46Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal 47 recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding 48 the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-49 limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in 50 understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we 51 generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited 52 PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured 53 skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in 54 CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of 55 wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response 56 to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts 57 (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers 58 or sizes as compared to controls. Lipid droplet phenotypes in PCYT1A-null HEK293 cells were 59 rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His human PCYT1A cDNAs. 60 While both edited cellular models had normal morphology and proliferation rates compared to 61 unedited controls, Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte 62 differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-63 200 lipid levels in PCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as 64 compared to wild-type controls. The specific lipids altered and extent of change varied by cell 65 type. Importantly, both PCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines 66 had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels 67 of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting 4 68 compensatory PC production through increased LPC remodeling by LPCAT or decreased 69 conversion of PC to LPC by phospholipase A 2 . Our results show that all tested PCYT1A alleles 70 associated with SMD-CRD are hypomorphic and suggest involvement of PCYT1A in chondrocyte 71 differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation. 72 Author Summary 73 Rare genetic disorders can reveal the function of genes on an organismal scale. When 74 normal gene activity is lost, patients can experience a range of symptoms, often dependent on the 75 residual activity of the encoded protein. Rare variants in the gene PCYT1A can cause multiple 76 inherited disorders, including a disorder of the skeleton and the retina characterized by short 77 stature, bone abnormalities, and blindness. PCYT1A is required for normal cellular function, 78 particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. 79 T...