Characterization of additional vitamin D binding protein variants

Area of Science:

• Genetics
• Biochemistry
• Molecular Biology

Background:

• The vitamin D binding protein (DBP) gene (GC) exhibits significant genetic variation.
• Common polymorphisms like p.D432E and p.T436K may affect vitamin D metabolism.
• Previous biochemical studies suggested the existence of less common, deleterious DBP variants.

Purpose of the Study:

• To identify underlying mutations in the GC gene responsible for previously observed biochemical variants.
• To characterize the identified mutant DBP proteins using mass spectrometry.
• To confirm the presence and nature of low-frequency DBP mutations in circulation.

Main Methods:

• Genetic screening of GC exons and boundaries using denaturing high-performance liquid chromatography (DHPLC).
• Sanger sequencing to identify specific mutations.
• Immuno-capture coupled with mass spectrometry to characterize protein variants in serum.

Main Results:

• Identified an alanine deletion at codon 246 (p.A246del) in 10 samples and a cysteine to phenylalanine substitution at codon 311 (p.C311F) in 1 sample.
• Mass spectrometry confirmed the presence of mutant DBP proteins and their post-translational modifications.
• Characterized the loss of a disulfide bond at cysteine-311 and formation of a novel mixed disulfide species.

Conclusions:

• Confirms the existence of additional deleterious GC mutations, including low-frequency variants.
• Demonstrates that mutant DBP proteins are secreted and detectable in the bloodstream.
• Highlights the utility of combining molecular screening with mass spectrometry for identifying and characterizing mutant DBP species.