Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a pleiotropic disease spectrum from adult neurodegeneration to severe developmental disorders

Abstract

FLVCR1 encodes Feline leukemia virus subgroup C receptor 1 (FLVCR1), a solute carrier (SLC) transporter within the Major Facilitator Superfamily. FLVCR1 is a widely expressed transmembrane protein with plasma membrane and mitochondrial isoforms implicated in heme, choline, and ethanolamine transport. While Flvcr1 knockout mice die in utero with skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia, rare biallelic pathogenic FLVCR1 variants are linked to childhood or adult-onset neurodegeneration of the retina, spinal cord, and peripheral nervous system. We ascertained from research and clinical exome sequencing 27 individuals from 20 unrelated families with biallelic ultra-rare missense and predicted loss-of-function (pLoF) FLVCR1 variant alleles. We characterize an expansive FLVCR1 phenotypic spectrum ranging from adult-onset retinitis pigmentosa to severe developmental disorders with microcephaly, reduced brain volume, epilepsy, spasticity, and premature death. The most severely affected individuals, including three individuals with homozygous pLoF variants, share traits with Flvcr1 knockout mice and Diamond-Blackfan anemia including macrocytic anemia and congenital skeletal malformations. Pathogenic FLVCR1 missense variants primarily lie within transmembrane domains and reduce choline and ethanolamine transport activity compared with wild-type FLVCR1 with minimal impact on FLVCR1 stability or subcellular localization. Several variants disrupt splicing in a mini-gene assay which may contribute to genotype-phenotype correlations. Taken together, these data support an allele-specific gene dosage model in which phenotypic severity reflects residual FLVCR1 activity. This study expands our understanding of Mendelian disorders of choline and ethanolamine transport and demonstrates the importance of choline and ethanolamine in neurodevelopment and neuronal homeostasis.

Keywords: Diamond-Blackfan anemia; FLVCR1; choline; ethanolamine; multiple congenital anomalies; neurodegeneration; neurodevelopmental disorders.

Figure 2:. Phenotypic features of FLVCR1-related developmental and neurodegenerative disorders

Photographs and magnetic resonance imaging (MRI) from each family are divided into separate boxes bordered by thin black lines. Per medrxiv requirements, patient photographs have been removed but are available upon request to the corresponding authors. A-E) Images of Individual 1 (I-1), Family 1 with homozygous FLVCR1 p.G464S variant. A-D show examples of self-mutilation including foot, ear, and arm ulcerations (yellow arrowheads) and digital amputation (red arrowhead). E shows sagittal T1-weighted brain MRI. Pontine thinning is indicated with yellow arrow. F) Image of Individual 8 (I-8), Family 7 with homozygous FLVCR1 p.G464S variant. Self-mutilation and digital amputation of the hand is shown. G, H) Images of Individual 2 (I-2), Family 2. G shows patient with triangular facies, microcephaly and severe developmental delay. H shows axial T2-weighted brain MRI demonstrating severe reduction in cerebral brain volume and a simplified gyral pattern. I, J) Images of Individual 5 (I-5), Family 5. I shows patient with microcephaly, broad nasal bridge, widely spaced eyes, and severe developmental delay. J shows axial T1-weighted brain MRI, demonstrating severe reduction in cerebral brain volume and a simplified gyral pattern. K, L) Images of Individual 3 (I-3), Family 3. K shows axial T2-weighted brain MRI with severe reduction in cerebral brain volume. L shows T2-weighted cervical spine MRI demonstrating posterior column T2 signal hyperintensity (red arrow). M-R) Images of Individuals 10 and 11 (I-10 and I-11), Family 8. M and N show individuals 10 and 11. Photographs demonstrating microcephaly, severe developmental delay, broad nasal bridge, and tracheostomy secondary to respiratory failure. O-R show axial T1-weighted brain MRI from Individual 11 demonstrating reduction in cerebral brain volume with relative cerebellar sparing and cystic encephalomalacia (red arrows). S-U) Images of Individual 7 (I-7), Family 6. Images show axial T2-weighted brain MRI with reduction in cerebral brain volume. V, W) Images of Individual 16 (I-16), Family 11. V and W show coronal T2-weighted brain MRI and axial T1-weighted brain MRI respectively demonstrating a severe reduction in brain volume with hydrocephalus ex vacuo. X-d) Images of Individual 17 (I-17), Family 12. X shows irregular undulation of the ear lobe. Y shows the left foot with ankle dislocation and absence of the great toe, 2^nd toe, and 3^rd toe. Z shows the right foot demonstrating cleft foot, absence of the 2^nd toe, syndactyly of toes 3–4, and proximal displacement of the great toe. a is a full body radiograph demonstrating ten ribs, long limbs, absent left tibia (red arrow), and bilateral ectrodactyly of the feet. b-d are fetal T2-weighted MRI images performed at 28 weeks gestation. b shows hydrocephalus with absence of midline structures (blue arrow) and hypertrophic kidney with pelviectasis (red arrow). c shows hydrocephalus with absence of some midline structures (red arrow) and thinning of the cortical mantle (blue arrow). d shows severe hydrocephalus with thinning of the cortical mantle (red arrow), small cerebellum and brainstem (blue arrow), and left kidney hypertrophy with absence of the right kidney (yellow arrow). e-g) Images of Individual 13 (I-13), Family 9. Postmortem images of Individual 13 (c-e) show joint contractures, long tapering figures. absent right thumb, hypoplastic left thumb, microcephaly, and cleft lip and palate.

Figure 3:. Summary of the FLVCR1 allelic series

A) Model of protein-coding portions of human FLVCR1 cDNA (NM_014053.4) showing location of novel and previously published start loss variants, splicing variants, nonsense variants, and frameshift variant alleles. Start loss and splicing variants are indicated by arrowheads above the model. Nonsense and frameshift variants are indicated by arrowheads below the model. Red arrowheads show variants associated with severe phenotypes, whereas light blue shading indicates the relatively milder neurological disease. B) Model of human FLVCR1 protein (Uniprot entry Q9Y5Y0) showing location of novel and previously published missense variants. Light gray rectangles indicate transmembrane domains, dark gray rectangles indicate intracellular domains, and dark blue rectangles indicate extracellular domains. Red ball and sticks indicate severe phenotypes, whereas light blue ball and sticks indicate mild phenotypes. Portions of FLVCR1 found within the plasma membrane isoform FLVCR1a and mitochondrial isoform FLVCR1b are indicated below the figure in green and purple, respectively. Red box indicates region deleted due to c.884–3C>G (r.884_1024del, p.A295_Y341del). ESM1b pathogenicity predictions are shown above the model. Increasing likelihood of pathogenicity is reflected by the blue (low) to yellow (high) gradient. C) Comparison of pathogenicity predictions (CADD, REVEL, MetaRNN) and conservation metrics (phyloP100way, GERP) between novel and previously reported pathogenic FLVCR1 variants and all variants within gnomAD v3.1.2 (76,156 human samples, 189 variants) and primateseq v1.0 (811 primate samples from 236 primate species, 209 variants). * p<0.05, unpaired t test with Welch’s correction. D) Comparison of pathogenicity predictions (CADD, REVEL) and conservation metric (GERP) between mild and severe FLVCR1-related phenotypes. Differences in transmembrane domains 9–11 and isoform clustering of FLVCR1 variants between mild and severe phenotypes is also displayed. Gray = variants located outside transmembrane domains 9–11, black = variants located inside transmembrane domains 9–11, purple = variants located inside mitochondrial isoform FLVCR1b, green = variants located inside plasma membrane isoform FLVCR1a but not FLVCR1b. ns = not significant, TM = transmembrane.

Figure 4:. Pathogenic missense variation in FLVCR1 reduces choline and ethanolamine transport

A-B) Choline transport activity of the FLVCR1 missense variants expressed as absolute values (DPM/well) or as percentage of wildtype (WT) FLVCR1. These missense variants and WT human FLVCR1 (hFLVCR1) were co-expressed with choline kinase alpha (CHKA) in HEK293 cells for activity assay with [3H] choline. Mock was transfected with CHKA alone. Experiments were performed at least twice in triplicates. Data are expressed as mean ± SD. ****P<0.0001; One-way ANOVA for A-B. ns, not significant. C-D) Ethanolamine transport activity of the FLVCR1 missense variants expressed as absolute values (DPM/well) or as percentage of WT FLVCR1. These missense variants and WT human FLVCR1 were co-expressed with ethanolamine kinase 1 (ETNK1) in HEK293 cells for activity assay with [14C] ethanolamine. Mock was transfected with ETNK1 alone. Experiments were performed at least twice in triplicates. Data are expressed as mean ± SD. ****P<0.0001; One-way ANOVA for C-D. ns, not significant. E) Western blot analysis of HEK293 cells overexpressed with WT or hFLVCR1 variants. Red vertical arrowhead marks M_r = 59 kD migration of FLVCR1; GAPDH loading control shown below. F) Immunostaining of WT or hFLVCR1 mutants overexpressed in HEK293 cells. Vertical yellow horizontal bar shows image scale. Arrows (yellow) show plasma membrane, where membrane GFP was used as a marker. Arrowheads show intracellular signals of hFLVCR1.