A homozygous deletion in the SLC19A1 gene as a cause of folate-dependent recurrent megaloblastic anemia

TO THE EDITOR:

Megaloblastic anemia resulting from ineffective hematopoiesis in the bone marrow (BM) is one of the main hematologic signs of folate or vitamin B₁₂ deficiency. Functional deficiencies of these 2 vitamins originate from nutritional, gastrointestinal, or genetic factors, and their clinical symptoms result from the impaired synthesis of nucleotides in hematopoietic cells and S-adenosylmethionine in the nervous system.¹  Folates are pteroyl(poly)glutamate derivatives with various 1-carbon moieties at the pterine ring, with the major circulating form being 5-methyltetrahydrofolate. Folates are delivered to tissues by at least 5 transporters with different kinetic properties and variable expression.^2,3  Two inherited disorders in folate transport, hereditary folate malabsorption and cerebral folate deficiency resulting from mutations in the SLC46A1⁴  and FOLR1⁵  genes, have been reported in humans.

Reduced folate carrier (RFC; FOLT1) is encoded by the SLC19A1 gene and facilitates the cellular uptake of anionic folates,⁶  of folate analogs such as methotrexate (MTX), and 2′3′-cyclic-GMP-AMP, a second messenger that activates the antiviral stimulator of interferon genes pathways.⁷  Loss of its function leads to MTX resistance in cancer cells.^8,9  A murine model of RFC deficiency showed embryonic lethality before E9.5. These mice could be partially rescued by supplementing the pregnant dams with folic acid; however, all liveborn mice died within 12 days because of the absence of hematopoiesis in BM, spleen, and liver.¹⁰  To our knowledge, no inherited disease caused by loss-of-function mutations in the SLC19A1 gene has been described in humans.

In our study, we presented the first case of recurrent severe megaloblastic anemia in a patient with a homozygous SLC19A1 mutation. The patient’s history was unremarkable with normal growth and development until age 15 years, when he presented with the first episode of severe anemia (hemoglobin [Hb], 5 g/dL), mild hemolysis, hyperhomocysteinemia of 34.7 μmol/L, low total vitamin B₁₂ levels of 138 ng/L, and normal serum folates of 10.7 nmol/L. He denied having any bleeding, limb numbness, any other neurologic symptoms, or symptoms of glossitis. He reported poor eating habits with decreased intake of food for 6 months preceding his presentation, avoidance of vegetables, and avoidance of morning meals. He responded well to treatment with cyanocobalamin and folate, but he returned at age 17 years with a second episode of severe anemia (Hb, 7.8 g/dL) and signs of hemolysis with elevated bilirubin (46 μmol/L), hyperhomocysteinemia, and low total vitamin B₁₂ levels (for laboratory and clinical details, see Table 1 and supplemental Data, available on the Blood Web site). BM aspiration showed 48% megaloblastic erythroid precursors with dysplastic signs, and the peripheral blood smear revealed sporadic macro-ovalocytes, frequent schistocytes, and hypersegmented neutrophils (supplemental Figure 1). Flow cytometry of the BM showed abundant erythropoietic precursors. Despite parenteral cyanocobalamin therapy, anemia and hyperhomocysteinemia persisted. The laboratory abnormalities and clinical signs normalized within a month but only after adding folic acid (10 mg/day) to the patient’s therapy. In contrast, carefully monitored withdrawal of folic acid resulted in an immediate increase in homocysteine to levels of 17 to 31 μmol/L, despite persisting normal folate levels in serum (Figure 1A).

Whole exome sequencing revealed a homozygous 3bp deletion, c.634_636delTTC (rs757838708), in the SLC19A1 gene inherited from heterozygous parents. This variant leads to a deletion of phenylalanine residue 212 (p.Phe212del) located in a highly conserved Lys204-Arg214 peptide sequence that is crucial for the function of RFC¹¹  (supplemental Figure 2).

To test the pathogenicity of the p.Phe212del mutation, we generated a model system using genome-editing techniques to produce a monoclonal homozygous SLC19A1^mut K562 cell line and 2 clones with full SLC19A1 gene knockouts (KOs) as a result of frameshift mutations (supplemental Figure 4C-D). First, we tested the capacity of the mutated RFC protein to transport radioactively labeled MTX into cells. The ability of the SLC19A1^mut clone to transport MTX into K562 cells was significantly decreased by ∼42% compared with the SLC19A1^wt clone (P < .001), and it was similar to that of the SLC19A1^KO clone (Figure 1B). The residual MTX transport into SLC19A1^mut and SLC19A1^KO cells represents the capacity of all other endogenously expressed folate transporters¹²  with the exception of RFC. Confocal microscopy using a monoclonal anti-RFC antibody showed the presence of antigen in the SLC19A1^mut clone, which indicated sufficient (although slightly decreased) expression of the p.Phe212del mutant (supplemental Figure 5). Next, we examined the resistance of these cell lines to the cytotoxic effects of MTX. As expected, the homozygous SLC19A1^mut clone showed an increased resistance to MTX compared with the SLC19A1^wt cells (50% inhibitory concentration [IC₅₀], 0.287 vs 0.036 µM). However, the IC₅₀ of the SLC19A1^mut clone was still about an order of magnitude lower compared with that of the 2 clones with a complete SLC19A1 KO (IC₅₀, 3.078 and 1.681 µM; Figure 1C). In summary, these experiments clearly and congruently showed that homozygous deletion of the Phe212 residue impairs transport of MTX and increases the resistance of genetically modified cells to this cytotoxic anti-folate drug, which uses RFC to enter the cells.

This patient presented a diagnostic conundrum that was resolved owing to next-generation sequencing analysis. The biochemical findings at the beginning of both episodes of anemia were dominated by low concentrations of serum vitamin B₁₂, probably caused by poor nutrition and/or increased demand for cobalamin in the remethylation pathway. The absence of serum folate deficiency obscured the role of folates in the patient’s anemia; however, folates were decreased in erythrocytes in the single analysis performed when the patient was not treated with folic acid. The classical metabolic consequences of folate deficiency such as decreased serum methionine and increased cystathionine were not present, but there was markedly increased total homocysteine and sarcosine in blood and the purine de novo synthesis intermediate 5-amino-4-imidazolecarboxamide riboside (AICAr) in urine,^13-15  as well as their normalization only after introducing folic acid treatment, which strongly indicates a functional folate deficiency in tissues.^13,16 

The episodic clinical course in the patient was milder than that observed in the KO mouse model.¹⁰  Because of the existence of multiple folate transporters and their complex biology,²  it is conceivable that under conditions of normal dietary folate intake, the other folate transporters may compensate for the impaired RFC transport activity.¹⁷  Indeed, an increased expression of the SLC46A1 and FOLR2 genes (7- to 14-fold messenger RNA increase in the patient compared with healthy controls) was observed in the patient’s bone marrow as well as in the gene-edited mutant and KO K562 cells (supplemental Figure 6). These data indicate a grossly increased total capacity of alternative (albeit less kinetically favorable) folate transporters that may compensate for the impaired function of the p.Phe212del RFC under conditions of sufficient folate intake and moderate cellular needs (see supplemental Results). However, inadequate folate intake combined with increased demand for folates reported in adolescent males^18,19  may have contributed to episodes of anemia in our patient.

Our study describes the first human patient with recurrent megaloblastic folate–dependent anemia resulting from a homozygous p.Phe212del mutation in the SLC19A1 gene. Several lines of evidence support the causal role of this mutation in the pathogenesis of anemia in this patient. First, the role of RFC in hematopoietic cells was clearly demonstrated in a KO mouse model.¹⁰  Second, the low frequency of this mutation in available population databases (see supplemental Results) and its location in the conserved cytosolic loop of RFC supports the hypothesis of its pathogenicity. Third, detailed functional testing in CRISPR/Cas9-edited K562 cells clearly showed the decreased ability of the mutant protein to transport 5-methyltetrahydrofolate analog MTX; because of the intrinsic limitations of the cellular transport studies, the degree of functional impairment could not be exactly quantitated and some residual activity of p.Phe212del RFC cannot be ruled out. Fourth, the clinical observation of severe anemia with megaloblastic changes in the patient’s BM suggestive of impaired maturation and ineffective hematopoiesis as well as the signs of demyelination on electromyography are indeed compatible with clinically significant tissue folate deficiency. Finally, the reversal of anemia and rapid drop of total homocysteine and AICAr concentration only after the addition of folic acid to cyanocobalamin therapy during the second episode is typical for folate deficiency.²⁰ 

In summary the above data support the key role of defective folate transport to hematopoietic cells in the development of anemia in this patient. We propose that there may be additional individuals with germline mutations in SLC19A1; however, their phenotypes could be masked either by mandatory folate fortification or by the commonly used combined treatment with vitamin B₁₂ and folic acid in those with unresolved megaloblastic anemia without properly elucidated primary cause.