Dihydrofolate Reductase Deficiency Due to a Homozygous DHFR Mutation Causes Megaloblastic Anemia and Cerebral Folate Deficiency Leading to Severe Neurologic.

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Dihydrofolate Reductase Deficiency Due to a Homozygous DHFR Mutation Causes Megaloblastic Anemia and Cerebral Folate Deficiency Leading to Severe Neurologic Disease Holger Cario, Desirée E.C. Smith, Henk Blom, Nenad Blau, Harald Bode, Karlheinz Holzmann, Ulrich Pannicke, Karl-Peter Hopfner, Eva- Maria Rump, Zuleya Ayric, Elisabeth Kohne, Klaus-Michael Debatin, Yvo Smulders, Klaus Schwarz The American Journal of Human Genetics Volume 88, Issue 2, Pages 226-231 (February 2011) DOI: 10.1016/j.ajhg.2011.01.007 Copyright © 2011 The American Society of Human Genetics Terms and Conditions

Figure 1 Identification of the DHFR c.458A>T Mutation in a Family and Tertiary Structure of Human DHFR (A) Pedigree of the family. Genetic analysis was performed in the affected siblings and their parents. (B) Sequence analysis of DHFR exon 5 detected a homozygous base change, c.458A>T, in all siblings and a heterozygous mutation in both parents. Amplifications were performed with the HotStar-Taq DNA Polymerase Kit (QIAGEN) with 35 cycles in a GeneAmp PCR System 9700 (Applied Biosystems). Sequencing reactions were performed with BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) and analyzed on an ABI 3100 DNA Genetic Analyzer (Applied Biosystems). (C) Crystal structure of human DHFR (yellow ribbon model with highlighted secondary structure, Protein Data Bank entry 2W3M) bound to NADPH (color-coded stick model with green carbons, white phosphors, red oxygens, and blue nitrogens) and DHF (ruby stick model). The mutation site Asp153Val is highlighted as a magenta stick model. Asp153 stabilizes one side of the “F-G” loop, indicating that the mutation alters DHFR activity by influencing the fold and dynamics of the catalytically important “F-G” and “Met20” loops. The figure was prepared with the program PyMOL (Schrödinger). The American Journal of Human Genetics 2011 88, 226-231DOI: (10.1016/j.ajhg.2011.01.007) Copyright © 2011 The American Society of Human Genetics Terms and Conditions

Figure 2 Functional Characteristics of the DHFR Mutant (A) DHFR activity in lymphoblastoid cells of control individuals (DHFR wild-type), the patients' mother (heterozygous for DHFR p.Asp153Val = p.D153V), and the three siblings (homozygous for DHFR p.D153V) as measured by the formation of THF out of DHF per hour and mg protein. A DHFR Kit (Sigma) was used for the assay (for details, see text). (B) Fluorescence-activated cell sorting (FACS) analysis of lymphoblastoid cells after incubation with 10 μM fluorescein-labeled methotrexate (FMTX; Invitrogen). Cells were additionally stained with 7-Aminoactinomycin D (7-AAD; 2,5 μg/ml) to sort vital cells for FMTX-FACS analysis. (C) DHFR RT-PCR after isolation of mRNA from lymphoblastoid cells of the heterozygous mother (lanes 2 and 9), the three homozygous siblings (lanes 3–5 and 10–12; patients 1–3), and a control individual (DHFR wild-type) (lanes 6 and 13) revealed a single ∼450 bp cDNA fragment (agarose gel, 1.5%). cDNA was synthesized with the use of random hexamers and Superscript II RNase HT (Invitrogen) at 42°C for 50 min. DHFR PCR was performed in 35 cycles at 55°C (for primer sequences, see Table S1). (D) DHFR protein expression in DHFR p.D153V heterozygous (lane 1, mother) and homozygous (lane 2, patient 2; lane 3, patient 3; lane 4, patient 1) lymphoblastoid cells (upper panel) and fibroblasts as compared to control cells (stepwise dilution of total blotted protein: lanes 5–9). In the lower panel, lane 4 is waste because fibroblasts from patient 1 were not available. After separation on a 15% SDS gel, proteins were blotted on an Immobilon-P membrane (Millipore). Primary antibodies (DHFR [E-18], MAPK1 [D-2], Santa Cruz Biotechnology) were diluted to a concentration of 1:200 and 1:10,000; secondary antibodies (anti-goat IgG-HRP, Santa Cruz Biotechnology; goat anti-mouse IgG [H+L]-HRP, BioRad), 1:2000 and 1:10,000, respectively. Blots were developed with the use of the Super Script West Pico Chemoluminescent Substrate Kit (Thermo Scientific). The American Journal of Human Genetics 2011 88, 226-231DOI: (10.1016/j.ajhg.2011.01.007) Copyright © 2011 The American Society of Human Genetics Terms and Conditions