Maternal Haplotypes in DHFR Promoter and MTHFR Gene in Tuning Childhood Acute Lymphoblastic Leukemia Onset-Latency: Genetic/Epigenetic Mother/Child Dyad Study (GEMCDS)

Abstract

Childhood acute lymphoblastic leukemia (ALL) peaks around age 2-4, and in utero genetic epigenetic mother-fetus crosstalk might tune ALL onset during childhood life. Folate genes variably interact with vitamin status on ALL risk and prognosis. We investigated DHFR and MTHFR gene variants in 235 ALL children and their mothers to disclose their role in determining ALL onset age and survival. Pyrosequence of DHFR 19bp ins/del (rs70991108; W/D), MTHFR C677T (rs1801133; C>T), and MTHFR A1298C (rs1801131; A>C) was assessed in children and in 72% of mothers for dyad-analysis comparison. DHFR DD-children had delayed ALL onset compared to WW-children (7.5 ± 4.8 vs. 5.2 ± 3.7 years; P = 0.002) as well as MTHFR 1298 CC-children compared to AA-children (8.03 ± 4.8 vs. 5.78 ± 4.1 years; P = 0.006), and according to the strong linkage disequilibrium between MTHFR 677 T-allele and 1298C-allele, MTHFR TT-children showed early mean age of onset though not significant. Offspring of MTHFR 677 TT-mothers had earlier ALL onset compared to offspring of 677 CC-mothers (5.4 ± 3.3 vs. 7 ± 5.3 years; P = 0.017). DHFR/MTHFR 677 polymorphism combination influenced onset age by comparing DD/CC vs. WW/TT children (8.1 ± 5.7 vs. 4.7 ± 2.1 years; P = 0.017). Moreover, mother-child genotype combination gave 5.5-years delayed onset age in favor of DD-offspring of 677 CC-mothers vs. WW-offspring of 677 TT-mothers, and it was further confirmed including any D-carrier children and any 677 T-carrier mothers (P = 0.00052). Correction for multiple comparisons maintained statistical significance for DHFR ins/del and MTHFR A1298C polymorphisms. Unexpectedly, among the very-early onset group (<2.89 years; 25th), DD-genotype inversely clustered in children and mothers (4.8% vs. 23.8% respectively), and accordingly ALL offspring of homozygous DD-mothers had increased risk to have early-onset (adjusted OR (odds ratio) = 3.08; 1.1-8.6; P = 0.03). The opposite effect DHFR promoter variant has in tuning ALL onset-time depending on who is the carrier (i.e., mother or child) might suggest a parent-origin-effect of the D-allele or a two-faced epigenetic role driven by unbalanced folate isoform availability during the in-utero leukemogenesis responsible for the wide postnatal childhood ALL latency.

Keywords: ALL onset; Childhood Acute Lymphoblastic Leukemia; DHFR; MTHFR; MTX; epigenetics; folate; mother-fetus in utero crosstalk; parent-origin effect (POE); polymorphism/gene variant.

Figure 1

ALL onset age distribution according to sex. Females (pink) and males (light blue) in the whole cohort.

Figure 2

DHFR genotype effect on ALL (acute lymphoblastic leukemia) onset age according to the carrier. Schematic representation summarizing the hypothesized axis observed between DHFR gene variant and ALL onset age leading to opposite directions according to the carrier (Upper Panel). Mean values of ALL onset age (mean ± SD) according to DHFR genotype in the whole cohort (black), in the dyad-children (blue), and mothers (red) subgroup (Lower Panel). The DHFR/MTHFR 677 genotype combinations are referred to children or mothers genotype as indicated.

Figure 3

DHFR/MTHFR genotype distribution and ALL onset age. In panel (a), whole cohort of patients (n = 235). In panel (b), mother-child dyad subgroup (n = 196 subjects each). Genotype stratification is shown for both children and mothers in black and red, respectively. Genotype frequencies are reported as a percentage for each age quartile. Vertical dashed lines delimitate the quartiles of ALL onset age (1st, 2nd, 3rd, 4th). The number of cases (children and mothers) stratified, by DHFR/MTHFR genotypes and onset age quartiles, is shown in Table S3.

Figure 3

DHFR/MTHFR genotype distribution and ALL onset age. In panel (a), whole cohort of patients (n = 235). In panel (b), mother-child dyad subgroup (n = 196 subjects each). Genotype stratification is shown for both children and mothers in black and red, respectively. Genotype frequencies are reported as a percentage for each age quartile. Vertical dashed lines delimitate the quartiles of ALL onset age (1st, 2nd, 3rd, 4th). The number of cases (children and mothers) stratified, by DHFR/MTHFR genotypes and onset age quartiles, is shown in Table S3.

Figure 4

Event-free survival (EFS) among high-risk patients stratified by DHFR/MTHFR 677 gene variant. EFS at 5-years survey in patients carrying DHFR DD- or WD-genotype, and/or MTHFR TT-genotype (Any variant) vs. the remaining patients (Others). Panel (a) indicates EFS, as specified in Methods section; Panel (b) indicates EFS including prednisone response. The inclusion of the MTHFR A1298C variant, or its coupling with DHFR ins/del variant, did not modify survival analysis.