Folate-Dependent Normocytic Anemia Caused By a Hypomorphic Mutation in SLC19A1 gene

Next generation sequencing has enabled rapid diagnosis of patients with monogenic diseases and discovery of novel disease-causing variants. However, the functional characterization of their role in disease pathogenesis still remains challenging. Reduced folate carrier (RFC) encoded by the SLC19A1 gene is one of the main folate transporters in mammalian cells and its deficiency has been shown to be embryonically lethal in a murine knockout model. In humans, only mutations in the proton-coupled folate transporter (PCFT) and folate receptor alpha (FOLR1) have been shown to cause hereditary folate malabsorption leading to folate deficiency, the latter being brain-specific. RFC is also crucial for the uptake of antifolates, such as methotrexate (MTX).

We present a case of 15-year-old male with severe normocytic anemia (Hb 5 g/dL), ineffective erythropoiesis with megaloblastic erythroid precursors in his bone marrow, elevated bilirubin (34 µmol/l), homocysteine (34.7 µmol/l) and mild esophagitis. He responded well to supplementation with vitamin B12 and folate. His blood count and biochemical signs normalized within one month and he experienced no symptoms until the age of 17, when he presented with a second attack of anemia (Hb 7.8 g/dl) that mirrored the previous one and new onset of mild neurological symptoms. He required erythrocyte transfusions every 6 weeks and did not respond to vitamin B12 therapy. Folic acid was added to his therapy at day 265 of his second episode and his blood count and biochemical results normalized within one month. His disease remains stable on regular supplementation with folic acid. Whole exome sequencing of the patient revealed a homozygous in-frame deletion of 3 nucleotides in SLC19A1 gene, leading to a deletion of phenylalanine residue 212 (F212del) located in the central cytosolic loop, that is crucial for the RFC transport function.

HEK293 cell lines transfected with GFP-tagged mutant SLC19A1 gene showed significantly reduced MTX transport ability compared to wild-type transfected cells, in accordance with previously published results showing that loss of RFC transport is one of the mechanisms of MTX resistance. Confocal microscopy did not show any disruption of membrane trafficking of the protein, suggesting the F212 deletion leads to decreased transport capacity.

K562 cell line was then used to create a permanent model of patient's variant by using CRISPR/Cas9 nuclease targeting the SLC19A1 gene at the respective locus and ssDNA exogenous donor carrying the deletion. Single-cell sorting was then used to generate monoclonal populations homozygous or heterozygous for patient's variant as well as populations with complete SLC19A1 knockout. Proliferation assay showed decreased sensitivity to MTX of cells homozygous for the deletion compared to wild type K562 cells (IC50=0.287 µM vs 0.036 µM). However, the sensitivity to MTX of the cells with SLC19A1 knockout was even lower (IC50=2.380 µM) suggesting partially but not fully inactivating character of patient's variant. Interestingly, the cells heterozygous for the variant showed similar sensitivity to MTX to that of wild type K562 cells (IC50=0.038 µM), in concordance with no observed impact on erythropoiesis of patient's parents, who were confirmed to be heterozygous carriers of the variant by Sanger sequencing.

We describe the first case of inborn RFC deficiency in human. Functional analysis of model cell lines with homozygous F212 deletion in RFC confirmed it impacts the function of RFC and causes the anemia in our patient. The partial reduction of the transporter capacity could explain the late onset of the disease and possibility to reverse the phenotype by regular supplementation of folic acid.

Supported by PRIMUS/17/MED/11 and GACR 17-04941Y