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Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease

Abstract

Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10−13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.

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Fig. 1: Collapsing analysis identifies PHGDH as a MacTel gene.
Fig. 2: PHGDH variants with disrupted enzyme function identified in patients with MacTel.
Fig. 3: PHGDH p.Gly228Trp variant decreases serine synthesis in RPE.
Fig. 4: HSAN1/MacTel-linked SPTLC1 p.Cys133Tyr and PHGDH p.Gly228Trp variants elevate deoxySA in RPE.

Data availability

The datasets generated and/or analysed during the current study, if not presented in the manuscript, are available at GitHub (https://github.com/igm-team/MacTel) and/or from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Lowy Family for funding support of the MacTel project, the Lowy Medical Research Institute, and this study. Funding for the LC–MS procedure used to measure lipids was provided by NIH grant no. R01CA234245 to C.M.M. Some genetic studies were supported, in part, by NIH grants nos. R01EY028203, R01EY029315, and P30EY019007 to R.A., and by unrestricted funds from the Research to Prevent Blindness to the Department of Ophthalmology, Columbia University. We thank the Moorfields Eye Hospital Reading Centre for their evaluation of clinical images. For discussions and expertise on measurement of PHGDH activity we thank K. Mattaini. We thank M. L. Moon and J. Orozco for administrative assistance, and J. Trombley for clinical oversight. We thank B. Hart and P. Bernstein at the Moran Eye Center for supplying us with patient monocytes from which we generated iPSCs. We thank P. Westenskow for his assistance in establishing iPSC-RPE differentiation protocols, and C. Bautista and J. Gleeson for their plasmids and assistance in establishing CRISPR editing protocols. We also thank T. Cherry for processing the differential expression analysis for selected genes in Extended Data Fig. 3.

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Authors

Contributions

Conception of the genetics part of the study was done by R.A. and D.B.G. Design of collapsing analysis was performed by D.B.G. Sample preparation and some sequence acquisition was performed by C.C. Data analysis and interpretation was performed by T.N., J.A.H., E.H.B., C.J.W., R.A. and D.B.G. PHGDH enzymatic assay was designed and interpreted by M.L.G., R.B.B. and R.F. Execution of enzymatic assay was performed by R.B.B., R.F. and S.H.-P. iPSC-RPE metabolite measurement experiments were designed by K.E., M.L.G., M.W. and C.M.M.. CRISPR editing and iPSC-RPE on patients with HSAN1 were designed by K.E. and generated by K.E., S.H.-P. and S.G. Metabolite measurements on MS were performed by M.W., M.B. and E.W.L. Interpretation of metabolite measurements was performed by M.W. and K.E. The interpretation of cumulative results, writing of the manuscript and manuscript revision were performed by R.A., M.F., C.M.M., D.B.G., K.E., T.N., M.L.G., R.B.B., L.S. and M.I.D.

Corresponding author

Correspondence to Rando Allikmets.

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The authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks Stylianos Antonarakis, James Hurley, Xihong Lin, Jason Locasale and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Pooja Jha; Isabella Samuelson.

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Extended data

Extended Data Fig. 1 Pedigrees segregating possibly pathogenic PHGDH variants.

Three variants, given for each group, were analyzed for segregation with the disease in 5 families. The specific number, age, and result of genetic analysis is given for all family members who were available for clinical and genetic analyses. Filled, black symbols represent affected individuals, white symbols define unaffected family members and grey symbols depict family members with ambiguous diagnoses (maybe or maybe not affected). Ages of family members at the time of recruitment, when clinical diagnosis was determined, are given below each symbol. Wt, wild type allele; mut, the allele with the specific PHGDH variant.

Extended Data Fig. 2 Example of western blots.

a, Example western blots of overexpressed variants. Arrows indicated overexpressed protein (upper band, shifted from the FLAG-HA tag) and endogenous PHGDH protein (lower band). b, Relative expression of each PHGDH variant calculated from three independent experiments and normalized to corresponding WT expression in each blot. c) PHGDH enzymatic activity after correcting for endogenous activity and without normalizing for overexpressed variant protein abundance. Data for variants that retain less than 25% activity or expression are shown in red, between 25-75% activity or expression are shown in pink, and more than 75% activity or expression are shown in grey. Data are shown as the mean +/- SEM, n ≥ 3 independent experiments. Source data

Extended Data Fig. 3 Relative gene expression.

a, qPCR showing relative gene expression of enzymes from serine biosynthesis pathway. b, Schematic of the serine biosynthesis pathway from glucose showing metabolites (black) and enzymes/regulators (red). Data shown as the mean of three independently derived clones of wildtype (WT) and PHGDH p.Gly228TrpHET iPSC-RPE assayed in triplicate. Error bars +/- SEM. *p<0.05, **p<0.01 with unpaired two tailed t-test. c, Western blot of PHGDH and beta-actin loading control in WT and Gly228TrpHET iPSC-RPE clones. d, Relative protein levels of PHGDH normalized to beta-actin. Data shown as mean of 3 clones. Error bars +/- SEM. Unpaired two tailed t-test shows no difference. Source data

Extended Data Fig. 4 Metabolite tracing.

a, Schematic illustrating key metabolites in central carbon metabolism. b, % labeling of central carbon metabolites from [U-13C] glucose in cell pellet of iPSC-RPE. Points are the mean of three separately run wildtype iPSC-RPE samples. Error bars are +/- SEM. c, Schematic showing basal and apical secretion of metabolites from iPSC-RPE in transwells. d, Apical (blue) and basal (red) media measurements of serine from iPSC-RPE. e, Mean intracellular abundance of serine and glycine in wildtype iPSC-RPE. n=3 independent clones of wildtype iPSC-RPE. Error bars are +/- SEM. Source data

Extended Data Fig. 5 Isotopologue distribution.

a, Isotopologue Distribution of U-13C from glucose in serine and glycine. b, Relative abundance of 13C isotope in fully labeled serine (M3) and glycine (M2) from [U-13C6] glucose in cell culture media (secreted) between WT and PHGDH p.Gly228TrpHET iPSC-RPE over a period of 24 hours. a,b, Data shown as the mean of nine WT and eight PHGDH p.Gly228Trp replicates from three independently derived clones. Error bars +/- SEM. *p>0.05, **p>0.01 with unpaired two-tailed T-test. a, serine: M0 p=0.02, M2 p=0.04, M3 p=0.03; glycine: M0 p=0.0002, M1 p=0.052, M2 p=0.002. b, serine p=0.03. Source data

Extended Data Fig. 6 deoxySA/SA ratios.

a, DeoxySA/SA ratios following 2, 4, and 8 days of culturing control iPSC-RPE in serine and glycine free media. Each time point run in triplicate. Error bars SEM. b, Relative intracellular deoxySA/SA ratios in WT and PHGDH p.Gly228Trp iPSC-RPE following 2 days in serine and glycine free media. Data represented as mean of nine WT and eight PHGDH p.Gly228Trp replicates from three independently derived clones. c, Relative intracellular deoxySA/SA ratios in control patient and HSAN1 patient iPSC-RPE following 2 days in serine and glycine free media. Data represented as the mean of five independently derived iPSC-RPE clones from two control patients and six independently derived iPSC-RPE clones from two HSAN1 patietns. Error bars SEM. **p<0.01 unpaired two-tailed T-test. b, p=0.0002. c, p=0.0015. Source data

Supplementary information

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Eade, K., Gantner, M.L., Hostyk, J.A. et al. Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease. Nat Metab 3, 366–377 (2021). https://doi.org/10.1038/s42255-021-00361-3

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