Identification and Characterization of an Inborn Error of Metabolism Caused by Dihydrofolate Reductase Deficiency
2011; Elsevier BV; Volume: 88; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2011.01.004
ISSN1537-6605
AutoresSiddharth Banka, Henk J. Blom, John H. Walter, Majid Aziz, Jill Urquhart, Christopher M. Clouthier, Gillian Rice, Arjan P.M. de Brouwer, Emma Hilton, Grace Vassallo, Andrew Will, Desirée E.C. Smith, Yvo M. Smulders, Ron A. Wevers, Robert Steinfeld, Simon Heales, Yanick J. Crow, Joelle N. Pelletier, Simon Jones, William G. Newman,
Tópico(s)Biochemical and Molecular Research
ResumoDihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease. Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease. Folates are critical cofactors for single-carbon metabolism in biological processes, including DNA synthesis, regulation of gene expression, and synthesis of amino acids, neurotransmitters, and myelin.1Djukic A. Folate-Responsive Neurologic Diseases.Pediatr. Neurol. 2007; 37: 387-397Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Systemic folate deficiency, most frequently due to dietary insufficiency, manifests as biochemical, hematological and neurological disturbance. Six inherited disorders of folate transport and metabolism have been described (Table S1 available online). Of particular note, mutations in FOLR1 (MIM 136430), encoding folate receptor alpha, cause a brain specific folate-transport defect that manifests between 2 and 3 years of age and is characterized by irritability, slow head growth, psychomotor retardation, cerebellar ataxia, pyramidal tract signs, dyskinesia, and seizures.2Steinfeld R. Grapp M. Kraetzner R. Dreha-Kulaczewski S. Helms G. Dechent P. Wevers R. Grosso S. Gärtner J. Folate Receptor Alpha Defect Causes Cerebral Folate Transport Deficiency: A Treatable Neurodegenerative Disorder Associated with Disturbed Myelin Metabolism.Am. J. Hum. Genet. 2009; 85: 354-363Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar The only consistent biochemical abnormality observed in children affected with FOLR1 mutations is a low level of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF) with normal serum and erythrocyte folate levels. Cerebral folate deficiency (CFD) can also result from impaired transport of 5-MTHF across the blood-CSF barrier due to antibodies to folate receptors.3Ramaekers V.T. Rothenberg S.P. Sequeira J.M. Opladen T. Blau N. Quadros E.V. Selhub J. Autoantibodies to Folate Receptors in the Cerebral Folate Deficiency Syndrome.N. Engl. J. Med. 2005; 352: 1985-1991Crossref PubMed Scopus (212) Google Scholar Here, we describe the clinical features and molecular mechanism of an inborn error of folate metabolism characterized by infantile-onset megaloblastic anemia and/or pancytopenia, severe CFD, and moderate cerebral BH4 deficiency. Our proband (family 1, II:6 in Figure 1A ) is the fourth child of first-cousin British Pakistani parents (I-1 and I-2), born at full term after an uneventful pregnancy and with no neonatal problems. His weight and head circumference (HC) at birth were 2.83 kg (9th–25th centile) and 33 cm (2nd–9th centile), respectively. He presented at 4 mo of age with increasing pallor, poor feeding, and secondary microcephaly with an HC of 36.8 cm (< 0.4th centile). Investigations revealed severe anemia (Table 1), and his blood film showed a dual red cell population with oval macrocytes and microcytes and hypersegmented neutrophils (Figure S1A). Within 2 days, he was pancytopenic. His bone marrow demonstrated megaloblastic erythropoesis (Figure 1B). Serum folate, vitamin B12, and ferritin levels were normal. A working diagnosis of transcobalamin II deficiency (MIM 275350) was made, and hydroxocobalamin was started. After 1 week, he started having generalized tonic-clonic and right-sided focal seizures that were refractory to phenytoin, benzodiazepines, and pyridoxine. His hematological profile remained unresponsive to hydroxocobalamin. Partial seizure control was achieved with phenobarbitone and levetiracetam. His brain MRI showed severe cerebellar and cerebral atrophy (Figures 1C, 1D, and 1E). CSF neurotransmitters were analyzed by high-performance liquid chromatography with electrochemical and/or fluorescence detection4Hyland K. Surtees R.A.H. Heales S.J.R. Bowron A. Howells D.W. Smith I. Cerebrospinal fluid concentrations of pterins and metabolites of serotonin and dopamine in a pediatric reference population.Pediatr. Res. 1993; 34: 10-14Crossref PubMed Scopus (111) Google Scholar and revealed markedly decreased CSF 5-MTHF, with low CSF BH4 and normal dihydrobiopterin (BH2) levels (Table 1). Metabolic investigations showed that there was no methylmalonic aciduria and that plasma homocysteine and phenylalanine levels were normal. Table S2 provides details of all other metabolic investigations that were performed.Table 1The Results of Hematological and CSF Investigations of the ProbandPretreatmentAfter Folinic Acid TreatmentInvestigation(Reference Range)At Presentation2 Days after PresentationAfter TransfusionAfter 1 Week of TreatmentAfter 1 Month of TreatmentAfter 4 Months of TreatmentAge in months3.53.54.5569.5Haemoglobin(10.0–13.0 g/dl)5.2 ↓3.7 ↓8.2 ↓8.6 ↓10.612.7Red cell count(3.80–4.90 × 109/l)1.9 ↓1.31 ↓3.03 ↓3.18 ↓3.914.65Mean corpuscular volume (73–88 fl)858281848286White cell count(6.0–17.0 × 109/l)8.42.3 ↓3.6 ↓5.8 ↓9.28.0Neutrophils(1.00–6.00 × 109/l)2.70.95 ↓1.76 ↓2.472.632.78Lymphocytes(3.30–11.50 × 109/l)4.21.11 ↓1.34 ↓2.25 ↓5.524.19Monocytes(0.20 −1.30 × 109/l)0.70.17 ↓0.240.560.760.92Eosinophils(0.05–1.10 × 109/l)0.60.08NA0.550.31NABasophils(0.02–0.20 × 109/l)0.20 ↓0 ↓0.01 ↓0.01 ↓0.01 ↓Platelets(150–560 × 109/l)240138 ↓316423390358CSF 5-MTHF(72–305 nmol/l)NANA9 ↓98NA81CSF total neopterin(7–65 nmol/l)NANA3620NANACSF tetrahydrobiopterin(∗nmol/l)NANA23 ↓(27–105)13 ↓(23–55)NANACSF dihydrobiopterin(0.4–13.9 nmol/l)NANA8.95.3NANACSF pyridoxal phosphate(∗nmol/l)NANA52(44–89)51(23 – 87)NANACSF homovanillic acid(∗nmol/l)NANA288 ↓(324–1098)298 ↓(362–955)NA330(176–851)CSF 5-hydroxyindoleacetic acid (∗nmol/l)NANA198 ↓(199–608)142(63–503)NA165(68–451)Reference ranges for investigations marked with ∗ are age dependent and are given with individual measurements. NA, not available; ↓, results below normal. Open table in a new tab Reference ranges for investigations marked with ∗ are age dependent and are given with individual measurements. NA, not available; ↓, results below normal. Oral folinic acid (30 mg daily) was initiated, after which anemia (Figure 1F and Figure S1B), seizure control, and CSF 5-MTHF level showed rapid and significant improvement, although the BH4 level dropped further (Table 1). Now, at the age of 19 months, the patient remains profoundly delayed with central hypotonia, poor head control, and inability to fix and follow. He has two to three short focal seizures daily, is fed via gastrostomy, and suffers from frequent lower respiratory tract infections. An older sibling (II:3) died at 28 weeks of age with a history of anemia and intractable seizures of undefined cause. Postmortem examination showed a small brain, weighing 480 g (normal 615 g), with ventricular dilatation and white matter atrophy of the cerebrum. There was neuronal loss in the cerebellar folia with a reduction of both the internal and external granular cell layers. Neuronal and vascular calcifications were seen in the basal ganglia and subcortical white matter, respectively, with gliosis in the periventricular white matter and possible optic atrophy. The cause of death was Klebsiella aerogenes pneumonia. The proband's two remaining older siblings (II-2 and II-5) and parents (I-1 and I-2) are healthy. CFD and megaloblastic anemia in the presence of normal serum folate excluded the possibility of defects in any of the known folate transporters.2Steinfeld R. Grapp M. Kraetzner R. Dreha-Kulaczewski S. Helms G. Dechent P. Wevers R. Grosso S. Gärtner J. Folate Receptor Alpha Defect Causes Cerebral Folate Transport Deficiency: A Treatable Neurodegenerative Disorder Associated with Disturbed Myelin Metabolism.Am. J. Hum. Genet. 2009; 85: 354-363Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 5Qiu A. Jansen M. Sakaris A. Min S.H. Chattopadhyay S. Tsai E. Sandoval C. Zhao R. Akabas M.H. Goldman I.D. Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption.Cell. 2006; 127: 917-928Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar Absence of hyperhomocysteinemia indicated that the defect did not involve the methylation cycle but was most likely to be in the DNA-synthesis arm of folate metabolism. Ethical approval for the study was obtained from the University of Manchester (06138) and NHS ethics committees (06/Q1406/52). Informed consent was obtained from all participants or their parents. We undertook autozygosity mapping and genotyped the proband (II:6) and two unaffected siblings (II:2 and II:5) by using the Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA) as described previously.6Daly S.B. Urquhart J.E. Hilton E. McKenzie E.A. Kammerer R.A. Lewis M. Kerr B. Stuart H. Donnai D. Long D.A. et al.Mutations in HPSE2 Cause Urofacial Syndrome.Am. J. Hum. Genet. 2010; 86: 963-969Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Genotype calls were generated with the use of the Birdseed V2 algorithm within the same software, and the results were analyzed by AutoSNPa.7Carr I.M. Flintoff K.J. Taylor G.R. Markham A.F. Bonthron D.T. Interactive visual analysis of SNP data for rapid autozygosity mapping in consanguineous families.Hum. Mutat. 2006; 27: 1041-1046Crossref PubMed Scopus (135) Google Scholar Multiple homozygous regions, equivalent to approximately 180 Mb and unique to the affected individual, were identified (Table S3). This included a 3 Mb region at chromosome 5q14.1 flanked by rs4521453 and rs10059759, containing DHFR. Primers for PCR and sequencing were designed with Primer3 on the basis of NCBI reference sequence NM_000791.3, covering the coding regions and intron-exon boundaries of exons 1 to 6 of the DHFR and ensuring no amplification of the pseudogenes. Primer sequences and PCR conditions are provided in Table S4. Sequence variants were named in accordance with HGV nomenclature, and NM_000791.3 was used as the reference sequence. DNA sequence analysis of DHFR revealed a homozygous missense mutation, c.238C>T (p.Leu80Phe), in exon 3 (Figure 2A ). The mutation segregated with the disorder in the family (Figure 2B) and, importantly, was homozygous in DNA extracted from a neonatal blood spot available from the affected deceased sibling (patient 2, II:3). The mutation was absent in 292 chromosomes from ethnically matched Pakistani controls (Figure 2C). Another child (family 2) with CFD, megaloblastic anemia, and normal serum folate was ascertained. He is the first child born to first-cousin British Pakistani parents, delivered at full term with a birth HC of 33.5 cm (25th centile). He presented at 10 weeks of age with reduced oral intake, icterus, and hepatomegaly. His hemoglobin (Hb) level was 6.1 g/dl, with a mean corpuscular volume of 95 fl. His HC was 37 cm (0.4th–2ndcentile). Within 5 days, he developed leucopenia (5.4 × 109/l) and thrombocytopenia (71 × 109/l). A diagnosis of megaloblastic anemia of unknown cause was made, and folic acid was started. His anemia resolved and his neurodevelopment was satisfactory. Folic acid was stopped at 7.5 months of age, and within 6 weeks he presented in status epilepticus. His brain MRI demonstrated marked hypoplasia of the cerebellar vermis. At 11 months of age, his CSF 5-MTHF level was extremely low, at 3 nmol/l. Treatment 5 mg folinic acid twice daily was begun. Brain MRI, repeated at 23 months, showed atrophy of the cerebellar hemispheres, patchy high signal in the subcortical white matter, and delayed myelination. Currently, at 5 years of age, he is microcephalic (HC < 0.4th centile), with severe developmental delay and cerebellar ataxia. DHFR mutation analysis in this child, apparently unrelated to the proband from family 1, revealed the same homozygous missense change, p.Leu80Phe. We screened 20 additional patients with CFD of unknown etiology: 19 without anemia and one with anemia but with a low serum folate. No variants in the DHFR coding sequence were identified in these patients. Human, mouse, chicken, zebrafish, and Drosophila DHFR protein sequences were aligned by ClustalW and demonstrated conservation of the leucine residue at position 80 across species to Drosophila (Figure S2). We assessed the expression profile of DHFR in human tissues by quantitative real-time PCR analysis (qPCR), using SYBR Green and Taqman (both from Applied Biosystems, Foster City, CA, USA) techniques. Total RNA for qPCR analyses from different adult human brain areas was purchased from Stratagene (La Jolla, CA, USA), except for hippocampus, thalamus, and spinal cord total RNA, which was ordered from Biochain (Hayward, CA, USA). RNA was available from a human embryonic brain panel of forebrain, telencephalon, diencephalon, midbrain, hindbrain, and optic vesicles (MRC Newcastle Brain Tissue Resource, Newcastle, UK) from Carnegie stage 16 (week 6) and stage 21 (week 8) human embryos.6Daly S.B. Urquhart J.E. Hilton E. McKenzie E.A. Kammerer R.A. Lewis M. Kerr B. Stuart H. Donnai D. Long D.A. et al.Mutations in HPSE2 Cause Urofacial Syndrome.Am. J. Hum. Genet. 2010; 86: 963-969Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Total RNA from different human adult and fetal tissues was purchased from Stratagene Europe (Amsterdam, The Netherlands), except for cochlear RNA, isolated from an 8-week-old embryo with the use of the NucleoSpin RNA II Kit (Macherey-Nagel, Düren, Germany). Whole-blood RNA was isolated from the proband, his parents, and three healthy control individuals with the use of the PAXgene Blood RNA Kit (QIAGEN, Venlo, The Netherlands). Whole-blood and cochlear RNA was treated with DNase I (Invitrogen, Leek, The Netherlands), and the concentration and purity were determined by optical densitometry. SYBR Green-based QPCR expression analysis was performed on a 7500 Fast Real-Time PCR System with the use of Power SYBR Green PCR Master Mix (Applied Biosystems). Primers were designed with the use of the primer3 program, and GUSB and PPIB were used as reference genes (Table S5). Five micrograms of total RNA was transcribed into cDNA with the use of the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), and cDNA was purified with the NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany). QPCR quantifications were performed in duplicate on the equivalent of 12.5 ng total RNA input. Differences in expression of a gene of interest between two samples were calculated by the comparative Ct or 2ΔΔCt method.8Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25511) Google Scholar TaqMan-based QPCR analysis was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Predesigned, validated primers and probe mix for DHFR were purchased from Applied Biosystems. 18S and HPRT1 were used as reference genes. Four hundred nanograms of RNA was transcribed to cDNA with the use of the high-capacity RNA-to-cDNA Kit (Applied Biosystems). qPCR quantifications were performed in three triplicate experiments with water controls. The results were analyzed by StepOne software (Applied Biosystems). Differences in expression of a gene of interest between two samples were calculated by the comparative 2ΔΔCt method. SYBR Green and Taqman real-time PCR analyses demonstrated that DHFR is widely expressed in fetal and adult tissues, including throughout the fetal and adult brains and whole blood (Figure 2B). Of note, expression was higher in the adult brain than in the fetal brain. Levels of DHFR transcripts from lymphocytes from the proband and his heterozygous parents were not significantly different from each other (data not shown). We performed an immunoblot on fibroblasts from a control line and identified very faint bands, even with high-protein loading, showing that DHFR is expressed relatively poorly in the fibroblasts (data not shown). Therefore, we undertook immunoblot analysis of cellular and nuclear lysates from EBV-immortalized lymphoblastoid cells, which revealed that DHFR was undetectable in the proband and was present at lower levels in his parents compared to controls (Figure 2C). DHFR activity in EBV-immortalized lymphoblastoid cells was measured by the formation of tetrahydrofolate from dihydrofolate. A DHFR activity kit (Sigma-Aldrich, St. Louis, MO, USA) was used for the assay. Lymphoblastoid cells were incubated with 300 μM NADPH and 250 μM DHF for 30 min. Samples were stabilized with 10 mM mercaptoethanol and frozen immediately. Lysates were prepared from cells with the use of three freeze-thaw cycles. After centrifugation, the supernatant was acidified with 10 mM formic acid, and an internal standard ([13C5]-5-methenyltetrahydrofolate) was added. Proteins were removed by a molecular weight cut-off filter (Millipore, molecular weight < 10,000). Tetrahydrofolate concentration was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).9Smith D.E.C. Kok R.M. Teerlink T. Jakobs C. Smulders Y.M. Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.Clin. Chem. Lab. Med. 2006; 44: 450-459Crossref PubMed Scopus (57) Google Scholar DHFR activity in the proband's immortalized lymphoblasts was about 100-fold lower compared to controls, confirming the diagnosis of DHFR deficiency (Table 2). We were unable to determine whether the remaining activity in the proband's sample was residual DHFR activity or background activity due to unexpected processes influencing the signal in LC-MS/MS that we considered as folates. The obligate heterozygote parents showed an intermediate reduced DHFR activity compared to controls.Table 2The Results of Measurement of DHFR Activity and Folate Vitamers in the Proband on Folinic Acid Therapy and in His Heterozygous ParentsAssay (Units)Reference rangePatientMotherFatherControl 1Control 2DHFR activity (nmol THF/hr/mg protein)NA1.5 ↓↓59.7 ↓52.9 ↓154.7149.6MTHF (nmol/l)95–47016911743 ↓NANANonmethylTHF (nmol/l)measured as THF + methylene-THF + methenyl-THF + formyl-THF0–6777 ↑9.845NANADihydrofolate + folic acid (nmol/l)031 ↑↑00NANANA, not available; THF, tetrahydrofolate; ↑, values above the reference range; ↓, values below the reference range. Double arrows indicate values that are severely abnormal. Open table in a new tab NA, not available; THF, tetrahydrofolate; ↑, values above the reference range; ↓, values below the reference range. Double arrows indicate values that are severely abnormal. The effect of the DHFR mutation on the concentrations of folate vitamers in erythrocytes in vivo was studied with the use of a previously described method.9Smith D.E.C. Kok R.M. Teerlink T. Jakobs C. Smulders Y.M. Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.Clin. Chem. Lab. Med. 2006; 44: 450-459Crossref PubMed Scopus (57) Google Scholar Methylfolate and nonmethylfolate metabolites were determined by LC-MS/MS, which distinguishes 5-MTHF, folic acid (sum of folic acid and dihydrofolate, which converts to folic acid during assay), nonmethyltetrahydrofolates, and unsubstituted tetrahydrofolate. This showed an accumulation of folic acid and dihydrofolate in the proband, but not in his heterozygous parents (Table 2). To explore the mechanism of loss of enzyme activity, a homology model of the p.Leu80Phe variant was constructed with the MOE molecular modeling program, version 2009.10 (Chemical Computing Group, Montreal, Canada). Energy minimizations, homology models, and protein-geometry analysis were performed with the compute module with the use of the CHARMm22 force field and a distance-dependent dielectric (ɛ = 1) as implemented in MOE. The wild-type crystallographic structure of human DHFR (Protein Data Bank [PDB] coordinates 2W3M) was used for all calculations. The crystallographic waters along with the NADPH and folate were removed from the structure. Hydrogens were added at the normal ionization state of amino acids at pH 7.0, and atomic partial charges were fixed to the CHARMm22 atom types. To eliminate potential steric clashes, the backbone atoms of the protein were tethered to their initial positions by application of a tethering force constant of 1 kcal·mol−1, and the initial structure was minimized with the use of a conjugate gradient minimization until a convergence of 0.001 kcal·mol−1 was attained (Figure 3B ). To construct a homology model of the p.Leu80Phe variant, the wild-type human DHFR template structure (PDB 2W3M) was modified to include the mutation. After removal of water and ligands and the addition of hydrogens and charges as for the wild-type, ten intermediate models were constructed and scored with the use of a generalized Born/volume integral internal fitness function within the MOE homology modeler.10Labute P. The generalized Born/volume integral implicit solvent model: Estimation of the free energy of hydration using London dispersion instead of atomic surface area.J. Comput. Chem. 2008; 29: 1693-1698Crossref PubMed Scopus (338) Google Scholar The model with the best internal fitness score was selected and subjected to a conjugate gradient minimization with a tethering force as described above, until a convergence of 0.001 kcal·mol−1 was attained. The structures for the wild-type and the p.Leu80Phe mutant of DHFR revealed that introduction of a phenylalanine induces a shift in the position of the critical lysine 55 residue, predicting a significant steric clash and disrupting cofactor binding for the p.Leu80Phe mutant relative to wild-type DHFR (Figure 3C). Homology modeling holds the inherent disadvantage of exploring only the local conformational space, thus potentially remaining within a local energy minimum that does not represent the lowest energy structure. Therefore, we performed a molecular dynamics simulated annealing, which allowed the backbone and side-chains to sample the accessible conformational space much more widely for comparison of the effects of the variant on the structural integrity and dynamic behavior of DHFR. All molecular dynamics simulated annealing calculations were performed with the InsightII package, version 2000.1 (Accelrys, San Diego, CA, USA). The Biopolymer, Analysis, and Decipher modules were used for analysis of molecular dynamics simulated annealing trajectories. Simulated annealing calculations were performed with the Discover module employing the constant valence force field (CVFF), and calculations were run on an Origin 2000 Silicon Graphics Fuel Server. For the minimized structures of wild-type and mutant p.Leu80Phe DHFR, atomic potentials were fixed to the default CVFF atom types. The wild-type and mutant p.Leu80Phe DHFR were then subjected to a molecular dynamics simulated annealing protocol according to the following iterative procedure: The structure was heated to 1000 K over 5 ps, equilibrated at 1000 K for 3 ps, then cooled down to 300 K in 5 ps, with the use of an exponential rate constant (timtmp) of 0.7 ps for both the heating and cooling steps. The time step of the molecular dynamics simulations was set to 1 fs, and a distance-dependent dielectric constant of 1 was applied to the system. During the simulated annealing protocol, the backbone atoms of the protein were tethered to their initial positions by applying a force constant of 1200 kcal·mol−1·Å2. This force constant sufficiently restricts the protein backbone to limit local unfolding of secondary structural elements while allowing for subtle backbone movements that permit broad side-chain conformation exploration. After each simulated annealing cycle, the resulting cooled structures were subjected to a final energy minimization step involving 1000 steps of steepest descent followed by conjugate gradient minimization until a convergence of 0.01 kcal·mol−1·Å2 was reached. This cycle was repeated nine times for a total simulation time of 130 ps, yielding ten minimized structures for the wild-type DHFR. The protocol was repeated for mutant p.Leu80Phe, resulting in ten minimized conformations for the p.Leu80Phe mutant. This analysis suggested that the p.Leu80Phe mutation could result in potential destabilization of the protein and/or a disruption of NADPH binding (Figures 3D and 3E). DHFR catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential step in the synthesis of precursors of DNA, including glycine and purines, and the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate.11Trimble J.J. Murthy S.C. Bakker A. Grassmann R. Desrosiers R.C. A gene for dihydrofolate reductase in a herpesvirus.Science. 1988; 239: 1145-1147Crossref PubMed Scopus (48) Google Scholar It is also the only enzyme that reduces folic acid, a synthetic vitamin not found in nature, to dihydrofolate. DHFR is a key enzyme in all prokaryotes and eukaryotes, being present in all dividing cells. Cell lines or animal models deficient in DHFR are considered not viable without supplemented glycine, purines, and thymidine, indicating its critical role in cellular function. Variants in DHFR, including a 19 base pair deletion in intron 1, have been studied in numerous disorders, including breast cancer,12Xu X. Gammon M. Wetmur J. Rao M. Gaudet M. Teitelbaum S. Britton J. Neugut A. Santella R. Chen J. A functional 19-base pair deletion polymorphism of dihydrofolate reductase (DHFR) and risk of breast cancer in multivitamin users.Am. J. Clin. Nutr. 2007; 85: 1098-1102PubMed Scopus (59) Google Scholar neural-tube defects,13van der Linden I.J.M. Afman L.A. Heil S.G. Blom H.J. Genetic variation in genes of folate metabolism and neural-tube defect risk.Proc. Nutr. Soc. 2006; 65: 204-215Crossref PubMed Scopus (92) Google Scholar and preterm delivery.14Johnson W.G. Scholl T.O. Spychala J.R. Buyske S. Stenroos E.S. Chen X. Common dihydrofolate reductase 19-base pair deletion allele: a novel risk factor for preterm delivery.Am. J. Clin. Nutr. 2005; 81: 664-668PubMed Scopus (64) Google Scholar However, no molecularly confirmed cases of DHFR deficiency have been described to date. In 1967, Walters reported a child with megaloblastic anemia and proposed that he may have DHFR deficiency (T.R. Walters, 1967, Midwest Society for Pediatric Research, abstract). Skin fibroblasts from this child were later demonstrated to have normal enzyme activity.15Erbe R.E. Inborn errors of folate metabolism.N. Engl. J. Med. 1975; 293: 753-757Crossref PubMed Scopus (29) Google Scholar In 1976, Tauro et al. reported two neonates with congenital deficiency of DHFR.16Tauro G.P. Danks D.M. Rowe P.B. Van der Weyden M.B. Schwarz M.A. Collins V.L. Neal B.W. Dihydrofolate Reductase Deficiency Causing Megaloblastic Anemia in Two Families.N. Engl. J. Med. 1976; 294: 466-470Crossref PubMed Scopus (29) Google Scholar However, subsequent analysis revealed that the first child had methionine synthase reductase deficiency (MIM 602568),17Rosenblatt D.S. Fenton W.A. Inherited disorders of folate and cobalamin transport and metabolism.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill, New York2001: 3903-3904Google Scholar and the second patient was shown to have transcobalamin II deficiency (MIM 275350).18Hoffbrand A. Tripp E. Jackson B. Luck W. Frater-Schröder M. Hereditary Abnormal Transcobalamin II Previously Diagnosed as Congenital Dihydrofolate Reductase Deficiency.N. Engl. J. Med. 1984; 310: 789-790PubMed Google Scholar The phenotype of the patients we describe with biallelic mutations in DHFR is distinct from patients with the other recognized disorders of folate metabolism or transport. Megaloblastic anemia was the presenting feature in our patients with DHFR deficiency, which resolved completely with folic acid or folinic acid replacement. In contrast, CSF 5-MTHF levels normalized and seizures responded better to folinic acid. The neurodevelopmental improvement wa
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