts with C677T SNP had a significant association with OS, with the Hmz mutant genotype (T/T) presenting a shorter median OS (59.7mo for C/C Hmz, 41.3 for Htz and 11.4 for T/T Hmz, p<0.01, with 10yOS of 30%, 8.4% and 0%). Median OS for A1298C was not statistically different.

Methylation of p15 (compared to unmethylated p15) associated with: 1. lower blast percentages (4.4% vs 39.6% of pts with excess blasts, p<0.001); 2. less aggressive clinical subtypes (86.7% in MDS-Low Blasts vs 2.2% in MDS with Increased Blasts 2, p=0.001); and 3. lower-risk IPSS-R (9.8% vs 34.5% of pts with high-risk, p=0.04). p15 methylation also associated with a lower likelihood of transformation to AML (7.5% vs 40.7%, p=0.002) maintaining its independent prognostic value in multivariate modeling, but with no survival impact. No associations of relevance were observed for the other tumor suppressors.

Conclusions: Our findings suggest that p15 methylation, through its association with lower blast burdens and less aggressive disease, could be an epigenetic initiating event, or a protective event against transformation; this raises relevant questions regarding the role for the use of hypomethylating agents in pts with p15 methylation.

The demonstration of the independent predictor value of p15 methylation (for transformation to AML) and of MTHFR SNPs (for OS) also add to the complex prognostication background in MDS, supporting the integration of epigenetic markers and gene SNPs into existing risk indices, to further improve the risk stratification of pts.

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2025
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