Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination Tojo Nakayama, Almundher Al-Maawali, Malak El-Quessny,

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Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination Tojo Nakayama, Almundher Al-Maawali, Malak El-Quessny, Anna Rajab, Samir Khalil, Joan M. Stoler, Wen-Hann Tan, Ramzi Nasir, Klaus Schmitz-Abe, R. Sean Hill, Jennifer N. Partlow, Muna Al-Saffar, Sarah Servattalab, Christopher M. LaCoursiere, Dimira E. Tambunan, Michael E. Coulter, Princess C. Elhosary, Grzegorz Gorski, A. James Barkovich, Kyriacos Markianos, Annapurna Poduri, Ganeshwaran H. Mochida The American Journal of Human Genetics Volume 96, Issue 5, Pages 709-719 (May 2015) DOI: 10.1016/j.ajhg.2015.03.003 Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 1 Pedigrees Studied and Findings from Brain MRI of the Affected Individuals (A) Pedigree structure of the studied families (MR900 and MC27800). Shaded symbols indicate affected individuals. (B) Facial pictures of the affected individuals in family MR900. (C) MRI findings of the affected individuals. T2-weighted axial images (top row) and T1-weighted sagittal images (bottom row) are shown. The ages at imaging were 4 years and 0 months (MR901), 3 years and 4 months (MR902), 2 years and 7 months, (MR27801), 1 year and 10 months (MC27802), and 3 years and 1 month (control subject). All affected individuals showed microcephaly, hypomyelination, reduced white-matter volume, a thin corpus callosum, and a mildly thin brain stem. The scale bar represents 5 cm. The American Journal of Human Genetics 2015 96, 709-719DOI: (10.1016/j.ajhg.2015.03.003) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 2 Linkage Mapping and Identification of PYCR2 Mutations (A) Genome-wide LOD scores combined for the two families show the only significant linkage region to be 1q42, which has a maximum multipoint LOD score of 3.72. (B) Schematic representation of the PYCR2 variants. Residue Arg119 is in the NAD(P)-binding domain, and residue Arg251 is in the dimerization domain. (C) Sanger sequencing confirmed the identified variants in family MR900 (c.355C>T [p.Arg119Cys]) and family MC27800 (c.751C>T [p.Arg251Cys]). These variants segregated with the disease phenotype in each family. Positions of variants are according to RefSeq NM_013328.3 (cDNA) and RefSeq NP_037460.2 (protein). The American Journal of Human Genetics 2015 96, 709-719DOI: (10.1016/j.ajhg.2015.03.003) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 3 Effect of PYCR2 Mutations (A) Immunoblot analysis of PYCR2 levels in lymphoblastoid cell lines. Cells from an affected individual (MR901) showed a marked reduction of PYCR2, whereas cells from his father (MR904) showed a mild reduction. (B) Quantification of PYCR2 levels. The PYCR2/β-actin intensity ratios for MR901 and MR904 were measured in relation to that of the control subject. (C) Abundance of wild-type (WT) and variant (p.Arg119Cys and p.Arg251Cys) PYCR2 in transfected HEK293FT cells. The cells were transfected with a wild-type or mutant human PYCR2 cDNA construct with a C-terminal V5 tag, along with a separate EGFP construct. Immunostaining for V5 showed an almost undetectable level of the p.Arg119Cys variant protein and mildly decreased level of the p.Arg251Cys variant protein. The scale bar represents 20 μm. (D) Immunoblot analysis of PYCR2 and EGFP levels in transfected HEK293FT cells showed a dramatic reduction of the p.Arg119Cys variant protein and a mild reduction of the p.Arg251Cys variant protein. EGFP served as a transfection control. (E) Immunoblot analysis was quantified by comparison of the PYCR2-V5/EGFP intensity ratio between the variants proteins and the wild-type protein. The data were averaged from three independent experiments. The p.Arg119Cys variant showed less than 2% of the wild-type abundance. Although the p.Arg251Cys variant did not reach statistical significance, it showed a trend toward abundance lower than wild-type abundance. Error bars represent the SD. ∗∗∗p < 0.001. The American Journal of Human Genetics 2015 96, 709-719DOI: (10.1016/j.ajhg.2015.03.003) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 4 PYCR2 Loss of Function Increases Apoptosis under Oxidative Stress (A) HEK293FT cells were treated with 400 μM H2O2 for 1 hr, followed by 24 hr in culture. The mutant cells, harboring a homozygous frameshift mutation (PYCR2 mutant), showed more apoptosis than did wild-type cells, as detected by TUNEL assay. The scale bar represents 200 μm. (B) Quantification of TUNEL assay revealed a statistically significant increase in apoptosis of the mutant cells treated with H2O2. Data were averaged from two independent experiments. Error bars represent the SD. ∗∗p < 0.01. The American Journal of Human Genetics 2015 96, 709-719DOI: (10.1016/j.ajhg.2015.03.003) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 5 Morpholino-Based Knockdown of Zebrafish pycr1b Recapitulates the Human Microcephaly Phenotype (A) Compared to zebrafish injected with control MO (control MO), 4-dpf zebrafish larvae injected with 3.5 ng pycr1b MO at the 1- to 2-cell stage (pycr1b MO) showed a small head size (horizontal white bars represent the maximum head width measured) and a curved tail. Co-injection of human PYCR2 mRNA (hPYCR2 WT; 20 pg) rescued the phenotype. The scale bar represents 100 μm. Representative images are shown. (B) Measurements of the maximum head width of pycr1b morphants were significantly smaller than those of the control. Co-injection of human PYCR2 mRNA (hPYCR2 WT) rescued the phenotype, but mutant mRNAs (hPYCR2 c.355C>T and hPYCR2 c.751C>T) failed to show a full rescue effect. Twenty larvae for each category were randomly selected for measurement of the maximum head width illustrated in (A). Bars represent the mean value and SEM. ∗∗∗p < 0.001. (C and D) Sagittal brain sections of larvae injected with control MO (C) or pycr1b MO (D) at 4 dpf were stained with toluidine blue. The size of the forebrain, midbrain, and hindbrain was significantly smaller in the pycr1b morphant than in the control. Scale bars represent 100 μm. The American Journal of Human Genetics 2015 96, 709-719DOI: (10.1016/j.ajhg.2015.03.003) Copyright © 2015 The American Society of Human Genetics Terms and Conditions