TO THE EDITOR:

Vitamin B12 (VB12), or cobalamin, is a water-soluble vitamin. It serves as an enzyme cofactor in the 1-carbon metabolism pathway, which plays a key role in a wide range of biological processes, including red blood cell (RBC) formation, DNA synthesis, and myelination of the central nervous system.1-3  VB12 deficiency is clinically associated with megaloblastic anemia and neurodegenerative disorders and is also linked to cardiovascular diseases, which is thought to be mediated via hyperhomocysteinemia.1,3  Uptake of VB12 from the diet in the gastrointestinal tract depends on intrinsic factor (encoded by GIF).3,4  VB12 in blood is bound to haptocorrin (HC; encoded by TCN1) and transcobalamin (encoded by TCN2) and circulates as holohaptocorrin (holoHC) and holotranscobalamin, respectively.3,5,6 

Although VB12 deficiency often results from poor gastrointestinal absorption,7  genetic factors also contribute to phenotypic variation. Previous genome-wide association studies (GWASs), primarily focusing on populations of European and Asian ancestry, have identified 13 loci (FUT2, CUBN, TCN1, MUT, MS4A3, FUT6, PRELID2, CLYBL, CD320, TCN2, ABCD4, MMAA, and MMACHC) associated with VB12.8-15  GWASs in Chinese11  and Indians14  identified VB12-associated shared genetic variants across ancestry groups, as well as population-specific variants. VB12 and holoHC concentrations are higher in blacks than in whites,16  yet no GWAS has examined the genetic determinants of VB12 specifically in African Americans (AAs). In the current study, we used whole-genome sequencing (WGS) from the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) Program to assess genetic variants associated with VB12 in an unselected population-based sample of AAs from the Jackson Heart Study (JHS).17 

The JHS is a population-based longitudinal study based in Jackson, Mississippi17,18 ; additional information is reported in supplemental Methods (available on the Blood Web site). The JHS was approved by the Institutional Review Board of the University of Mississippi Medical Center, and participants provided written informed consent. At the baseline examination, VB12 was measured in plasma in a subset of 1851 JHS participants using a homogenous enzyme immunoassay system and Hitachi 911 equipment (Roche Diagnostics, Indianapolis, IN).19  A total of 3406 JHS participants underwent 30× coverage WGS through the TOPMed project at the Northwest Genome Center at the University of Washington, and genotype calling was performed by the Informatics Resource Center at the University of Michigan, as previously described.20  After quality control (supplemental Methods), a total of 29 665 030 variants (minor allele frequency > 0.001) and 1280 participants (20.6-91.6 years of age, 65.5% female, VB12 = 639.9 ± 289.3 pg/mL) were included in the discovery analysis (phs000964.v2.p1 for sequencing data and phs000286.v5.p1 for VB12 data on the Database of Genotypes and Phenotypes; https://www.ncbi.nlm.nih.gov/gap).

Association of each single variant with rank-based inverse-normal–transformed VB12 concentration was tested in JHS participants adjusted for age, sex, and the first 10 principal components (PCs) of genetic ancestry using a linear mixed model approach to account for familial relationships, as implemented in EPACTS 3.2.6 on the University of Michigan ENCORE server (https://encore.sph.umich.edu). Genome-wide significance was predefined as P < 1.69 × 10−9, or 0.05/29 665 030 variants tested; 33 variants across 2 genomic regions (TCN1 and FUT2) reached genome-wide significance (P < 1.69 × 10−9) for VB12 (supplemental Figure 1).

The index variant rs34530014 on chromosome 11 is a 1-bp deletion of TCN1 (minor allele frequency = 0.036; P = 6.48 × 10−15; Table 1; Figure 1A) associated with lower VB12. It is a loss-of-function (LoF) frameshift mutation (p.Val58Cysfs) prevalent only among African ancestry populations, originally reported in 2 Afro-Caribbean pedigrees with HC deficiency.21  Replication of the novel LoF variant TCN1-rs34530014 was performed using genomic data from 2 AA electronic medical record–linked biobanks: the Biobank of Vanderbilt University (BioVU)22  and BioMe of Mount Sinai23  (supplemental Methods). Because TCN1-rs34530014 was not directly genotyped or imputed in BioVU or BioMe, we used its linkage disequilibrium (LD) proxy rs11822978 (r2 = 0.98 in JHS) and confirmed its association with lower VB12 in 3924 additional AAs from BioVU and BioMe (Table 1). TCN1-rs34530014 is distinct from other VB12-associated TCN1 variants (including 2 missense variants, rs34324219 and rs34528912) identified in Europeans and Indians.9,12-14  Of the TCN1 variants previously associated with lower VB12, only rs34324219 was nominally associated with lower VB12 in the JHS (P = .009; supplemental Table 1). Together, these results demonstrate an ancestry-specific role for rs34530014 in lower VB12 concentration in African ancestry populations and substantial allelic heterogeneity in the genetic architecture of VB12 at the TCN1 locus.

Table 1.

Association of TCN1-rs34530014 with VB12 concentration

Trait Study SNP CHR:POS EA/NEA EAF BETA (SE) P
VB12 JHS rs34530014 11:59631467 1280 A/AC 0.036 −0.817 (0.104) 6.48 × 10−15 
VB12 BioVU rs11822978 11:59626896 725 T/C 0.034 −0.158 (0.038) 3.28 × 10−5 
VB12 BioMe rs11822978 11:59626896 3199 T/C 0.032 −0.272 (0.037) 2.76 × 10−13 
Trait Study SNP CHR:POS EA/NEA EAF BETA (SE) P
VB12 JHS rs34530014 11:59631467 1280 A/AC 0.036 −0.817 (0.104) 6.48 × 10−15 
VB12 BioVU rs11822978 11:59626896 725 T/C 0.034 −0.158 (0.038) 3.28 × 10−5 
VB12 BioMe rs11822978 11:59626896 3199 T/C 0.032 −0.272 (0.037) 2.76 × 10−13 

CHR, chromosome; EA, effect allele; EAF, effect allele frequency; NEA, noneffect allele; POS, position; SE, standard error; SNP, single nucleotide polymorphism.

*

Genome-wide significance was predefined as P < 1.69 × 10−9 or 0.05/29 665 030 variants tested.

Figure 1.

Locus-zoom plots.TCN1-rs34530014 (A) and FUT2-rs507766 (B). Genetic coordinates are displayed along the x-axis (Build 37/hg19), and genome-wide association significance level is plotted against the y-axis as −log10(P value). The purple diamond indicates the top hit. LD is generated using JHS WGS data and is indicated by the color scale in relationship to the top hit, with red for strong LD (r2 > 0.8) and navy blue for weak LD (r2 < 0.2).

Figure 1.

Locus-zoom plots.TCN1-rs34530014 (A) and FUT2-rs507766 (B). Genetic coordinates are displayed along the x-axis (Build 37/hg19), and genome-wide association significance level is plotted against the y-axis as −log10(P value). The purple diamond indicates the top hit. LD is generated using JHS WGS data and is indicated by the color scale in relationship to the top hit, with red for strong LD (r2 > 0.8) and navy blue for weak LD (r2 < 0.2).

The identification of this naturally occurring LoF mutation prevalent in AAs allowed us to further investigate additional hematologic and clinical consequences of mild HC deficiency, beyond the association with lower VB12 concentration. The association of rs34530014 with additional quantitative traits, including RBC indices and homocysteine, was assessed in the JHS and the Women’s Health Initiative SNP Health Association Resource24  (supplemental Methods). An elevated concentration of methylmalonate is a more sensitive and specific marker for VB12 deficiency1,3 ; unfortunately, it was not available in our data sets. In addition, a phenome-wide association study (PheWAS) of TCN1-rs11822978 was performed in BioVU and BioMe. A total of 340 traits was scanned for association using logistic regression adjusted for age, sex, and the first 10 PCs, including metabolic, neurological, and hematologic outcomes and disorders. Individual study association results were combined using fixed-effect inverse-variance–weighted meta-analysis, and P values were corrected for multiple testing. Although marginal associations (P < .05) were observed for mean corpuscular hemoglobin and mean corpuscular volume, as well as diagnosis codes Vitamin Deficiency and Other Deficiency Anemia, none of these reached the Bonferroni-corrected threshold (P > .006, or 0.05/8 traits for RBC indices, supplemental Table 2; and P > 1.47 × 10−4, or 0.05/340 traits for PheWAS, supplemental Table 3).

In contrast to mutations of TCN2, which result in transcobalamin II deficiency (OMIM #275350) with severe clinical manifestations,25  the clinical impact of HC deficiency is poorly defined. The distinct consequences of TCN1 and TCN2 mutations are consistent with the fact that holotranscobalamin is the bioactive form of VB12 that is taken up by all tissues, whereas the biological role of holoHC is unclear.6  Despite the relatively large overall sample, the small number of homozygotes (4 in JHS) limited our ability to accurately assess the recessive effect of the TCN1-rs34530014 on VB12 and related traits and disorders. Therefore, further investigation, including very large phenotypic data sets of African ancestry individuals, will be needed to draw firm conclusions about the clinical impact of HC deficiency.

The index single nucleotide polymorphism at the other genome-wide significant association peak in the JHS on chromosome 19 was rs507766 (located in the 3′ untranslated region of FUT2; P = 5.51 × 10−11) (Figure 1B). This noncoding variant is in only moderate LD (r2 = 0.55 in JHS) with the common secretor variant FUT2-rs601338 (P = 1.49 × 10−7 for association with VB12 in JHS), which was previously identified in Europeans and Indians.8,9,14  Thus, we found evidence for 2 functional alleles involving FUT2: 1 with an additive genetic effect (rs507766) and 1 with a dominant effect (rs601338) on VB12 (supplemental Table 4). We confirmed 4 previously identified loci (MUT, MMACHC, FUT6, and CD320; supplemental Table 1) at P < .05 with consistent association directions of effects.

We report a novel association between the African ancestry–specific LoF variant TCN1-rs34530014 and VB12 concentration and an additional FUT2 variant rs507766 associated with VB12 specific to AAs, highlighting the ancestry heterogeneity of genetic factors that influence VB12. Another population-specific VB12-associated LoF variant, CLYBL-rs41281112 (p.Arg259Ter), is found in ∼3% of apparently healthy non-African individuals (but is rare in Africans).11  Further elucidation of the genetics and role of HC-bound VB12 may lead to improved diagnosis and differentiation of true bioavailable VB12 deficiency from total VB12 deficiency.

The online version of this article contains a data supplement.

Acknowledgments

The authors thank the staff and participants in the JHS. The contributions of the investigators of the NHLBI TOPMed Consortium (https://www.nhlbiwgs.org/topmed-banner-authorship) are gratefully acknowledged. The authors also gratefully acknowledge the studies and participants who provided biological samples and data for TOPMed.

The JHS is supported and conducted in collaboration with Jackson State University (HHSN268201300049C and HHSN268201300050C), Tougaloo College (HHSN268201300048C), and University of Mississippi Medical Center (HHSN268201300046C and HHSN268201300047C) contracts from the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) and the National Institute for Minority Health and Health Disparities. The analysis was also funded by NIH NHLBI grants R01 HL129132 and R01 HL130733. WGS for the TOPMed program was supported by the NIH NHLBI. WGS for “NHLBI TOPMed: The Jackson Heart Study” (phs000964.v1.p1) was performed at the University of Washington Northwest Genomics Center (HHSN268201100037C). Centralized read mapping and genotype calling, along with variant quality metrics and filtering, were provided by the TOPMed Informatics Research Center (3R01HL-117626-02S1). Phenotype harmonization, data management, sample-identity quality control, and general study coordination were provided by the TOPMed Data Coordinating Center (3R01HL-120393-02S1). L.M.R. is supported by NIH NHLBI grant T32 HL129982. The BioVU data set used in the analyses described was obtained from the Vanderbilt University Medical Center, which was supported by institutional funding and by NIH National Center for Advancing Translational Science Clinical and Translational Science Awards grant ULTR000445. Genome-wide genotyping was funded by NIH National Institute of General Medical Sciences/OD grant RC2GM092618 and NIH National Human Genome Research Institute (NHGRI)/National Institute of General Medical Sciences grant U01HG004603. Analysis of BioVU data was partially funded by NHGRI grant U01HG009086, which supports the Vanderbilt Analysis Center for the Genome Sequencing Project. The Mount Sinai Institute for Personalized Medicine Biobank Program is supported by The Andrea and Charles Bronfman Philanthropies. Work in BioMe was also supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. R.J.F.L. is supported by NIH, National Institute of Diabetes and Digestive and Kidney Diseases grants R01DK110113, R01DK101855, and R01DK107786; and NIH, NHGRI grant U01HG007417.

The views expressed in this article are those of the authors and do not necessarily represent the views of the NHLBI, the NIH, or the US Department of Health and Human Services.

Authorship

Contribution: A.P.R. and P.L.A. were responsible for the study concept and design; J.G.W. and A.C. acquired the phenotypic and genotypic data in the JHS; A.P.R., P.L.A., Y.H., L.M.R., and L.M.P. performed the statistical analyses in the JHS and the Women’s Health Initiative SNP Health Association Resource, the meta-analysis, and the PheWAS; A.M., G.N., M.H.P., and R.J.F.L. performed replication analysis in BioMe; X.Z., Q.W., and B.L. performed replication analysis in BioVU; Y.H. drafted the manuscript; and all authors contributed to the interpretation of the results and critical revision of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

A complete list of the members of the NHLBI Trans-Omics for Precision Medicine Consortium appears in the online appendix.

Correspondence: Alex P. Reiner, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109; e-mail: apreiner@u.washington.edu.

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