DOI: 10.5061/dryad.6djh9w18j
title: Transcobalamin receptor antibodies in autoimmune vitamin B12 central deficiency

description: 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.
description: <em>Programmable Phage Display</em>
 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 10<sup>10</sup>
 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.
 <em>CRISPRi Screen</em> 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
 &lt; 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 &gt;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.
description: # Transcobalamin Receptor Antibodies in Autoimmune Vitamin B12 Central
 Deficiency
 [https://doi.org/10.5061/dryad.6djh9w18j](https://doi.org/10.5061/dryad.6djh9w18j) ## Description of the data and file structure NID0063_peptide.csv -&gt; normalized RPK file of peptide enrichments for mock IP (BEAD), GFAP commercial Ab IP, and Case 1 CSF IP (NID0063) NID0063_gene.csv -&gt; normalized RPK file of gene-level enrichments for mock IP (BEAD), GFAP commercial Ab IP, and Case 1 CSF IP (NID0063) B12_K562_combo.csv -&gt; CRISPRi screen hit list; negative effect size indicates that the knockdown impaired holotranscobalamin uptake; positive effect size indicates that the knockdown promoted uptake; confidence score and FDR calculated using caSTLE (Morgens et al, 2016). REP1_NEG_S15_L003_R1_001.fastq.gz -&gt; fastq file of replicate 1 of CRISPRi screen, bottom 5% B12 uptake REP1_POS_S16_L003_R1_001.fastq.gz -&gt; fastq file of replicate 1 of CRISPRi screen, top 5% B12 uptake REP2_NEG_S17_L003_R1_001.fastq.gz -&gt; fastq file of replicate 2 of CRISPRi screen, bottom 5% B12 uptake REP2_POS_S18_L003_R1_001.fastq.gz -&gt; fastq file of replicate 2 of CRISPRi screen, top 5% B12 uptake PEP43_Plate3_BEAD***.fastq.gz -&gt; fastq file of mock immunoprecipitations from PhIP-seq experiment PEP43_Plate3_REP***_NID-0063 ***.fastq.gz -&gt; fastq file of Case 1 CSF immunoprecipitations from PhIP-seq experiment