Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on vitamin B12 measurement in the blood which may not accurately reflect levels in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro. Despite normal serum levels, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased CSF B12 levels and clinical improvement. Optofluidic screening enabled rapid isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier. Autoantibodies targeting the same epitope of CD320 were identified in 7 other patients with neurologic deficits of unknown etiology, in 6% of healthy controls, and in 21.4% of a neuropsychiatric lupus cohort. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we discovered that the LDL receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue-specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.

Programmable Phage Display

We adapted a previously published protocol for phage immunoprecipitation sequencing (PhIP-seq). A library containing 731,724 49-amino acid peptides with 25-amino acid overlaps was cloned into T7 bacteriophage. Patient CSF was incubated with 10¹⁰ plaque forming units of the phage library, antibodies were enriched with protein A/G magnetic beads, and antibody-bound phage was amplified in E. coli before a second round of immunoprecipitation. Enriched phage lysates were adaptor ligated and barcoded prior to pair-end sequencing on an Illumina Novaseq to a depth of 2 million reads per sample. Reads were trimmed, aligned at the amino-acid level using RAPSearch, and normalized to sequencing depth to generate reads per 100,000 bases (RPK) for each sample. Enriched peptides were identified by calculating the fold-change of normalized counts between samples immunoprecipitated with CSF or magnetic beads only. The Benjamini-Hochberg method was used to correct for multiplicity and calculate false discovery rates.

CRISPRi Screen

The 10-sgRNA-per-gene CRISPRi repression library was synthesized, cloned, and infected into dCas9-KRAB K562 cells as previously described. Briefly, ~300 million K562 cells stably expressing EF1alpha-dCas9-KRAB were infected with the 10 guide/gene sgRNA library at a multiplicity of infection < 1. Infected cells underwent puromycin selection (1 ug/ml) for 5 days after which point puromycin was removed and cells were resuspended in normal growth medium without puromycin. After selection, sgRNA infection was confirmed by flow cytometry, which indicated >90% of cells expressed the mCherry reporter. Sufficient sgRNA library representation was confirmed by deep sequencing after selection. Cells were cultured and maintained at 1000× coverage for 1 week. Staining reagent was prepared by conjugating human transcobalamin II to a pH-sensitive fluorescent dye (pHrodo Deep Red iFL NHS ester) at a dye:protein molar ratio of 4:1. Free dye was removed using a 40kDa MWCO Zeba Spin Desalting Column. pHrodo-conjugated transcobalamin was incubated with a 3X molar ratio of cyanocobalamin at room temperature for 1 hour to form pHrodo-holotranscobalamin. This complex (100ug) was incubated with 300 million library cells per replicate (x2) in 150mL medium at 37 C for 4 hours prior to sorting. Cells were then washed and prepared for FACS. Cells that internalized the top 5% (15 million cells) and bottom 5% (15 million cells) of phrodo-holoTC were sorted for downstream processing. Genomic DNA was extracted for all populations separately using a QIAGEN Blood Midi Kit. Deep sequencing of sgRNA sequences on an Illumina NextSeq was used to monitor library composition. Guide composition was analyzed and compared to the plasmid library and between conditions using casTLE.