Risk of retinoblastoma is associated with a maternal polymorphism in dihydrofolatereductase (DHFR) and pre-natal folic acid intake

Abstract

Background

Incidence of unilateral retinoblastoma varies globally suggesting possible environmental contributors to disease incidence. Maternal intake of naturally occurring folate from vegetables during pregnancy is inversely associated with risk of retinoblastoma in offspring.

Methods

Using a case-control study design, we examined the association between retinoblastoma risk and maternal variations in the folate-metabolizing genes, methylenetetrahydrofolate reductase (MTHFR677C>T, rs1801133) and dihydrofolate reductase (DHFR 19base pair deletion of intron 1a [DHFR19bpdel], rs70991108). In central Mexico, we enrolled 103 mothers of children with newly diagnosed unilateral retinoblastoma and 97 control mothers who had healthy children in an IRB approved study.

Mothers were interviewed regarding perinatal characteristics including use of prenatal vitamin supplements and gave peripheral blood samples used for PCR-based genotyping of rs1801133 and rs70991108.

Results

The risk of having a child with unilateral retinoblastoma were associated with maternal homozygosity for DHFR19bpdel (OR=3.78, 95%CI:1.89,7.55; p=0.0002), even after controlling for child’s DHFR19bpdel genotype (OR=2.81, 95%CI:1.32,5.99; p=0.0073). In a subgroup of 167 mothers with data on prenatal intake of supplements containing folic acid (a synthetic form of folate), DHFR19bpdel-associated risk was significantly elevated only among those who reported taking folic acid supplements. Maternal MTHFR genotype was unrelated to risk of having a child with retinoblastoma.

Conclusion

Maternal homozygosity for a polymorphism in the DHFR gene necessary for converting synthetic folic acid into biological folate is associated with increased risk for retinoblastoma. Prenatal ingestion of synthetic folic acid supplements may be associated with increased risk for early childhood carcinogenesis in a genetically susceptible subset of the population.

Introduction

Retinoblastoma, a malignant primitive neuroectodermal tumor, arises in immature retinal cells. Incidence of retinoblastoma varies geographically. The highest rates of incidence are found in some populations of Africa, South America, India and Scandinavia^1,2, while in the US, comparisons by ethnicity show that incidence is highest among Latinos.³

A US based case-control study in the 1980s suggested that factors associated with perinatal poverty such as lack of prenatal vitamins and lower maternal education were associated with having a child with retinoblastoma.⁴ Global variation in incidence of retinoblastoma appears to be primarily among the sporadic unilateral form², which, in contrast to the bilateral form, involves only one eye and is not associated with a family history of the disease. Given that the median age at diagnosis for unilateral retinoblastoma is 23 months, the initiating events for tumor development are likely to occur during pregnancy and early infancy. In 85% of cases, the unilateral form is also associated with mutations in RB1 occurring in somatic (but not germ) cells. Although the origin of these somatic RB1 mutations is unknown, the majority involves methylated cytosines in CpG islands and results in stop codons and truncated pRb.^5,6 A recent report has documented imprinting of a maternal RB1 gene⁷. In a case-control study in Mexico (before folic acid fortification), we found that maternal consumption of vegetable derived folate during pregnancy was protective against having a child with unilateral retinoblastoma.⁸

Folate provides precursors for nucleotide biosynthesis and methyl groups, via methyl folate, for methylation reactions including DNA methylation. The potential role of folate in development of pediatric tumors derived from embryonal layers has not been well studied. Polymorphisms in genes encoding enzymes involved in metabolism of one-carbon donors have been studied in relation to development of birth defects such as neural tube defects (NTD) as well as cancer incidence in adults.^9–11 Because retinoblastoma is a primitive neuroectodermal tumor arising in the retina and therefore embryologically derived from the neural tube, we hypothesized that dysregulation of folate metabolism during early retinal development might influence tumorigenesis.

In the present study we examined the relationship between retinoblastoma risk and prevalence of maternal polymorphisms in two genes that encode two enzymes of folate metabolism, MTHFR 677C>T (rs1801133) and the DHFR 19basepair deletion of intron 1a, (rs70991108). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, which is used for homocysteine methylation. The MTHFR 677C>T (rs1801133) polymorphism has been studied extensively regarding increased susceptibility for development of NTD when folate availability is low⁹ as well as for risk of cancer incidence.¹¹ In addition to its role in thymidylate synthesis, DHFR is responsible for the conversion of orally ingested folic acid (found in supplements and fortified foods) into folate (reduced), which can then be incorporated into the body’s folate pool. The 19bp deletion in intron 1a of the human DHFR gene (DHFR19bpdel) was shown to be associated with an increased risk for NTD¹² as well as an increased risk for breast cancer among women taking folic acid supplements.¹⁰ Homozygosity for DHFR19bpdel is associated with decreased efficiency in using folic acid^13,14 and is present in 17–19% of populations studied.^10,13,14

We carried out a case-control study in central Mexico to examine whether the risk for having a child with retinoblastoma is associated with homozygosity for polymorphisms in MTHFR and DHFR. A second objective was to examine whether prenatal folate intake would be associated with risk of retinoblastoma.

Methods

Between January 2000 and December 2009, 108 mothers of children with newly diagnosed unilateral retinoblastoma at two adjacent referral hospitals in Mexico City were invited to participate in this case-control study, which was approved by IRBs of all participating institutions. Mothers of children with a known family history of retinoblastoma were not eligible to participate in the study. The study was designed to recruit 100 cases and 100 controls. Two mothers declined to participate. Case mothers (N=106) were enrolled during their child’s initial visits to the treating hospital. Enrolled mothers were asked to refer a friend (not related by blood to the case mother) who had a child of the same age as their child (range ±1 year) to serve as a control mother. Eligibility criteria for control mothers also included not having a family member with retinoblastoma. Among the 97 control mothers, more than 81% were the first friend the case mother approached, while the remaining mothers were the second friend approached. Control mothers were enrolled during home visits in 18 states in central and southern Mexico. At the time of enrollment, all participating mothers gave signed consent. Blood samples were then obtained from all mothers and from most case and control children. Mothers were interviewed regarding supplement intake during the first trimester of pregnancy using a validated questionnaire.¹⁵ If women reported taking any supplements, they were asked about the supplement’s brand, dose and form of administration (tablet, powder etc.). Supplemental intake of folic acid was noted as present or absent after analyzing the folic acid content of the supplements reported using a nutrient content database for supplements available in Mexico.¹⁶ Blood was drawn into Becton Dickinson CPT (Becton Dickinson, NJ) vials and kept at 4°C until centrifugation. Buffy coat were stored at −80°C until DNA extraction using standard non-organic methods (Qiagen, Valencia, CA). Genotyping for the MTHFR 677C>T and DHFR 19bpdel polymorphisms were performed by PCR amplification using RFLP and allelic specific methods, respectively.^9,10,12 Resulting PCR products were separated on 3% agarose gels and were visualized with ethidium bromide. About 20% of samples were randomly selected to be run in duplicate with 99% concordance. Laboratory personnel were blinded to sample origin. Batches contained equivalent proportions of case and control samples. Samples and questionnaires were linked through de-identified and bar-coded labels.

Statistical analysis

Quantitative variables

Maternal age and number of years of schooling completed were examined as continuous variables. Genotyping was examined by comparing the homozygous variant genotype with the genotypes containing at least one wild type allele based on results from analyses done with the Framingham Offspring Cohort demonstrating impaired function with homozygous 19bp deletion for DHFR^13,14 and data demonstrating impaired MTHFR function with the homozygous TT genotype.¹⁷ Vitamin supplement use was defined as whether or not mothers reported taking vitamins containing synthetic folic acid during their first trimester.

Statistical methods

We calculated summary statistics to describe sample characteristics and used Chi-square test and t-test to detect group differences in categorical variables and continuous variables, respectively. Variables with skewed distribution were transformed to meet assumptions for t-test. Based on distribution of mothers’ genotype, we estimated allele frequency and tested for Hardy-Weinberg equilibrium in the control mothers. A logistic regression model was used to assess the association between maternal genotype and the odds of having a child with disease, with and without adjusting for control variables of child’s genotype. The child’s genotype variable had three categories with one category for “missing genotype” on 33 children. Odds ratios and 95% confidence intervals were derived from the model parameters and standard errors were used to aid interpretation.

Results

We recruited 106 mothers of children with unilateral retinoblastoma at time of initial diagnosis, of which, 103 were eligible for participation in the study (the child of one mother did not have retinoblastoma on histopathologic examination, while the children of two mothers were discovered to have family members with retinoblastoma and were thus excluded). All references to “case mothers” refer to mothers of children with unilateral retinoblastoma. All references to “control mothers” refer to mothers who had healthy children. All control mothers were healthy and met eligibility criteria to participate.

Analyses were performed on a dataset that contained measurements from 200 mothers (97 control mothers and 103 case mothers) who had data from their perinatal characteristics interviews as well as blood samples, and had genotype results for DHFR19bpdel (198 mothers) or MTHFR677C>T (197 mothers). Case mothers did not differ from control mothers in demographic characteristics such as age at delivery of the index child, child’s birth weight, and maternal education or in the proportion reporting smoking during pregnancy, or folic-acid supplement use in their first trimester of pregnancy (Table 1). Supplement intake was missing for 31 mothers because they were lost to follow up before completing their dietary interview. DHFR and prenatal supplement data were thus available for 167 mothers (96 controls and 71 cases). Among mothers missing data on supplement use there was a significantly higher proportion who were mothers of case children, were homozygous for DHFR19bpdel, and had lesser education when compared with mothers that had data on supplement intake.

Table 1.

Comparison of frequency of genetic polymorphisms, demographic characteristics and vitamin supplement intake between mothers of controls and unilateral cases

	Control (n=97)	Unilateral case (n=103)
	%(n affected/n total informative)	%(n affected/n total informative)
DHFR19bpdel/del*	14.58 (14/96)	39.22 (40/102)
MHTFR677TT	26.04 (25/96)	29.70 (30/101)
Smoked during pregnancy	9.37 (9/96)	3.06 (3/98)
Folic acid supplement use during first trimester	46.39 (45/97)	50.00 (36/72)
	Median (range)	Median (range)
Age at delivery (years)	24.4 (14.4, 39.5)	25.2 (13.17, 38.66)
Years of schooling	9 (0,17)	9 (0,17)
Child’s weight at delivery (kg)	3.3 (1.4, 4.8)	3.3 (2.1, 5.0)

^*p = 0.0002, case control difference detected by Chi-square test.

There was no significant departure from Hardy-Weinberg equilibrium detected in the 96 control mothers for either DHFR (p=0.99) or for MTHFR (p=0.06). Estimated allele frequency of DHFR19bpdel (rs70991108) was 0.61458. Genotypes for DHFR and MTHFR were not correlated in either control mothers (p=0.99) or in case mothers (p=0.42). The proportion of control mothers homozygous for DHFR19bpdel is similar to that reported in US cohorts.^10,14

A higher proportion of case mothers were homozygous for DHFR19bpdel (DHFR19bpdel/del) (39.2%) compared with control mothers (14.5%); OR was 3.78, 95%CI: (1.89, 7.55), p=0.0002. After controlling for the child’s genotype, the OR for the association between DHFR and having a child with retinoblastoma was 2.81, 95%CI (1.32, 5.99); p=0.0075).

The distribution of the homozygous genotype for MTHFR677C>T did not differ between case and control mothers (p=0.68).

Because we were interested in folate intake during initial retinal development, we included questions regarding folic acid supplement intake during their first trimester of pregnancy and during the three months prior to pregnancy. Case children were diagnosed at a median age of 24 months; thus, mothers were interviewed approximately three years after their pregnancy began. Other work done by Mejia et al. has shown that reliable information on vitamin intake can be assessed from Mexican mothers four to six years post-partum.¹⁵ Only four mothers reported use of any supplements during the three months prior to their pregnancy. Therefore, we could not evaluate the impact of supplement use during this period. However, among the 169 mothers who were interviewed about use of vitamin supplements during their first trimester, 46.9% (n=81) took folic acid supplements in the first trimester. The proportion of folic acid supplement use in the first trimester among the mothers of unilateral cases was 50%, close to 46.4% among the mothers of controls, p=0.76 (Table 1), which is similar to the proportion (53%) reported for pregnant women surveyed as part of the 1999 Mexican national nutrition survey in Mexico.¹⁶ Use of folic acid supplements in the first trimester was unrelated to DHFR19bpdel or maternal age at delivery, but those who used supplements had more schooling than those who did not, p=0.006 (Table 2).

Table 2.

Comparison of genotypes and demographic characteristics of mothers by use of supplements containing synthetic folic acid in the 1^st trimester

	Used folic acid containing supplements in the first trimester	Did not use folic acid containing supplements in the first trimester
	n = 81	n = 88
Characteristic	Proportion affected % (n affected/n total informative)	Proportion affected % (n affected/n total informative)
Homozygous for DHFR19bp del	20.25 (16/79)	20.45 (18/88)
Homozygous for MTHFR677C>T	31.65 (25/79)	21.84 (19/87)
Had child with unilateral retinoblastoma	44.44 (36/81)	41.91 (36/88)
	Median (range)	Median (range)
Age at delivery (years)	25.6 (14.4, 39.5)	24.5 (14.9, 39.5)
Years of schooling*	9 (4,17)	9 (0,17)

^*p= 0.006, group mean difference detected by t-test.

Among those who reported taking folic acid containing supplements during the first trimester, women with the DHFR19bpdel/del genotype were more likely to have a child with unilateral retinoblastoma, OR= 3.58, 95%CI=(1.11, 11.55), p=0.03; while the association was insignificant among those not taking folic acid supplements in the first trimester, even after adjusting for the child’s genotype (Table 3). However, there was no significant difference in the odds ratios for the DHFR-retinoblastoma risk association between mothers taking or not taking supplements (p=0.18). Odds of having a child with retinoblastoma were not associated with homozygosity of MTHFR 677 C>T, regardless of folic acid supplement use during pregnancy (Table 3).

Table 3.

Odds ratios (OR) and 95%CI for gene-disease association by first trimester use of supplements containing synthetic folic acid

Genotype	All cases vs controls	Folic acid supplement intake in 1st trimester (n=79)	No Folic acid supplement intake in 1st trimester (n=88)	Difference between ORs
	OR (95%CI)	OR (95%CI)	OR (95%CI)	p-value
Maternal Homozygosity forDHFR19bpdel+	3.78 (1.89,7.55)**	3.58 (1.11,11.55)*	1.59(0.56,4.51)	0.31
Maternal Homozygosity for DHFR19bpdel+†	2.81(1.32,5.99)**	3.31 (0.95,11.50)***	0.98 (0.27,3.53)	0.18
Maternal Homozygosity for MTHFR677C>T+	1.20 (0.64, 2.24)	0.77 (0.30, 2.03)	1.45 (0.52,4.05)	0.38

⁺compared with heterozygotes, or homozygous non-variant allele;

^†adjusted for Child’s DHFR genotype;

^*p=0.03;

^**p<0.01;

^***p<0.06.

Mothers of children born after 2001 were more likely to take folic acid supplements than those born before, which may be secondary to the greater attention given to promoting folic acid intake coinciding with the implementation of folic acid flour fortification. Adjusting for before and after full implementation did not affect the strength or direction of our results.

In summary, we have found that maternal homozygosity for DHFR19bpdel is associated with an increased risk of having a child with unilateral retinoblastoma. The DHFR associated risk is significantly elevated only among those who reported taking folic acid containing vitamin supplements during pregnancy.

Discussion

Results of this case control study suggest that risk for development of unilateral retinoblastoma is associated with maternal homozygosity for the 19bp deletion polymorphism in the intron 1a of the gene for DHFR.¹⁴ When stratified by folic acid supplement use during the first trimester of pregnancy, DHFR associated risk for retinoblastoma is elevated only in those mothers taking supplements containing synthetic folic acid.

Folic acid, the synthetic form of naturally occurring folate, is found in fortified foods and vitamin supplements. Folic acid must be reduced to tetrahydrofolate (THF) by DHFR in order to enter the cellular metabolic pathway. It has been shown in humans that the capacity of DHFR to convert ingested folic acid into reduced folate is limited.¹⁸ Ingestion of 200 micrograms (mcg) of folic acid in a single dose results in the appearance of circulating unmetabolized folic acid (cUMFA).¹⁹

The effect of the DHFR19bpdel polymorphism on the enzyme or its activity has not been characterized. Recent studies of the Framingham Offspring Cohort have shown that homozygotes for DHFR19bpdel have normal concentrations of plasma and red blood cell (RBC) folate, as well as plasma homocysteine, indicating that this polymorphism does not affect regeneration of THF from dihydrofolate (DHF).¹⁴ But, when daily folic acid intake was below 250 mcg, homozygosity for the DHFR19bpdel was associated with significantly lower RBC folate compared to those without the polymorphism. In addition, among individuals whose daily folic acid intake exceeded 500 mcg, the prevalence of high concentrations of cUMFA was two-fold higher in DHFR19bpdel homozygotes than in those without the polymorphism.¹⁴ Together, these Framingham data strongly imply that homozygosity for the DHFR19bpdel results in a diminished capacity of the body in converting ingested folic acid into reduced folate. This threshold level of intake is well below both the tolerated UL and the Dietary Reference Intake (DRI) for pregnant women (600 mcg daily).¹³ Women who are homozygous for DHFR19bpdel have an increased risk for breast cancer, but this risk is increased only among women taking vitamin supplements, suggesting that the effect of the polymorphism on cancer incidence be apparent only when intake surpasses a threshold.¹⁰

The possibility that excessive intake of folic acid or presence of cUMFA is associated with adverse effects has been documented both in humans and in animal models. Presence of cUMFA is inversely related to natural killer cell cytotoxicity in older women.²⁰ In pregnant mice extreme folic acid intake (20 times the DRI) has been associated with embryonic delay and growth retardation²¹ and with increased incidence of NTD.²² Undoubtedly, the increased intake of folic acid through both food fortification and supplements has been beneficial particularly for preventing NTD, however, potential adverse effects even if involving rarer diseases need to be identified in order to inform future policies.

Because cUMFA results from the interaction between DHFR19bpdel and high folic acid intake, it is plausible that the increased retinoblastoma risk may be associated with cUMFA. Mothers who are homozygous for DHFR19bpdel and consume higher amounts of folic acid during pregnancy might have resulting high levels of cUMFA during a critical period of retinal development, potentially increasing tumorigenic risk. Increased levels of cUMFA have been associated with decreased Natural Killer (NK) cell cytotoxicity in older women.²⁰ Maternal NK cells are particularly active during the first trimester of pregnancy, when they contribute to building decidual tissue and formation of new vessels as well as to cytolytic immune surveillance.^23,24 Consequently, the developing fetus might be particularly susceptible to factors decreasing NK function.

Risk of retinoblastoma may be associated with less effective use of ingested folic acid during a key period in formation of these retinal tumors. There is contradictory evidence suggesting that it acts as both a protectant and a promoter of cancer. Both insufficient folate intake as well as increased folic acid intake has been associated with development of carcinomas in adults and with promotion of neoplastic lesions and genetic damage in rodent models.^25–28 Vitamin supplementation with folic acid has been associated with increased risk of breast cancer.^10,23 Prospective studies have shown that ingestion of folic acid at the tolerated upper limit (UL) of 1,000 mcg may contribute to tumor progression in colonic adenomas and to increased incidence of breast²⁹, prostate³⁰ and lung³¹ cancer.

Flour fortification with folic acid is mandatory in approximately 42 countries, though levels of fortification vary and remain controversial.³² Fortification has led to higher than predicted ingestion of folic acid, in part, because consumption patterns for flour-containing foods have changed and are higher than envisioned when fortification was initially planned.^33,34 In Mexico, since 2001, wheat flour is fortified with 2,000 mcg folic acid per kilogram flour.^35,36 Supplements routinely contain high amounts of folic acid and even fortified foods often contain more than the required amount.³³ In the1999 pre-fortification Mexican national nutrition survey (ENSANUT), among non-pregnant women of child bearing age, 12% were consuming a daily dose above the UL.^13,16 Data from the 2006 ENSANUT demonstrated that poor Mexican women consume excessive levels of carbohydrates.³⁷ Some of these carbohydrates would likely originate from foods containing fortified flour. Together these data suggest that consumption at levels present in the diet of some Mexican women may plausibly be above levels associated with increased cancer risk.^29–31 Mothers of children with unilateral retinoblastoma may be consuming elevated quantities of folic acid, potentially through dietary consumption of foods made with folic acid fortified flour.

Our prior study in Mexico did not have access to parental bio-specimens.⁸ Both that study and the one by Bunin in the US were performed before flour fortification with folic acid.⁴ All the data suggesting geographical variations in incidence of retinoblastoma were compiled prior to widespread adoption of fortification.^1,2 Since fortification has begun, no new worldwide incidence data on retinoblastoma have been published though efforts are currently underway to collect such data and estimates are expected to be available in 2013.³⁸

An inherent limitation of our study is that it is a retrospective study, a necessity because of the rarity of retinoblastoma. Because of the case-control design, there may be recall bias in the maternal recollection of prenatal vitamin intake; however, this recall bias would not be expected to vary with genotype. As shown in Table 1, our case and control mothers did not differ in the proportion consuming vitamins containing synthetic folic acid. One additional limitation is that we are unable to fully account for dietary intake because the nutrient database currently available in Mexico was updated in 2003, and reflects fortification of some industrialized foods, but does not reflect the synthetic folic acid content of non-industrialized products containing fortified wheat flour. Because these non-industrialized foods are consumed frequently by our population, accurate calculation of folic acid intake is not possible without accounting for the content of these foods. We are consequently unable to fully examine a threshold effect of synthetic folic acid intake with DHFR. We limited our examination of DHFR to the 19bpdel polymorphism in intron 1A because of its functional significance in the Framingham Offspring Cohort¹⁴, its vitamin-specific carcinogenic effect in breast cancer¹⁰ and its association with NTDs¹². However, if there is linkage disequilibrium between DHFR19bpdel in intron1A and other DHFR polymorphisms, this might partially account for our findings. We were unable to fully examine the role of the child DHFR genotype or interactions between child and maternal DHFR genotypes because of our current sample size. Future plans to validate our findings include an expansion of our study population and a determination of dietary folic acid content in non-industrialized fortified foods, as well as collaboration with other groups to examine our hypotheses in other populations and expansion to include additional MTHFR and DHFR polymorphisms.

In summary, our study is the first to suggest that maternal metabolism of folic acid may affect the risk of a child developing retinoblastoma, and that maternal genotype for DHFR predicts risk for unilateral retinoblastoma. Our data is consistent with the hypotheses arising from the US and European trials showing increased incidence of cancer among adults taking folic acid supplements.