Characterization of additional vitamin D binding protein variants

Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 54–59

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Characterization of additional vitamin D binding protein variants Lei Fua,b , Chad R. Borgesc , Douglas S. Rehderc , Betty Y.L. Wonga , Rashida Williamsb , Thomas O. Carpenterd,e, David E.C. Colea,b,f,g,* a

Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada c Department of Chemistry & Biochemistry and Center for Personalized Diagnostics at the Biodesign Institute, Arizona State University, Tempe, AZ, USA d Department of Pediatrics (Endocrinology), Yale University School of Medicine, New Haven, CT, USA e Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA f Department of Pediatrics (Genetics), University of Toronto, Toronto, ON, Canada g Department of Medicine, University of Toronto, Toronto, ON, Canada b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 June 2015 Received in revised form 7 December 2015 Accepted 22 February 2016 Available online 23 February 2016

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. ã 2016 Elsevier Ltd. All rights reserved.

Keywords: Vitamin d binding protein Genetic variants Molecular screening Immuno-capture coupled mass spectrometry

1. Introduction Vitamin D binding protein (DBP), also known as group specific component (Gc), is a 51–52 kDa multi-functional plasma glycoprotein. The physiological functions include transport of vitamin D and its metabolites, macrophage activation, actin scavenging, and fatty acid binding [1]. The protein comprises three domains with

Abbreviations: DBP, vitamin D binding protein; DHPLC, denaturing high performance liquid chromatography; GC, group specific component; dbSNP, single nucleotide polymorphism database; 25OHD, 25-hydroxyvitamin D; LC-ESI-TOFMS, liquid chromatography-electrospray ionization-time of flight-mass spectrometry; MAF, minor allele frequency. * Corresponding author at: Department of Clinical Pathology, Sunnybrook Health Sciences Centre, Room E346, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada. E-mail address: [email protected] (D.E.C. Cole). http://dx.doi.org/10.1016/j.jsbmb.2016.02.022 0960-0760/ ã 2016 Elsevier Ltd. All rights reserved.

overlapping properties. The first domain (aa. 1–191, based on the mature peptide) contains the peptide sequence responsible for binding vitamin D (aa. 35–49) as well as cell binding and actin binding regions. The second domain (aa. 192–378) is responsible for actin binding and mediates the largest number of interactions. The third domain acts similarly to the first in its interaction with the cell and with actin molecules; however, it does not interact directly with vitamin D [2–4]. The GC gene (NCBI GENE ID2638) for the DBP is found on chromosome 4 at 4q11–q13 and consists of 13exons and 12 introns, and shows significant variation [5,6]. Two missense variants (D432E and T436 K) are the major common polymorphisms, and both may influence vitamin D metabolism [7,8]. These single nucleotide polymorphisms (SNPs) in exon 11 encode electrophoretically distinguishable proteins called Gc1F, Gc1S and Gc2, respectively. However, many other less common

L. Fu et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 54–59

variants, identified by isoelectric focusing techniques, have been reported in the past [10–12]. The functional consequences of these additional variants have not yet been delineated. We have previously demonstrated that the GC genotypes are correlated with differences in circulating DBP levels, and strikingly, that GC genotypes are correlated with total circulating 25hydroxyvitamin D (25OHD) levels [7]. This relationship between genotypes and serum levels of vitamin D metabolites has broader implications when genetic background is considered [9]. Yet to be determined is the presence of less common genetic variants in the circulation and the question as to whether they too exert an effect on serum levels. In order to identify additional less-common variants, various molecular and spectrometric techniques might be employed. We used an immuno-capture coupled mass spectrometry technique that has been developed to characterize large-mass proteins. It can be used to detect the microheterogeneity (polymorphisms, transcript variants and posttranslational modifications) that exists for specific proteins within individual samples as well as at the population level [13–15]. These techniques were not only able to identify common DBP alleles (Gc1F, Gc1S and GC2) by their unique m/z profiles, but also detect novel posttranslationally modified proteoforms. In addition, these mass spectrometric procedures have been used previously to examine the glycosylation status of DBP in cancer patients [14]. In this study, we aimed to identify new mutations of DBP by molecular screening and to characterize the mutant proteins in serum by mass spectrometry. 2. Methods 2.1. Study population Over 770 healthy children (6–36 months old) were recruited during well-child visits to one of four neighborhood health clinics in a midsized northeastern U.S. urban community (New Haven, CT, USA). As previously reported [16], biochemical parameters in serum samples were measured by clinical laboratories according to the standard procedures. Assignment of ancestry for the study subjects was based on self-reporting by the parents as well as analysis of ancestry informative markers. The study was approved by the Yale University Human Investigation Committee and written informed consent was obtained from the appropriate parent or guardian. 2.2. Genetic screening All the GC exons including the exon-intron boundaries and the hypersensitive site 4 region were PCR amplified and genetic variants in the amplified DNA fragments were screened by denaturing high performance liquid chromatography (DHPLC). Short exons with similar predicted melting curves were assembled into one amplified fragment (exon 2 with exon 7 and exon 10 with exon 12) using Meta-PCR which was made up of two PCR reactions as illustrated in Supplementary Fig. S1 [17]. The DHPLC protocol was performed as previously described [18]. The PCR product was heated to 96  C for 5 min and allowed to cool slowly to room temperature for 30 min, with a decrease in temperature of 3  C per minute. Analysis was carried out in the Transgenomic WAVE1 system equipped with a DNASepTM column (Transgenomics, Omaha, NE, USA). Five microlitres of PCR product were injected and eluted at the partial melting temperature with an acetonitrile gradient of 9% in 4.5 min and a constant flow rate of 0.9 mL/min. The gradient was created by mixing Buffer A (0.1 M Triethylamine acetate buffer (TEAA) pH7, 0.1 M Na2EDTA) and Buffer B (25% acetonitrile, 0.1 M TEAA, 0.1 M Na2EDTA). The initial and final concentrations of Buffer B were adjusted according to the

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length of the fragment. Eluted DNA fragments were detected by UV at 260 nm. After each analysis, the column was cleaned with 100% acetonitrile for 0.5 min and reconditioned in Buffer B (5% below the initial concentration) for 1.5 min before injecting next sample. 2.3. Genotype confirmation The specific mutations detected by DHPLC screening were characterized by Sanger sequencing. The two common genetic variants D432E and T436K were genotyped by an allele specific PCR-RFLP method as previously described [8]. The details of allele frequencies, genotypes and diplotypes of D432E and T436K for this specific cohort are found in an earlier publication [16]. 2.4. Protein mass spectrometry profiling DBP from serum/plasma samples was first isolated using the immuno-capture pipette tips derivatized with the DBP antibodies. Then, a Liquid Chromatography Electrospray Ionization Time of Flight Mass Spectrometry (LC-ESI-TOF-MS) system was used for detection and characterization of DBP protein variants as previously described [13–15]. Polyclonal rabbit anti-human DBP antibodies (Cat. No. A0021, DAKO Denmark A/S), were used in making immuno-capture pipette tips for isolation of DBP from serum/plasma samples. 25 mL of serum or EDTA plasma sample was required for the affinity extraction. Half of the eluted sample (5 mL) was injected into the LC-TOF-MS system within 10 min of elution. A trap-and-elute form of sample concentration/solvent exchange was used for the liquid chromatography/mass spectrometry analyses. The Bruker MicroOTOF-Q (QTOF) mass spectrometer was operated in positive ion, TOF-only mode. Agilent G1385A capillary nebulizer was the ion source. The ESI charge-state envelope was deconvoluted with Bruker Data Analysis v3.4 software. 2.5. Biochemical measurements Serum total 25OHD was measured by radioimmunoassay (DiaSorin, Stillwater, MN, USA). DBP was measured by an immunonephelometry method (Behring Diagnostics, Inc., Westwood, MA, USA) as described in previous publications [16,19]. To further validate the DBP method, a comparison study was performed against a commercial ELISA kit (Immundiagnostik AG, Germany; distributed by ALPCO, Salem NH, USA). The results yield an acceptable correlation (Pearson r = 0.61, p < 0.016). Serum calcium, phosphate and albumin levels were measured according to standard clinical laboratory procedures. 2.6. Statistical analysis GraphPad Prism 5 (GraphPad Software Inc., San Diego CA) and SPSS v20.0 (SPSS Inc., Chicago IL) software packages were used for data analysis. Comparisons of the means between groups were performed by one-way ANOVA. p < 0.05 was considered significant. 3. Results 3.1. GC variants Twenty four different genetic variants were found in our sample population. Twenty two were documented in the SNP database (dbSNP) and two were not. In Table 1, only the five variants causing amino acid changes are listed. All the other variants were silent mutations or located in non-coding regions. In addition to the two common variants, p.D342E and p.T436K, we identified ten samples

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L. Fu et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 54–59

Table 1 Summary of the functional genetic variants. Nucleotide changes

Amino acid changes

Exon number

SNP rs

c.737_739delCTG c.757C > T c.932G > T c.1296T > G c.1307C > A

p.A246del p.P253S p.C311F p.D432E p.T436K

7 7 8 11 11

rs.529043158

*

*

rs.780473721 rs.7041 rs.4588

Not reported in dbSNP.

(out of 761) containing an alanine deletion at codon 246 (c.737_739delCTG, p.A246del, rs.529043158) in exon 7. Finally, there was one sample (out of 761) containing a proline to serine substitution at codon 253 (c.757C > T, p.P253S) in exon 7, and one sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (c.932G > T, p.C311F, rs.780473721). The rare variant, p.P253S, is not reported in the dbSNP (as last accessed on September 10, 2015). Fig. 1 shows the comparison between samples with and samples without the A246del mutation in respect to DBP and 25 OHD levels. The concentrations of DBP and 25OHD in the samples with A246del were 174  19 mg/L (mean  SD) and 55  18 nmol/L, which were lower than those in the samples without A246del (197  29, and 65  16, respectively) (p < 0.05, ANOVA). These differences were not caused by seasonal effect of specimen collection nor daily vitamin D intake. Moreover, serum calcium and phosphate levels were not significantly different between the two groups (2.48  0.19 vs. 2.51  0.12 mmol/L for calcium; 1.71  0.21 vs.1.74  0.20 mmol/L for phosphate). Additional characterization of these DBP variants in comparison with wild-type (without A246del mutation) can be found in Supplementary table S1. 3.2. Mass spectra profiles Three representative serum samples were chosen for protein mass spectrometry profiling based on their genotypes. Their mass spectra profiles analyzed by LC-ESI-TOF-MS are shown in Fig. 2. Panel A shows the deconvoluted mass spectra of specimen 305 which was genotyped as homozygous D432, homozygous T436 and heterozygous A246del. The major peak (m/z = 51192) was Gc1F allele product (D432/T436). The smaller peak to the left (m/z = 51120) with mass shifted by 72 Da represents the mutant allele product Gc1F with an A246del mutation (A246del-Gc1F). The two peaks to the right (m/z = 51777 and 51848) show a signature mass shift (Dm) of 656.6 Da from their corresponding base peaks (m/z = 51120 and 51192). They constitute the glycosylated forms of the mutant protein product NeuNAc-Gal-GalNAcA246del-Gc1F, and the normal allele product NeuNAc-Gal-GalNAcGc1F, respectively. Panel B shows the deconvoluted mass spectra of specimen 207 which was genotyped as homozygous D432, heterozygous T436K and heterozygous A246del. The major peak (m/z = 51219) was Gc2 allele product (D432/436K). The smaller peak to the left (m/z = 51122) is identified as the mutant allele product A246delGc1F. The peak to the far right (m/z = 51778) is identified as the glycosylated form of mutant allele product NeuNAc-Gal-GalNAcA246del-Gc1F. Glycation of Gc2 allele product was evidenced by the small peak (m/z = 51382) with a signature mass shift (Dm) of approximately 162 Da [13]. Panel C shows the deconvoluted mass spectra of specimen 642 which was genotyped as homozygous D432, homozygous T436 and heterozygous C311F. On the left side, there were three peaks. The major peak (m/z = 51192) was the unmodified Gc1F allele product. The two minor peaks (m/z = 51240 and 51357) were

mutant allele product Gc1F with C311F mutation (C311F-Gc1F) and the S-cysteinylated mutant allele C311F-Gc1F, respectively. The right-hand three peaks (m/z = 51849, 51894 and 52013) are the glycosylated forms – NeuNAc-Gal-GalNAc-Gc1F, NeuNAc-GalGalNAc-C311F-Gc1F, and S-Cysteinylated NeuNAc-Gal-GalNAcC311F-Gc1F – corresponding to the three peaks on the left, as evidenced by their mass shift. 4. Discussion The alanine-246 deletion (A246del) is a recurrent mutation with a minor allele frequency (MAF) of 0.0066 (10 out of 1522 chromosomes screened) in our population. This is consistent with the frequency reported in dbSNP (rs.529043158, MAF = 0.0056) [6]. The 10 carriers in our dataset are of African-american and/or Hispanic ancestry. The other two missense mutations found in this cohort, P253S and C311F, were seen only once. These carriers too were of mixed Hispanic and African-American ancestry. All three identified mutations (A246del, P253S and C311F) are localized in domain II (aa. 192–378, based on the mature peptide)— not in domain I (aa. 1–191) where the vitamin D-binding site is located, nor in domain III (aa. 379–458) where the two common variants D432E and T436K are found [2,3]. According to the published crystal structures for DBP with bound 25OHD or actin [2,3], the C311 residue localizes to the last of the three regions interacting with actin (i.e., aa. 302–316) and participates in typical disulfide bridging. That the C311F mutation effectively prevents such disulfide bridging in the mature DBP molecule suggests that the remaining unpaired cysteine-299 sulfhydryl will be susceptible to reaction with other low molecular weight sulfhydryls. The detection of the S-cysteinylated DBP isoform (m/z = 51357, as shown on Fig. 2, Panel C) in the C311F mutant serum sample supports that supposition. As to the mass spectral analysis of the common SNPs, the mature gene product of D432 allele found in serum has an electrophoretic phenotype of Gc1F, the product of 432E allele a phenotype of Gc1S, and the product of the 436K allele a phenotype of Gc2 [13]. The nascent Gc2 protein without the threonine-436 is not recognized as an O-glycosylation site and is found in serum without the trisaccharide [20]. The mass spectra profiles shown in Fig. 2 are consistent. In addition, as shown in Fig. 2, the mutant allele proteins and posttranslational modified products (e.g. glycosylated, glycated or S-cysteinylated isoforms) were distinguishable from the wild-type or unmodified proteins in heterozygosity. Thus, our immuno-capture coupled mass spectrometry method allowed the characterization of specific protein species found in the circulation as well as the phasing in any of the double heterozygous. Furthermore, our results confirm the presence of mutant proteins in the circulation containing additional deleterious GC mutations. Early studies using isoelectric focusing techniques, reported more than 120 GC polymorphisms in different populations [12]. Other than the three common phenotypic variants Gc1F, Gc1S and Gc2, most of those variants have not been investigated in terms of their molecular nature or their impact on DBP structure and function. The molecular screening and mass spectrometry methods we report here may be helpful tools to identify and characterize additional mutant DBP species. In recent years, there has been a great interest in vitamin D and its health benefits for skeletal and non-skeletal related diseases [21–23]. DBP is the major binding protein and transporter for vitamin D and its metabolites, and plays an important role in vitamin D metabolism and its biological functions [7,24]. It is commonly accepted that vitamin D status is assessed by measuring total serum levels of 25OHD. However, recent evidence offers

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Fig. 1. Comparison between the samples with or without A246del mutation for DBP (Panel A) and 25OHD levels (Panel B).

increasing support for the “free hormone concept” similar to other steroids [25–27]. Although mathematical models have been developed to estimate free 25OHD levels [25–29], direct measurement is now an attractive alternative. The relationship of common DBP variants to vitamin D binding affinities is controversial. So too is the apparent relationship between DBP genotypes and serum

DBP concentrations [30–34]. Our work suggests that a further complexity may arise with associations between other less common mutants and altered serum concentrations of DBP and 25-hydroxyvitamin D. We found that both analytes were significantly lower in our small cohort with the A246del mutation. A larger study with a wider range of populations and ages,

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51192

A

Sample 305 Genotype: homozygous D432, homozygous T436, heterozygous A246del (A246-Gc1F/Gc1F)

Gc1F allele product

NeuNAc-Gal-GalNAc-Gc1F

NeuNAc-Gal-GalNAcA246del-Gc1F

51120 Mutant allele product: A246del-Gc1F

51000

51848

51777

51200

51400

51600

51800

Sample 207 Genotype: homozygous D432, Heterozygous T436K, heterozygous A246del (A246del-Gc1F/Gc2)

51219

B

52000 m/z

Gc2 allele product

51122

NeuNAc-Gal-GalNAcA246del-Gc1F

Mutant allele product: A246del-Gc1F

Glycation of Gc2 allele product 51778 51382

51000

51200

51400

51600

51800

Sample 642 Genotype: homozygous D432, homozygous T436, heterozygous C311F (C311F-Gc1F/Gc1F)

51192

C

52000 m/z

Gc1F allele product

NeuNAc-Gal-GalNAc-Gc1F 51849

Mutant allele product: C311F-Gc1F

NeuNAc-Gal-GalNAcC311F-Gc1F

51240 51894

S-Cysteinylated1 mutant C311F-Gc1F

S-Cysteinylated NeuNAc-GalGalNAc-C311Gc1F

51357 52013

51000 1

51200

51400

51600

51800

52000 m/z

Calculated Δm of S-Cysteinylation is 119 Da

Fig. 2. Mass spectra of intact DBP isolated directly from serum samples. The base peaks at approximately 51200 Da represent unmodified DBP (which varies in exact mass according to DBP genotypes). A peak at Dm + 656.6 represents an O-linked glycoform, a (NeuAc)1(Gal)1(GalNAc)1trisaccharide. Panel A: Homozygous Gc1F, with A246del on one allele. Panel B: Heterozygous A246del-Gc1F/Gc2. A peak at Dm + 163 represents glycated DBP. Panel C: Homozygous Gc1F, with C311F on one allele. Loss of 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 mutant protein.

particularly one that includes well-characterized adult groups, may help to resolve some of the uncertainty as to the wider significance of this finding. Thus, it remains to be seen how the additional genetic variants, such as the ones we describe here, may affect overall vitamin D

metabolism. Given the wide spectrum of electrophoretic variants identified in early studies and the additional DBP variants described here, it seems likely that further DBP genotype analyses across a variety of genetically and geographically distinct populations will also lead to new insights [9–11,35]. Study of

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such genetic variants may also lead to a better understanding of genetically defined differences in vitamin D metabolism and help explain some of the inter-individual responses to this important hormone. The role of DBP variants in vitamin D status deserves further investigation. Acknowledgements This study was supported in part by Dairy Farmers of Canada (DECC, LF, BYLW, RW), Gerber Foundation (TOC), and a Thrasher Research Fund Award 02829-4 (TOC). The authors are grateful for the assistance of Dr. Zhongsheng You at Washington University, St. Louis, MO, USA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jsbmb.2016.02.022. References [1] M. Speeckaert, G. Huang, J.R. Delanghe, Y.E. Taes, Biological and clinical aspects of the vitamin D binding protein (Gc-globulin) and its polymorphism, Clin. Chim. Acta 372 (October (1–2)) (2006) 33–42 (Epub 2006 May 12, PMID: 16697362). [2] C. Verboven, A. Rabijns, M. De Maeyer, H. Van Baelen, R. Bouillon, C. De Ranter, A structural basis for the unique binding features of the human vitamin Dbinding protein, Nat. Struct. Biol. 9 (February (2)) (2002) 131–136 (Erratum in: Nat Struct Biol 2002 Apr;9(4):316. PMID: 11799400). [3] L.R. Otterbein, C. Cosio, P. Graceffa, R. Dominguez, Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system, Proc. Natl. Acad. Sci. U. S. A. 99 (June (12)) (2002) 8003–8008 (Epub 2002 Jun 4. PMID: 12048248). [4] J. Zhang, D.M. Habiel, M. Ramadass, R.R. Kew, Identification of two distinct cell binding sequences in the vitamin D binding protein, Biochim. Biophys. Acta 1803 (May (5)) (2010) 623–629, doi:http://dx.doi.org/10.1016/j. bbamcr.2010.02.010 (Epub 2010 Mar 6, PMID: 20211661). [5] Y.H. Song, A.K. Naumova, S.A. Liebhaber, N.E. Cooke, Physical and meiotic mapping of the region of human chromosome 4q11–q13 encompassing the vitamin D binding protein DBP/Gc-globulin and albumin multigene cluster, Genome Res. 9 (June (6)) (1999) 581–587 (PMID: 10400926). [6] NCBI SNP, database for GC, homo sapiens. Available at: http://www.ncbi.nlm. nih.gov/snp and http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi? rs=529043158 [10 September, 2015] [7] T.O. Carpenter, J.H. Zhang, E. Parra, B.K. Ellis, C. Simpson, W.M. Lee, J. Balko, L. Fu, B.Y. Wong, D.E. Cole, Vitamin D binding protein is a key determinant of 25hydroxyvitamin D levels in infants and toddlers, J. Bone Miner. Res. 28 (January (1)) (2013) 213–221, doi:http://dx.doi.org/10.1002/jbmr.1735 (PMID:22887780). [8] L. Fu, F. Yun, M. Oczak, B.Y. Wong, R. Vieth, D.E. Cole, Common genetic variants of the vitamin D binding protein (DBP) predict differences in response of serum 25-hydroxyvitamin D [25(OH)D] to vitamin D supplementation, Clin. Biochem. 42 (July (10–11)) (2009) 1174–1177, doi:http://dx.doi.org/10.1016/j. clinbiochem.2009.03.008 (Epub 2009 Mar 18, PMID: 19302999). [9] A. Gozdzik, J. Zhu, B.Y. Wong, L. Fu, D.E. Cole, E.J. Parra, Association of vitamin D binding protein (VDBP) polymorphisms and serum 25(OH)D concentrations in a sample of young Canadian adults of different ancestry, J. Steroid Biochem. Mol. Biol. 127 (November (3–5)) (2011) 405–412, doi:http://dx.doi.org/ 10.1016/j.jsbmb.2011.05.009 (Epub 2011 Jun 12. PMID: 21684333). [10] M.I. Kamboh, R.E. Ferrell, Ethnic variation in vitamin D-binding protein (GC): a review of isoelectric focusing studies in human populations, Hum Genet. 72 (April (4)) (1986) 281–293 (Review. PMID:3516862). [11] J. Constans, M. Viau, Group-specific component: evidence for two subtypes of the Gc1 gene, Science 198 (December (4321)) (1977) 1070–1071 (PMID: 73222). [12] H. Cleve, J. Constans, The mutants of the vitamin-D-binding protein: more than 120 variants of the GC/DBP system, Vox Sang. 54 (4) (1988) 215–255 (PMID: 3388819). [13] C.R. Borges, J.W. Jarvis, P.E. Oran, S.P. Rogers, R.W. Nelson, Population studies of intact vitamin D binding protein by affinity capture ESI-TOF-MS, J. Biomol. Tech. 19 (July (3)) (2008) 167–176 (PMID:19137103). [14] D.S. Rehder, R.W. Nelson, C.R. Borges, Glycosylation status of vitamin D binding protein in cancer patients, Protein Sci. 18 (October (10)) (2009) 2036–2042, doi:http://dx.doi.org/10.1002/pro.214 (PMID:19642159). [15] C.R. Borges, D.S. Rehder, J.W. Jarvis, M.R. Schaab, P.E. Oran, R.W. Nelson, Fulllength characterization of proteins in human populations, Clin. Chem. 56

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