Characterization of additional vitamin D binding protein variants

The gene (GC) for the vitamin D binding protein (DBP) shows significant genetic variation. Two missense variants, p.D432E and p.T436K, are common polymorphisms and both may influence vitamin D metabolism. However, less common variants, identified biochemically, have been reported previously. This study aimed to identify the underlying mutations by molecular screening and to characterize the mutant proteins by mass spectrometry. Denaturing high performance liquid chromatography (DHPLC) was used for screening genetic variants in GC exons and exon/intron boundaries of genomic DNA samples. Sanger sequencing identified the specific mutations. An immuno-capture coupled mass spectrometry method was used to characterize protein variants in serum samples. Initial molecular screening identified 10 samples (out of 761) containing an alanine deletion at codon 246 in exon 7 (p.A246del, c.737-739delCTG), and 1 sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (p.C311F, c.932G > T). The mutant allele proteins and posttranslational modified products were distinguishable from the wild-type proteins by mass spectrum profiling. Loss of a disulfide bond due to loss of cysteine-311 was accompanied by the appearance of a novel mixed disulfide species, consistent with S-cysteinylation of the remaining unpaired cysteine-299 in the mutant protein. We confirm earlier biochemical studies indicating that there are additional deleterious GC mutations, some of which may be low-frequency variants. The major findings of this study indicate that additional mutant proteins are secreted and can be identified in the circulation. By combining molecular screening and mass spectrometric methods, mutant DBP species can be identified and characterized.