Elevated Serum Folic Acid Concentrations Were Associated with Higher mRNA Expression of DHFR Gene in Patients with Hereditary Spherocytosis
Authors: Paniz Clovis , Lucena Maylla Rodrigues , Bertinato Juliano Felix , Amorim Patricia Mendonça da Silva , Gomes Guilherme Wataru , Figueiredo Maria Stella , Green Ralph , Cancado Rodolfo , Domingos Claudia R Bonini , Bláia-D'Avila Vera , Cliquet Marcelo , Guerra-Shinohara Elvira Maria 
Published: October 2019

Abstract
Background
Folic acid (FA) is the synthetic form of folate, a B complex vitamin which plays an important role in several reactions in the body. Folate deficiency produces several clinical complications including anemia and is associated with an increased risk of cancer, cardiovascular disease and neural tube defects. With the advent of FA and iron fortification of wheat and corn flour (150 µg of folic acid and 4.2 mg of iron/100 grams) in Brazil since 2004, the population was exposed to amounts of folate beyond that normally present in foods. The form present in fortified food is taken up by cells and reduced by the enzyme dihydrofolate reductase (DHFR) first to dihydrofolate (DHF) and then tetrahydrofolate (THF). DHF is the preferred natural substrate. Amounts of FA higher than the defined tolerable upper intake of 1 mg/day could impair the ability of DHFR to convert DHF to THF. Some patients with hemolytic anemia, such as hereditary spherocytosis (HS), need larger amounts of FA to compensate for their increase in erythropoiesis and have been receiving 5mg/day of supplemental FA, the only formulation available in Brazil, in addition to being exposed to compulsory food fortification with FA.
Objective
The aim of this study was to compare the effects of 5mg/day FA on serum folate levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes; and cytotoxicity of lymphocytes and NK cells in patients with HS and healthy individuals not receiving supplemental FA.
Material and Methods
Twenty-five patients with HS exposed to mandatory fortification and in use or not of 5 mg/day of FA were included in this study. Forty-five healthy people were recruited as a control group, and matched with HS patients according to age, gender, body mass index and self-reported skin color. Blood count, including reticulocytes, C-reative protein and lactic dehydrogenase (LDH) were performed. Serum folate (SF) and vitamin B12 were determined by a microbiological method. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed in duplicate, using Real Time PCR. Cytotoxicity of lymphocytes and NK cells were also carried out using a flow cytometric assay.
Results
Eight HS patients did not use FA 5 mg everyday (4 declared no use anytime and 4 reported intermittent use) and none of control group used FA supplementation. Reticulocytes and LDH were higher in HS group (P<0.05), however no difference was found between levels of C-reative protein (P=0.173) and vitamin B12 (P= 0.699) when compared with control group. The HS group had higher SF levels and elevated mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 when compared with controls (P<0.05, Figure 1). It is not clear whether FA use or underlying disease was be responsible for increasing of mRNA expression of these genes. To verify the effect of SF levels on HS patients, this group was classified into two subgroups according to median SF (< 46.6 and ≥ 46.6 nmol/L). Interestingly, the subgroup with higher SF levels showed significantly elevated DHFR mRNA expression but no difference was found in the mRNA expression of the other genes studied. The two subgroups were similar according to WBC, RBC, hemoglobin, MCV, reticulocytes, LDH, C-reative protein, vitamin B12 and cytotoxic capacity of lymphocytes and NK cells.
Conclusions
Elevated SF concentrations were associated with higher mRNA expression of DHFR gene in HS patients, suggesting that the use of higher amounts of FA might influence the expression and activity of DHFR and thus affect folate metabolism in these patients. It is not known whether normal subjects receiving similar high doses of FA show the same effects.
Financing: FAPESP 2012/12912-1 and CNPq 4826412012-6
Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C). Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C).
The line in each graphic is the median.
The median and percentiles 25 and 75 (P25 – P75) for control and hereditary spherocytosis groups were, respectively: A- serum folate: 22.1 (14.5 – 36.6 nmolL) and 51.5 (19.5 – 95.2 nmolL), B- DHFR mRNA expression: 1.59 (1.00 – 2.48) and 35.8 (19.0 – 52.7). In Figure C, the median and percentiles 25 and 75 (P25 – P75) for DHFR mRNA expression, according to serum folate levels (SF <46.6 and SF ≥ 46.6 nmol/L), were, respectively: 19.6 (1.4 – 38.6) and 50.3 (23 – 66.8).

Disclosures
No relevant conflicts of interest to declare.