The common H202D variant in GDF-15 does not affect its bioactivity but can significantly interfere with measurement of its circulating levels

Abstract

Background: There is growing interest in the measurement of growth-differentiation factor 15 (GDF-15) in a range of disorders associated with cachexia. We undertook studies to determine whether a common histidine (H) to aspartate (D) variant at position 202 in the pro-peptide (position 6 in the mature peptide) interfered with its detection by three of the most commonly used immunoassays.

Methods Three synthetic GDF-15-forms (HH homo-, HD hetero- and DD homodimers) were measured after serial dilution using Roche Elecsys®, R&D Quantikine ELISA and MSD R&D DuoSet immunoassays. GDF-15 concentrations were measured by the Roche and the MSD R&D immunoassays in 173 genotyped participants (61 HH homozygotes, 59 HD heterozygotes and 53 DD homozygotes). For the comparative statistical analyses of the GDF-15 concentrations we used non-parametric tests, in particular Bland-Altman difference (bias) plots and Passing-Bablok regression. The bioactivity of the two different homodimers was compared in a cell based assay in HEK293S-SRF-RET/GFRAL cells.

Results The Roche assay detected H and D-containing peptides similarly but the R&D reagents consistently underreported GDF-15 concentrations in the presence of the D variant. DD dimers had recoveries of ~45% while HD dimers recovered to 62-78%. In human serum samples, the GDF-15- concentrations reported by the R&D assay were a median of 4% lower in HH, a median of 36% lower in HD and a median of 61% lower in DD compared to the Roche assay. The bioactivities of the HH and DD peptides were indistinguishable. Conclusions The D variant of GDF-15 substantially affects its measurement by a commonly used immunoassay, a finding which has clear implications for its interpretation in research and clinical settings.