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Association of a transcobalamin II genetic variant with falsely low results for the holotranscobalamin immunoassay
Author(s) -
SobczyńskaMalefora Agata,
Pangilinan Faith,
Plant Gordon T.,
Velkova Aneliya,
Harrington Dominic J.,
Molloy Anne M.,
Brody Lawrence C.
Publication year - 2016
Publication title -
european journal of clinical investigation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.164
H-Index - 107
eISSN - 1365-2362
pISSN - 0014-2972
DOI - 10.1111/eci.12617
Subject(s) - medicine , homocysteine , vitamin d binding protein , methylmalonic acid , exon , vitamin b12 , western blot , cobalamin , gastroenterology , vitamin , endocrinology , gene , genetics , biology
Background The clinical use of holotranscobalamin (holoTC) testing to evaluate vitamin B 12 status has increased in recent years. We present two patients (African Caribbean and Indian heritage), in which the holoTC assay indicated severe B 12 deficiency (< 5 pmol/L). Additional clinical tests revealed that these patients had normal levels of total vitamin B 12 in blood and unremarkable levels of two other markers of vitamin B 12 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‐B 12 (Axis‐Shield Diagnostics Ltd). Design 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 B 12 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 B 12 . 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‐B 12 test. Laboratories should be aware of this variant and use confirmatory tests when applicable.