title: Association of a transcobalamin II genetic variant with falsely low results for the holotranscobalamin immunoassay.
abstract: BACKGROUND
  The clinical use of holotranscobalamin (holoTC) testing to evaluate vitamin B12 status has increased in recent years. We present two patients (African Caribbean and Indian heritage), in which the holoTC assay indicated severe B12 deficiency (< 5 pmol/L). Additional clinical tests revealed that these patients had normal levels of total vitamin B12 in blood and unremarkable levels of two other markers of vitamin B12 status, homocysteine and methylmalonic acid. We hypothesized that these patients carry a variant in the transcobalamin gene (TCN2) that influences the most widely commercially available holoTC test - Active-B12 (Axis-Shield Diagnostics Ltd).
  
  
  METHODS
  Exon sequencing of the TCN2 gene was carried out. Protein characterization included total transcobalamin (TCN2) detection by Western blot, and holoTC by (57) Co-labelled B12 binding followed by size fractionation.
  
  
  RESULTS
  Exon sequencing of TCN2 revealed both patients were homozygous for the minor allele of rs35838082 (p.R215W). Western blot and chromatographic analyses revealed that the serum of these patients contains intact transcobalamin and that this variant-containing protein binds vitamin B12 . The variant is rare in Caucasians (minor allele frequency (MAF) < 0·01) but more common in South Asians (MAF ~ 0·02) and those of African origin (MAF ~ 0·25).
  
  
  CONCLUSIONS
  The impeded ability to detect normal levels of holoTC in these two patients may be due to this variant interfering with the detection of holoTC by one or both of the monoclonal antibodies currently employed in the Active-B12 test. Laboratories should be aware of this variant and use confirmatory tests when applicable.
authors.affiliation: Nutristasis Unit (St. Thomas' Hospital), Viapath, London, UK.
authors.forename: Agata
authors.initials: A
authors.lastname: Sobczyńska-Malefora
authors.affiliation: Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, Bethesda, MD, USA.
authors.forename: Faith
authors.initials: F
authors.lastname: Pangilinan
authors.affiliation: Medical Eye Unit, Guy's and St. Thomas' NHS Foundation Trust, Moorfields Eye Hospital, The National Hospital for Neurology and Neurosurgery, London, UK.
authors.forename: Gordon T
authors.initials: GT
authors.lastname: Plant
authors.affiliation: Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, Bethesda, MD, USA.
authors.forename: Aneliya
authors.initials: A
authors.lastname: Velkova
authors.affiliation: Nutristasis Unit (St. Thomas' Hospital), Viapath, London, UK.
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Harrington
authors.affiliation: School of Medicine, Trinity College Dublin, Dublin, Ireland.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Molloy
authors.affiliation: Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, Bethesda, MD, USA.
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Brody
mesh_terms: D000328:Adult; D044383:Black People; D015153:Blotting, Western; D016022:Case-Control Studies; D005189:False Positive Reactions; D005260:Female; D014644:Genetic Variation; D006720:Homozygote; D006801:Humans; D007118:Immunoassay; D008403:Mass Screening; D017422:Sequence Analysis, DNA; D014155:Transcobalamins / Q000235:genetics* / Q000378:metabolism*; D014806:Vitamin B 12 Deficiency / Q000175:diagnosis* / Q000378:metabolism; D055815:Young Adult
chemical_list: D014155:Transcobalamins
keywords: Holotranscobalamin; cobalamin; monoclonal antibody; single nucleotide polymorphism; transcobalamin; vitamin B12