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Purpose: FLVCR1 encodes a solute carrier protein implicated in heme, choline, and ethanolamine transport. Although Flvcr1 -/- mice exhibit skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia (DBA), biallelic FLVCR1 variants in humans have previously only been linked to childhood or adult-onset ataxia, sensory neuropathy, and retinitis pigmentosa., Methods: We identified individuals with undiagnosed neurodevelopmental disorders and biallelic FLVCR1 variants through international data sharing and characterized the functional consequences of their FLVCR1 variants., Results: We ascertained 30 patients from 23 unrelated families with biallelic FLVCR1 variants and characterized a novel FLVCR1-related phenotype: severe developmental disorders with profound developmental delay, microcephaly (z-score -2.5 to -10.5), brain malformations, epilepsy, spasticity, and premature death. Brain malformations ranged from mild brain volume reduction to hydranencephaly. Severely affected patients share traits, including macrocytic anemia and skeletal malformations, with Flvcr1 -/- mice and DBA. FLVCR1 variants significantly reduce choline and ethanolamine transport and/or disrupt mRNA splicing., Conclusion: These data demonstrate a broad FLVCR1-related phenotypic spectrum ranging from severe multiorgan developmental disorders resembling DBA to adult-onset neurodegeneration. Our study expands our understanding of Mendelian choline and ethanolamine disorders and illustrates the importance of anticipating a wide phenotypic spectrum for known disease genes and incorporating model organism data into genome analysis to maximize genetic testing yield., Competing Interests: Conflict of Interest James R. Lupski has stock ownership in 23andMe, is a paid consultant for Genome International, and is a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, genomic disorders, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories. Amber Begtrup and Houda Elloumi are employees of GeneDx, LLC. All other authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily 1 . Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome 2-7 . Earlier studies concluded that FLVCR1 may function as a haem exporter 8-12 , whereas FLVCR2 was suggested to act as a haem importer 13 , yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters 14-16 . Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle., (© 2024. The Author(s).)

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c -/- ) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c -/- embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c -/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome., (© 2024. The Author(s).)