Loss of function variants inPCYT1Acausing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation
Authors: Jurgens Julie , Chen Suming , Sobreira Nara , Robbins Sarah , Anzmann Arianna Franca , Dastgheyb Raha , Khuder Saja S. , Hoover-Fong Julie , Woods Courtney , Collins Felicity , Christodoulou John , Yamamoto Guilherme Lopes , Bertola Débora Romeo , Baratela Wagner A. R. , Curie Sophie D. , Haughey Norman , Cornell Rosemary , Valle David 
Published: December 2019

Spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants inPCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase α (CCTα), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences ofPCYT1Avariants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-editedPCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCTα steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). WhilePCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers or sizes as compared to controls. Lipid droplet phenotypes inPCYT1A-null HEK293 cells were rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His humanPCYT1AcDNAs. While both edited cellular models had normal morphology and proliferation rates compared to unedited controls,Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-200 lipid levels inPCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as compared to wild-type controls. The specific lipids altered and extent of change varied by cell type. Importantly, bothPCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting compensatory PC production through increased LPC remodeling by LPCAT or decreased conversion of PC to LPC by phospholipase A2. Our results show that all testedPCYT1Aalleles associated with SMD-CRD are hypomorphic and suggest involvement ofPCYT1Ain chondrocyte differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation.

Author Summary

Rare genetic disorders can reveal the function of genes on an organismal scale. When normal gene activity is lost, patients can experience a range of symptoms, often dependent on the residual activity of the encoded protein. Rare variants in the genePCYT1Acan cause multiple inherited disorders, including a disorder of the skeleton and the retina characterized by short stature, bone abnormalities, and blindness.PCYT1Ais required for normal cellular function, particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. To determine consequences of genetic variants in patients with this disorder, we made and studied a series of cellular models, including cells cultured from patients and CRISPR-edited cell lines lacking normal copies ofPCYT1A. Here we show that patient variants lead to reducedPCYT1Aexpression and/or function and have adverse consequences on cell biology and lipid metabolism that are often cell-type specific. This work advances understanding of the role of lipid metabolism in skeletal and eye development.