Genetic, anatomic, and clinical determinants of human PNAS PLUS serum sterol and vitamin D levels

Ashlee R. Stilesa, Julia Kozlitinab, Bonne M. Thompsona, Jeffrey G. McDonalda, Kevin S. Kingc, and David W. Russella,1

Departments of aMolecular Genetics, bInternal Medicine, and cRadiology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Contributed by David W. Russell, August 7, 2014 (sent for review March 4, 2014) An unknown fraction of the genome participates in the metabo- are unknown, genome-wide association studies can be used to lism of sterols and vitamin D, two classes of lipids with diverse identify loci that are linked to serum sterol levels (3, 4). Sub- physiological and pathophysiological roles. Here, we used mass sequent genetic and functional studies then define the role of spectrometry to measure the abundance of >60 sterol and vitamin the product specified by the identified gene (5, 6). D derivatives in 3,230 serum samples from a well-phenotyped pa- In the current study, we used the analytical methods of tient population. Twenty-nine of these lipids were detected in a ma- McDonald et al. (2) to measure serum sterols in 3,230 individ- jority of samples at levels that varied over thousands of fold in uals from a clinically well-defined cohort, the Dallas Heart Study different individuals. Pairwise correlations between sterol and (DHS) (7). In this large patient population, 27 sterols and vita- vitamin D levels revealed evidence for shared metabolic path- min D derivatives were consistently detected and each showed ways, additional substrates for known enzymes, and transcrip- marked interindividual variation in their serum levels. Through tional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and further studies, we identified genetic, anatomic, and clinical age. A genome-wide association study identified 16 loci that were phenotypes that were associated with many of these lipids. associated with levels of 19 sterols and 25-hydroxylated deriva- Results tives of vitamin D (P < 10−7). Resequencing, expression analysis,

and biochemical experiments focused on one such locus (CYP39A1), The data of Table 1 show the 27 sterols and vitamin D metab- GENETICS n = revealed multiple loss-of-function alleles with additive effects on olites that were detected in the DHS ( 3,230) together serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate with their mean and median concentrations. Interindividual of the encoded enzyme. Body mass index, serum lipid levels, and variation in the levels of these analytes ranged from as little hematocrit were strong phenotypic correlates of interindividual as 31-fold (24S-hydroxycholesterol) to as large as 7,760-fold variation in multiple sterols and vitamin D metabolites. We con- (24-dihydrolanosterol). Fig. 1A shows the raw data generated for clude that correlating population-based analytical measurements one analyte, 24S-hydroxycholesterol. The mean concentration of with genotype and phenotype provides productive insight into this oxysterol in the population was 60 ng/mL, and the range human intermediary metabolism. was 10–314 ng/mL. Levels of 24S-hydroxycholesterol correlated with those of (Fig. 1B, r = 0.53). This association – human genetics | genotype phenotype correlation accounted for 31% of the observed variance in interindividual 24S-hydroxycholesterol levels. Most analytes showed log-normal ipids are an important component of serum and there play distributions as exemplified by 24S-hydroxycholesterol and the Lessential roles in energy metabolism, signaling, and transport. overlaid best-fit curves shown in Fig. 1 C and D. Comparisons A recent survey revealed an unexpectedly large complexity in the between all possible pairs of analytes revealed additional sig- human serum lipidome, which was found to be composed of nificant correlations between many sterols (Fig. 2). A majority of hundreds of different molecular species in each major lipid class correlations were positive, and in general these were stronger (1). For example, cholesterol and other sterols were detected in than the smaller number of negative correlations identified. concentrations ranging from milligrams per milliliter to nano- grams per milliliter, and over 200 different triglyceride species were found. A pooled plasma sample derived from multiple Significance individuals was analyzed in this study; thus, whether the observed complexity reflected functional diversity in the roles played by Cholesterol is the major sterol in blood and in excess causes different lipids, was environmentally driven or was genetically cardiovascular disease. In addition to cholesterol, numerous determined could not be ascertained. other sterols of unknown function and pathogenicity circulate To address these issues with respect to sterols and vitamin D in the bloodstream. Here, we use chemical methods to screen metabolites (secosteroids), we developed analytical methods to for over 60 different sterols and sterol derivatives in the sera of measure more than 60 different types of these lipids in small 3,230 clinically well-characterized individuals. Twenty-seven ste- volumes (<200 μL) of human serum (2). An initial analysis of 200 rols and two sterol derivatives (vitamin D2 and D3)wereroutinely human serum samples showed that 22 of the >60 compounds detected in vastly different amounts in a majority of individuals. were routinely detected and began to define the ranges and Genes, ethnicity, gender, age, clinical phenotype, and anatomy distributions of these analytes in the population (2). were identified as significant sources of interindividual varia- The steady-state concentration of a given lipid is determined tion in these lipid metabolites. by rates of formation and degradation, the kinetics of movement Author contributions: A.R.S., J.G.M., and D.W.R. designed research; A.R.S., J.K., B.M.T., into and out of cells and tissues, the levels of lipoproteins that and J.G.M. performed research; B.M.T. and J.G.M. contributed new reagents/analytic transport sterols through the bloodstream, their availability in tools; A.R.S., J.K., J.G.M., K.S.K., and D.W.R. analyzed data; and A.R.S., J.K., and D.W.R. the diet, and the physiological state of the individual. For some wrote the paper. sterols, the individual contributions of these variables can be de- The authors declare no conflict of interest. termined from known metabolic pathways, clinical measurements 1To whom correspondence should be addressed. Email: [email protected]. of lipoprotein levels, and nutritional and health information. In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. other cases, where biosynthetic, catabolic, and transport pathways 1073/pnas.1413561111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1413561111 PNAS Early Edition | 1of9 Downloaded by guest on September 28, 2021 Table 1. Serum sterol and vitamin D levels in 3,230 subjects Metabolite LIPID MAPS ID Mean, ng/mL Median, ng/mL Range, ng/mL

25-Hydroxyvitamin D3 LMST03020246 43 39 5–160

25-Hydroxyvitamin D2 LMST03010030 5 2 0.1–232 LMST01010017 145 125 13–4,667 14-Desmethyl lanosterol LMST01010176 552 486 62–2,671 LMST01010066 41 30 2–843 LMST01010016 923 838 22–18,810 24-Dihydrolanosterol LMST01010087 34 17 2–15,333 Lathosterol LMST01010089 2,016 1,842 1–13,850 7-Dehydrocholesterol LMST01010069 642 529 7–21,143 8-Dehydrocholesterol LMST01010242 765 667 142–11,762 22R-Hydroxycholesterol LMST01010086 1 1 0.1–16 24S-Hydroxycholesterol LMST01010019 60 57 10–314 25-Hydroxycholesterol LMST01010018 10 8 1–56 24,25-Epoxycholesterol LMST01010012 2 1 0.1–56 27-Hydroxycholesterol LMST01010057 158 150 25–990 7α-Hydroxycholesterol LMST01010013 114 90 12–2,762 7α,27-Dihydroxycholesterol LMST04030081 10 10 2–90 Sitosterol LMST01040129 2,460 2,148 308–19,476 Campesterol LMST01030097 3,596 3,191 141–34,143 LMST01040123 131 116 3–10,000 Stigmastanol LMST01040128 23 19 1–1,138 Cholestanol LMST01010077 2,883 2,676 431–63,333 Cholestenone LMST01010015 110 53 4–1,457 7-Oxocholesterol LMST01010049 55 39 8–375 5α-Hydroxycholesterol LMST01010275 37 34 2–370 5,6α-Epoxycholesterol LMST01010011 99 90 9–388 5,6β-Epoxycholesterol LMST01010010 277 253 36–1,410 4β-Hydroxycholesterol LMST01010014 39 36 9–500 24-Oxocholesterol LMST01010133 6 5 0.4–116

The vitamin D derivatives and sterols routinely detected in the study population are listed together with their mean and median concentrations and ranges. Information regarding the structure and function of each analyte may be obtained by searching for the individual LIPID MAPS ID number at www.lipidmaps.org/data/structure. Information regarding quality assessment and control in the measurement process is available in ref. 2.

Additional factors contributing to interindividual variation in quence variations that contributed to variation, the exomes of sterol and vitamin D levels were identified by regression ana- the 3,230 individuals in the DHS cohort were genotyped for lyses. As shown in Fig. 3, sex, ethnicity, and age explained a large ∼240,000 single-nucleotide polymorphisms (SNPs) by chip-based fraction of the variation in multiple lipids. For example, three oligonucleotide hybridization (Illumina HumanExome Bead- intermediates of bile acid biosynthesis, 27-hydroxycholesterol, Chip). A majority of SNPs present on the genotyping chip were 7α,27-dihydroxycholesterol, and 7α-hydroxycholesterol were nonsynonymous sequence variations. Each genetic variant was significantly lower in females than in males, confirming an earlier tested for association with individual analytes as described in study (8). An intermediate in the Bloch pathway of cholesterol Materials and Methods. Variants in 16 different loci located on 10 synthesis, desmosterol, showed similar sexual dimorphism. For different chromosomes were associated with one or more of 19 these and other lipid species (Fig. 3 and Fig. S1), sex explained as sterols and vitamin D metabolites at exome-wide significance − − much as 24.7% of interindividual variability (R2). (P = 10 74 to 10 7) (Fig. 4 and Table S1). The levels of some Ethnicity was a significant determinant of variability for a differ- sterols were influenced by variants at several genomic loci (e.g., ent set of analytes. As noted (9), 25-hydroxyvitamin D3 levels were ABCG5/ABCG8, HSD3B7, and cholestanol), whereas other sterols highest in individuals of European-American descent, intermediate and secosteroids were associated with a single locus (e.g., EPHX2 in Hispanics, and lowest in African Americans (Fig. 3). Levels of and 24,25-epoxycholesterol, and GC and 25-hydroxyvitamin D3). 8-dehydrocholesterol showed a similar trend across these ethnic The rs2277119 variant in CYP39A1, which specifies an oxy- groups, whereas IgG showed an opposite trend (i.e., were highest sterol 7α-hydroxylase, was associated with elevated levels of 24S- − in African Americans). As with sex, ethnicity explained as much as hydroxycholesterol (P = 10 74) and is a G-to-A transition that 25% of interindividual variability in these traits (Fig. 3 and Fig. S2). alters codon 103 in the gene from arginine to histidine (R103H). Age was a weaker but significant determinant of variability, Given the reaction catalyzed by the encoded P450 (Fig. 5A), the with increasing age correlated with elevated levels of some association of this variant with higher serum levels of the oxysterol sterols such as 7-dehydrocholesterol and decreased levels of implied that R103H was a hypomorphic allele of CYP39A1. others such as 24S-hydroxycholesterol (10) and lathosterol (Fig. To determine whether there were other CYP39A1 variants as- 3 and Fig. S3). Additional principal-component analyses be- sociated with serum 24S-hydroxycholesterol levels, the 12 exons tween analytes showed that, overall, sex was the largest de- of this gene were sequenced in the 30 DHS individuals with the terminant of variation between individuals and that ethnicity was highest normalized 24S-hydroxycholesterol levels (Fig. 1D). This the second largest determinant (Fig. S4). resequencing revealed four additional nonsynonymous sequence The observed effects of ancestry on interindividual variation in variants: R23P, T288H, N324K, and K329Q (Fig. 5B and Table S2). sterol and secosteroids suggested that genetic factors were an The biochemical effects of these alterations and the R103H additional effector of analyte levels (11). To identify DNA se- variant were determined in transfection experiments. Fig. 5C

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1413561111 Stiles et al. Downloaded by guest on September 28, 2021 PNAS PLUS A 350 B

200 r=0.53 300

250 100

200 50 150

100 20 -Hydroxycholesterol (ng/mL) -Hydroxycholesterol

50 (ng/mL) S -Hydroxycholesterol 24 S 24 10 0 0 1 500 1000 1500 2000 2500 3000 3500 0 100 150 200 250 300 350 400 Subject Number Cholesterol (mg/dL) C D 700 500 1.5 5 600 400

4 500 1.0 300 400 3

300 GENETICS 200 2 0.5

Number of Individuals 200 Number of Individuals

100 1 100

0 0 0 0

0 0.2 0.4 0.6 0.8 1.0 1.2 −3.0 −2.5 −2.0 −1.5 −1.0 −0.5 0 24S-Hydroxycholesterol/Cholesterol log 24S-Hydroxycholesterol/Cholesterol

Fig. 1. Distribution of serum 24S-hydroxycholesterol levels in 3,230 DHS participants. (A) Raw data showing 24S-hydroxycholesterol levels in individual subjects. (B) Correlation between serum cholesterol and 24S-hydroxycholesterol levels in individuals. (C) Distribution of 24S-hydroxycholesterol levels after normalization to cholesterol levels. The red line shows the probability density function for the best-fitting log-normal distribution. (D) Normalized distri- bution following log transformation of 24S-hydroxycholesterol/cholesterol levels. The red line shows the probability density function for the best-fitting normal distribution.

shows averaged results from three separate experiments in which Earlier studies in a small number of subjects with cognitive enzyme activity encoded by each variant was assayed in triplicate impairment revealed a modestly significant association (P = 0.03) dishes. Relative to the normal enzyme, all sequence variants between gray matter volume and serum 24S-hydroxycholesterol reduced enzyme activity from ∼15% (rs17856332; Y288H) to levels (12), and based on indirect measurements, a similar re- 100% (rs41273654; K329Q), but did not have an obvious effect lationship was detected between the size of the brain and the on CYP39A1 mRNA or protein expression as judged by real- capacity of the liver to metabolize the oxysterol (13). These time PCR or immunoblotting (Fig. 5C, Inset), indicating that findings suggested that 24S-hydroxycholesterol was synthesized in these amino acid changes directly reduced enzyme activity. gray matter neurons, a hypothesis subsequently confirmed by To determine the association of these variants with serum 24S- histochemical studies (14). To determine whether brain anatomy hydroxycholesterol levels, the DHS population was genotyped influenced serum 24S-hydroxycholesterol levels, MRI was used to for the four CYP39A1 alleles identified by resequencing. Three measure total brain, gray matter, and white matter volumes in of the five alleles were independently associated with an increase 2,109 DHS participants. These analyses were adjusted for age, in serum 24S-hydroxycholesterol levels in the combined DHS race, sex, and cholesterol levels. Male and female values were cohort (African-American, European-American, and Hispanic separated to control for the known sexual dimorphism in brain participants), with two variants (rs2277119; R103H; and rs7761731; size (15). Total brain volume was modestly but significantly cor- N324K) making significantly larger contributions than the third related with oxysterol levels in men and women (r = 0.21 in men, − − (Table S2). The variants appeared to act additively, with no evi- r = 0.16 in women; P = 5.5 × 10 5 and 2.5 × 10 4, respectively). dence of a statistical interaction detected between alleles (P for Gray matter volume in both females and males was more strongly pairwise interactions, >0.05). As indicated in Fig. 5D, individuals correlated with serum 24S-hydroxycholesterol levels, whereas with one or more variant CYP39A1 alleles had progressively higher theassociationwithwhitemattervolume was weaker than that for serum 24S-hydroxycholesterol levels compared with those with no gray matter but statistically significant (Fig. S5). Gray matter vol- − variant alleles (P for trend, 1.5 × 10 34). Together, the CYP39A1 umes explained 1.75% of variance in serum 24S-hydroxycholesterol alleles explained ∼10.8% of the interindividual variation observed levels after adjustment for white matter volume. White matter was for serum 24S-hydroxycholesterol levels. not significantly associated with this trait after accounting for gray

Stiles et al. PNAS Early Edition | 3of9 Downloaded by guest on September 28, 2021 2 yl lanosterol xycholesterol o xycholesterol xycholesterol xycholesterol o ydrolanosterol ydrocholesterol h hydrocholesterol -Hydroxycholesterol R -Hydr S α ,27-Dihydroxycholesterol Cholestenone Campesterol Stigmastanol 7 Sitosterol Stigmasterol Cholestanol 7-Oxocholesterol 5 α -Hydro 5,6 α -Epoxycholesterol Cholesterol Lathosterol 7-Deh 14-Desmeth Desmosterol 24-Di 25-Hydro 24,25-Epoxycholesterol 5,6 β -Epoxycholesterol 4 β -Hydroxycholesterol 24-Oxocholesterol 22 24 8-De 7 α -Hydroxycholesterol Lanosterol 27-Hydr 25-Hydroxyvitamin D 25-Hydroxyvitamin Zymosterol 100 25-Hydroxyvitamin D3 3 0 11 4 4 7 -4 8 27 8 1 13 -4 4 8 1 5 5 4 -1 1 7 2 4 12 4 -3 8 10 25-Hydroxyvitamin D2 -4 3 4 -1 -7 1 8 13 2 3 4 4 3 -2 2 -1 4 1 -3 -1 0 4 2 -3 -1 6 -1 -1 Lanosterol -11 56 57 70 77 29 26 48 8 39 19 18 4 36 33 -9 -2 -11 -2 6 7 9 16 18 16 2 20 80 14-Desmethyl lanosterol -9 3 9 -23 14 21 27 4 17 4 -8 21 2 9 92 86 83 62 63 1 0 2 2 1 38 14

Zymosterol 43 44 54 34 33 32 -5 21 4 26 29 30 18 -7 -6 -14 -4 2 0 2 4 12 11 -9 24 Desmosterol 38 48 19 25 51 5 42 17 22 45 32 35 6 9 -1 4 15 2 5 8 17 14 5 22 60 24-Dihydrolanosterol 56 27 24 44 8 34 15 15 36 36 25 7 13 6 12 24 7 10 17 15 13 12 21 Lathosterol 34 24 43 3 35 16 16 34 37 30 -20 -14 -23 -11 -8 4 7 12 17 12 −11 19 7-Dehydrocholesterol 65 51 6 46 24 20 35 30 28 13 16 6 11 24 13 14 13 15 14 8 37 40 8-Dehydrocholesterol 46 4 33 12 23 27 3 25 18 22 11 13 25 4 6 13 14 12 8 24 Cholesterol 8 53 21 8 50 26 39 31 37 21 23 52 3 4 16 22 19 32 26 22R-Hydroxycholesterol 20 55 3 13 33 0 7 9 9 4 6 42 49 43 22 33 36 25 20 24S-Hydroxycholesterol 33 10 47 28 34 20 24 12 14 28 21 24 20 23 21 22 47

25-Hydroxycholesterol 16 39 53 23 6 12 11 4 13 46 52 47 37 40 34 24 24,25-Epoxycholesterol 22 29 25 -10 -10 2 1 -5 0 7 14 24 19 -13 20 0 27-Hydroxycholesterol 29 56 23 26 22 17 3 11 13 16 18 15 15 33

7α-Hydroxycholesterol 30 2 6 4 2 1 27 40 42 39 40 11 26

7α,27-Dihydroxycholesterol 9 14 13 8 12 -19 -15 10 23 12 1 4 −20 Sitosterol 93 84 62 63 2 0 2 3 4 41 17 Campesterol 84 61 66 4 3 6 7 6 43 16

Stigmasterol 63 60 2 2 6 6 4 38 7 −40

Stigmastanol 46 1 3 5 9 7 30 11 Cholestanol 3 2 8 8 8 45 16 −60 Cholestenone 87 37 12 32 33 34 7-Oxocholesterol 48 24 41 33 39 5α-Hydroxycholesterol 60 60 31 22 −80 5,6α-Epoxycholesterol 74 20 15

5,6β-Epoxycholesterol 24 22

4β-Hydroxycholesterol 18 −100

Fig. 2. Pairwise correlations between serum levels of analyte. Values are Spearman’s rank correlation coefficients (r) × 100 between the indicated pairs of analytes; r values for individual comparisons are depicted using a bipolar color progression as indicated by the scale on the right of the figure. Values greater than ±4 are statistically different from 0 at a 5% significance level.

matter volume. Total brain volume explained the same amount of exemplified by those between multiple clinical parameters and variance (1.75%) as did gray matter volume. Thus, gray matter 14-desmethyl lanosterol, 4β-hydroxycholesterol, and five plant volume is directly related to serum 24S-hydroxycholesterol levels, sterols (Fig. 6). whereas white matter is only indirectly associated through correla- tion to gray matter volume. Discussion Additional phenotypic determinants of interindividual varia- In the current study, we used mass spectrometry to quantify tion in other sterols and vitamin D metabolites were identified by vitamin D metabolite and sterol levels in sera from 3,230 un- correlating further clinical measurements in each participant selected subjects and then correlated interindividual variation with individual serum lipid levels. Using an arbitrary cutoff value in these lipids with genotype and phenotype. Screening for >60 of greater than or equal to ±10 to simplify presentation of these molecular species identified 29 that were consistently present data, positive and negative correlations were found between at widely varying levels in a majority of individuals. Variation many clinical parameters and different classes of lipids (Fig. 6). in specific lipids correlated with disparities in serum choles- Strong positive correlations existed within lipid classes, such as terol levels, ethnicity, sex, age, genetic variation, anatomy, and those between sterol synthesis intermediates and total serum clinical phenotypes. cholesterol and lipoprotein levels. Comparisons between classes For some analytes, such as 24S-hydroxycholesterol, the ob- revealed shared patterns of positive and negative correlations as served correlations were consistent with known metabolic path-

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1413561111 Stiles et al. Downloaded by guest on September 28, 2021 Gender PNAS PLUS

2 2 2 R = 20% R = 6% R = 5% R2 = 2 % -152 P = 7 x 10 P = 8 x 10-44 P = 1 x 10-36 P = 9 x 10-16 50000 5000 xycholesterol 50 200 1000 500 xycholesterol xycholesterol (ng/mL) (ng/mL) (ng/mL) (ng/mL) 1000 hydro Desmosterol 50 520 ,27-Di 27-Hydro 7 α -Hydro 50 7 α 2 10 20 50 200 1000 5000 FM FM FM FM Ethnicity

2 2 2 = 2 = 3 R = 20% R = 18% R 17% R 6.1% = -154 = -132 = -111 -43

P 4 x 10 200 P 4 x 10 P 4 x 10 P = 2 x 10 500 5000 IgG oxyvitamin D oxyvitamin (ng/mL) (ng/mL) (ng/mL) (mg/dL) 50 ydrocholesterol Stigmasterol h 2 5 10 50 5 8-De 25-Hydr 200 2000 20000 5 20 100 500 1 AA EA HIS AA EA HIS AA EA HIS AA EA HIS Age

2 2 2 2 R = 4% R = 3% R = 2.6% R = 2.4% GENETICS P = 9 x 10-30 200000 P = 3 x 10-22 P = 1 x 10-19 P = 6 x 10-18 5000 20000 200 1000 xycholesterol 500 (ng/mL) (ng/mL) (ng/mL) (ng/mL) Lanosterol Lathosterol 50 7-Dehydrocholesterol 24 S -Hydro 20 100 200 2000 20000 200 2000 10 <4040-50 50-60 60+ <4040-50 50-60 60+ <4040-50 50-60 60+ <4040-50 50-60 60+ Years Years Years Years

Fig. 3. Effects of sex, ethnicity, and age on analytes levels. Box and whisker plots depict median values for the indicated analyte (thick black bars), first-third quartile (interquartile range, box), 5th and 95th percentiles (thin horizontal lines), and outliers beyond this range (x). In sex comparisons: F, female; M, male. In ethnicity comparisons: AA, African American; EA, European American; HIS, Hispanic. R2 values indicate the percentage of variance explained by each of the indicated covariates. P values indicate significance of the observed relationship.

ways (Fig. 5). This oxysterol is synthesized in the brain by between 14-desmethyl lanosterol and other intermediates in the a neuronal enzyme (cholesterol 24-hydroxylase, CYP46A1) and cholesterol biosynthetic pathways such as lathosterol and lano- thereafter secreted into the circulation where it associates with sterol, and by weaker correlations between 4β-hydroxycholesterol circulating lipoproteins. Synthesis is required for normal brain and other ring-structure oxysterols such as 7α-hydroxycholesterol function (16, 17), and once synthesized, 24S-hydroxycholesterol (Fig. 2). is a potential ligand for the liver X receptor and a substrate for A common origin related to formation by spontaneous oxi- CYP39A1 and hepatic bile acid synthesis (18–20). dation may explain the positive associations between choles- Correlations between individual levels of different lipids tenone, 7-oxocholesterol, 5α-hydroxycholesterol, and the 5,6- revealed shared metabolic pathways (Fig. 2). Most correlations epoxycholesterols (Fig. 2) (23), as enzymatic pathways for the were positive, and these were usually stronger than the smaller formation of these sterols have not been defined. Similarly, posi- number of negative correlations detected. Common origins may tive correlations between these sterols and 22R-hydroxycholesterol explain many positive correlations such as those between most and 25-hydroxycholesterol suggest that some amount of these sterols and cholesterol, reflecting cotransport in serum lipo- two oxysterols reflects formation by spontaneous as opposed to protein particles (21), and those between the plant sterols si- enzymatic oxidation (24). tosterol, campesterol, stigmasterol, and stigmastanol, reflecting Precursor–product relationships explained several positive a shared dietary origin and their absorption and excretion via correlations, such as that between 27-hydroxycholesterol and ABCG5/ABCG8 (22). Unexpectedly, serum levels of a choles- 7α,27-dihydroxycholesterol, which are sequential intermediates in terol biosynthetic intermediate, 14-desmethyl lanosterol, and the the alternate pathway of bile acid synthesis (25), and that between ring-structure oxysterol 4β-hydroxycholesterol also correlated sig- 7-dehydrocholesterol and 8-dehydrocholesterol. Similarly, sterol nificantly (r > 0.38) with plant sterols (Fig. 2). These findings sug- intermediates that are unique to the Bloch pathway of cholesterol gested serum 14-desmethyl lanosterol and 4β-hydroxycholesterol biosynthesis (lanosterol, zymosterol, and desmosterol) were posi- may derive from the diet and/or that these sterols are ABCG5/ tively correlated as were intermediates in the Kandutsch–Russell ABCG8 substrates. A unique origin for these two sterols was pathway (lanosterol, 24,25-dihydrolanosterol, and lathosterol). also suggested by the negative or weak positive correlations These compounds are indices of whole-body cholesterol synthesis

Stiles et al. PNAS Early Edition | 5of9 Downloaded by guest on September 28, 2021 ABCG5 / ABCG8

DHCR24 SPRED2

PLA2G7 ABCC8 GC CYP39A1 EPHX2 FBXL19

HSD3B7 ZNF498 APOE CYP27A1 CRIP1 SDR42E1 1 2 4 6 7 8111 14 16 9

CAMPESTEROL 14-DESMETHYL LANOSTEROL 8-DEHYDROCHOLESTEROL 27-HYDROXYCHOLESTEROL CHOLESTANOL DESMOSTEROL 24(S)-HYDROXYCHOLESTEROL -HYDROXYCHOLESTEROL

SITOSTEROL LANOSTEROL 24-OXOCHOLESTEROL 25-HYDROXYVITAMIN D2

STIGMASTANOL 24,25- EPOXYCHOLESTEROL -HYDROXYCHOLESTEROL 25-HYDROXYVITAMIN D3 STIGMASTEROL 7- DEHYDROCHOLESTEROL ,27-DIHYDROXYCHOLESTEROL

Fig. 4. Chromosomal locations of genes significantly linked to individual lipid levels. Schematics of human chromosomes stained with Giemsa are shown together with the locations of genes significantly linked (P ≤ 10−7) to individual sterol and vitamin D metabolite levels, which are color-coded at the bottom of the figure.

(26), and levels of pathway-specific sterols may thus indicate rel- across hepatocyte and enterocyte membranes and that mutations ative outputs from Bloch versus Kandutsch–Russell pathways; in these genes underlie the genetic disease sitosterolemia in however, positive associations were also detected between inter- which plant sterols accumulate to pathologic levels (4, 22). mediates unique to each cholesterol biosynthetic pathway (e.g., Levels of 14-desmethyl lanosterol were associated with these between zymosterol and lathosterol). These associations may re- same ABCG5/ABCG8 variants, which confirmed the correlation flect crossover of intermediates between pathways (27), or regu- between plant sterols and 14-desmethyl lanosterol (Fig. 2), and lation of pathway genes by sterol regulatory element binding suggested that the latter sterol was an ABCG5/ABCG8 sub- protein (SREBP) transcription factors (28). strate. As in earlier studies (32), levels of 25-hydroxyvitamin D3 Exome-wide association studies revealed significant associa- were significantly associated with the serum vitamin D transport tions between levels of sterols and variants in genes encoding gene (GC) on chromosome 4. enzymes or proteins that are known to synthesize, metabolize, or Shared transport mechanisms may also underlie the strong transport the analytes to which they were linked (Fig. 4 and positive correlations between multiple clinical phenotypes and Table S1). For example, a variant in CYP27A1 (rs114768494), interindividual variation in different sterols (Fig. 6). Most serum which specifies sterol 27-hydroxylase (29), was significantly associ- sterols are associated with circulating lipoprotein particles (LDL, − ated (P = 6.9 × 10 20) with decreased serum 27-hydroxycholesterol very low-density lipoprotein, HDL) leading to strong correla- levels. Multiple variants in CYP39A1, which encodes an oxysterol tions between these lipids and cholesterol and triglyceride levels 7α-hydroxylase (20), were strongly associated with increased levels (8, 21, 22). Similarly, the positive correlations between some of the oxysterol 24S-hydroxycholesterol (Fig. 5). A variant sterols and hematocrit most likely represent the association and transport of these analytes within reticulocyte membranes. Sterols (rs751141) of EPHX2, which encodes a soluble epoxide hydrolase − are spontaneously transferred from donor membranes to red (30), was robustly associated (P = 7.5 × 10 39) with elevated blood cells (33), and in the mouse this movement may account for serum levels of 24,25-epoxycholesterol. Higher levels of an a substantial portion of reverse cholesterol transport (34, 35), the intermediate in the classic pathway of bile acid synthesis, movement of cholesterol and presumably other sterols from pe- 7α-hydroxycholesterol, and those of an intermediate in the al- α ripheral tissues to the liver. This pathway may underlie the cor- ternate pathway, 7 ,27-dihydroxycholesterol, were associated relation (r = 0.4; Fig. 6) between 27-hydroxycholesterol and P = × −21 P = × −40 ( 1.4 10 and 1.7 10 , respectively) with the same hematocrit in that a majority of this oxysterol in serum is formed HSD3B7 variant (rs34212827) of , which encodes an enzyme that in the lung (36), a tissue in which reticulocyte–cell membrane catalyzes an essential step in both pathways (31). Based on these interactions are frequent, and is thereafter converted to findings, other strong genetic associations shown in Fig. 4 may bile acids in liver. Plant sterol, 4β-hydroxycholesterol, and identify substrates of enzymes specified by variant alleles, in- 14-desmethylanosterol levels did not correlate with hematocrit, cluding 8-dehydrocholesterol and SDR42E1, which encodes a short suggesting that a shared origin, physiochemistry, or biology chain dehydrogenase/reductase, and 7-dehydrocholesterol and excludes these sterols from association with circulating cells. 24-oxocholesterol with CYP39A1. The experimental approaches taken here identify numerous With respect to transport, elevated levels of multiple plant relationships between serum levels of vitamin D metabolites sterols were strongly associated with variants in ABCG5/ABCG8 and sterols, genes, and clinical phenotypes, and in the case of (Fig. 4 and Table S1), confirming prior studies indicating the the oxysterol 24S-hydroxycholesterol and CYP39A1,identify encoded heterodimeric protein transports this class of sterols biochemical and anatomical bases for the observed relationship.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1413561111 Stiles et al. Downloaded by guest on September 28, 2021 A PNAS PLUS CYP39A1 24S-Hydroxy- 7α,24S-Dihydroxycholesterol cholesterol Oxysterol 7α-Hydroxylase B

NH2 1 2 3 4 567 8 910 11 12 COOH

R23P R103H Y288H N324K K329Q

C 300 MockWT Y288HR103HR23P N324KK329QkDa CYP39A1 54

a-tubulin 200 50

100 GENETICS 0 Dhdoyhlseo (ng/mL) 7 α ,24 S -Dihydroxycholesterol Mock WT Y288H R103H R23P N324K K329Q D P = 1.5 x 10-34 0.0

-0.5

-1.0

-1.5 Hydroxycholesterol/Cholesterol S -

-2.0 log 24 # of CYP39A1 SNPs 0 1234 5 6 # of Subjects 230 484 646 247 89 23 1

Fig. 5. Expression analysis of genetic variants linked to high serum 24S-hydroxycholesterol levels. (A) Biochemical reaction catalyzed by the CYP39A1 oxysterol 7α-hydroxylase. (B) Schematic of the 500-aa CYP39A1 protein showing the sequences and locations of the five variants (rs12192544, R23P; rs2277119, R103H; rs17856332, Y288H; rs7761731, N324K; rs41273654, K329Q) identified in individuals with high levels of 24S-hydroxycholesterol. (C) Expression analysis of normal and variant enzymes. Plasmids expressing normal (N) and the indicated variant CYP39A1 enzymes were transfected into cultured HEK 293 cells and assayed for oxysterol 7α-hydroxylase activity. Inset shows levels of CYP39A1 protein in transfected cells as determined by immunoblotting; α-tubulin served as a loading control. (D) Cumulative effects of multiple SNPs on serum levels of 24S-hydroxycholesterol. Log-normalized serum levels of the oxysterol (y axis) are indicated by whisker plots showing median values (thick black bars), first-third quartile (interquartile range, box), 5th and 95th percentiles (thin horizontal lines), and outliers beyond this range (open circles), and are plotted versus the total number of CYP39A1 SNPs present in an individual (x axis). The number of subjects who inherited a given number of SNPs is indicated at the bottom of the plot.

A key aspect of this study is the availability of a well-phenotyped and to determine whether analyte levels are therapeutically population cohort that allows useful information to be derived informative. from static measurements of serum analytes. A limitation is that this is an observational/cross-sectional study, and therefore we Materials and Methods cannot draw causal conclusions, only detect associations be- Materials and methods are described at length in SI Materials and Methods. tween clinical phenotypes and the analytes measured. Never- This description includes patient population, analytical chemistry, statistical theless, additional studies in this population may allow the analyses, genotyping, and biochemical and molecular biology assays, together definition of mechanisms underlying observed correlations with references. Analytical standards were from Avanti Polar Lipids (Alabaster, AL).

Stiles et al. PNAS Early Edition | 7of9 Downloaded by guest on September 28, 2021 ymphocytes it

rubin (direct) e DW SECOSTEROID BMI InsulinHOMA (uU/ml)Glucose IRSystolic (mg/dL)Diastolic BPAlbumin ALPBP ALT ASTGGTBili BilirubinCa (indirect)CK LDHMg TotalGFR ProteinBUN MDRDUric TotalAcidTG Cholesterol (mg/dL)VLDLLDL cholesterol (mg/dL) HDLCholesterol LDLCholesterol (mg/dL) PCSK9Density (mg/dL)IgA (ng/ml)(mg/dL)(mg/mL)IgG (mg/mL)AbsoluteAbsolute AbsoluteL WBCMonocytes HematocrNeutrophils CountHemoglobinF R 25−Hydroxyvitamin D3 -30 -20 -20 0 -10 -10 20 -10 10 10 -10 0 20 20 -10 -10 10 -10 -20 20 0 10 10 10 0 10 0 0 -10 -30 -10 0 10 0 20 20 20 -20 STEROL SYNTHESIS Lanosterol 10 20 20 10 10 10 10 10 20 10 20 -20 -20 0 0 10 0 10 10 0 10 50 30 30 40 -10 30 20 0 -10 10 0 10 10 10 10 0 0 14−Desmethyl lanosterol -30 -20 -20 -10 0 0 10 -10 -10 0 -10 0 0 10 10 0 0 0 -10 10 -10 30 -10 -10 20 20 20 10 0 0 -10 -10 -10 -20 0 0 10 0 Zymosterol 10 10 10 10 0 10 0 10 20 20 20 -10 -10 0 0 0 0 0 -10 0 10 30 30 30 20 -10 20 20 -10 -20 0 0 10 10 10 10 10 -10 Desmosterol 0 10 10 10 0 10 20 0 20 20 20 0 10 10 10 0 10 10 10 0 10 50 30 30. 50 -10 40 10 0 -10 0 0 0 0 30 30 20 -10 24−Dihydrolanosterol 0 0 0 0 10 10 0 10 10 10 20 -20 -10 0 0 0 10 0 0 0 0 40 20 20 40 0 40 20 0 -10 0 0 0 0 10 10 10 0 Lathosterol 20 20 20 10 10 20 10 10 20 10 20 -20 -10 0 0 0 10 0 10 -10 20 40 40 40 40 -10 30 10 0 0 10 0 10 10 10 10 0 0 7−Dehydrocholesterol 20 20 30 20 10 10 10 10 20 10 20 -10 -10 10 0 0 10 0 -20 10 20 50 40 40 40 -10 20 30 -10 -20 0 0 10 10 10 10 10 -10 8−Dehydrocholesterol -10 0 10 10 0 0 20 0 20 20 10 -10 10 10 -10 0 20 -10 -20 20 10 50 40 40 30 0 20 20 -10 -30 -10 -10 10 10 20 20 20 -20 OXYSTEROLS 24S−Hydroxycholesterol -10 10 0 0 0 0 20 10 10 10 10 -10 0 10 -10 10 10 10 0 0 0 50 30 30 50 0 40 20 0 0 0 0 0 0 10 10 10 -10 25−Hydroxycholesterol 10 10 10 10 10 10 0 10 10 10 20 -10 0 10 10 20 0 10 0 0 10 20 20 20 20 -10 20 10 10 10 10 10 0 10 20 20 0 0 24,25−Epoxycholesterol 0 20 20 10 0 0 20 20 20 20 20 -20 0 0 0 10 10 0 0 10 10 10 30 30 0 -20 -10 20 0 -10 20 20 20 20 20 30 10 0 27−Hydroxycholesterol -10 10 0 0 10 20 20 10 20 20 30 0 10 10 20 0 10 10 0 0 20 50 30 30 50 -10 40 10 0 0 10 10 0 0 40 40 20 -10 7α−Hydroxycholesterol 0 10 10 10 10 10 10 10 20 10 20 -10 0 0 0 10 0 0 0 0 20 30 30 30 20 -10 20 10 0 -10 10 10 10 10 20 20 20 0 7α,27−Dihydroxycholesterol 0 10 10 0 10 10 20 10 20 10 20 -10 10 10 10 20 10 20 10 0 20 40 30 30 40 -20 20 10 10 0 10 10 0 10 30 30 10 0 4β−Hydroxycholesterol -30 -30 -30 20 0 0 0 0 -10 0 -10 -20 0 10 0 0 0 10 0 0 -20 30 -10 -10 30 20 20 0 0 0 -10 -10 -10 -10 0 0 0 0 24−Oxocholesterol 0 10 10 0 0 0 0 0 10 10 10 -10 0 0 0 -10 10 0 -10 0 0 30 20 20 20 -10 30 10 -10 -20 0 0 0 0 10 10 10 -10 PLANT STEROLS Sitosterol -20 -20 -20 -10 0 0 10 -10 -10 0 -10 10 10 10 10 0 0 0 0 0 -10 30 -10 -10 30 20 20 0 0 0 -10 -10 -20 -20 0 0 0 0 Campesterol -20 -20 -20 -10 0 0 10 -10 -10 0 -10 0 0 10 10 0 0 10 0 0 -10 40 -10 -10 30 20 20 10 0 0 -10 -10 -10 -10 0 0 0 0 Stigmasterol -20 -20 -20 -10 0 0 10 0 -10 0 0 0 -10 10 10 0 0 10 0 0 -10 20 -10 -10 20 20 0 0 0 10 0 0 -10 -10 0 0 0 10 Stigmastanol -20 -20 -10 -10 0 0 0 0 -10 0 0 0 0 0 0 0 0 0 0 0 -10 20 0 0 20 10 10 0 0 0 0 -10 -10 -10 0 0 0 0 Cholestanol -20 -20 -20 -10 0 0 10 0 -10 0 0 -10 10 10 0 0 10 0 -10 0 0 50 0 0 50 20 40 10 0 -10 -10 0 -10 -10 0 0 0 0 OXIDIZED STEROLS 5α−Hydroxycholesterol 0 0 0 0 10 10 10 10 0 0 10 -10 10 10 0 20 0 0 0 0 10 20 10 10 10 0 10 0 0 0 0 10 10 10 10 10 10 0 5,6α−Epoxycholesterol 0 0 0 0 10 10 10 10 10 10 10 -10 10 10 0 20 0 10 0 0 10 20 10 10 20 0 10 0 0 0 0 10 0 0 20 20 10 0 5,6β−Epoxycholesterol 10 0 0 0 0 10 0 0 10 10 10 -10 10 0 0 10 0 0 0 0 10 20 10 10 20 0 30 0 0 0 0 0 0 0 20 20 10 0

−100 −50 0150 00

Fig. 6. Pairwise correlations between serum levels of analytes and clinical phenotypes. Values are partial correlation coefficients × 100 after adjustment for age, sex, and ethnicity between the indicated pairs of analytes and clinical parameters; r values for individual comparisons are depicted using a bipolar color progression as indicated by the scale below the figure. Only traits for which at least one r value was greater than or equal to ±10 are shown.

ACKNOWLEDGMENTS. We thank Jonathan Cohen, Helen Hobbs, and Jay by grants awarded to D.W.R. from the National Institutes of Health Horton for critically reading the manuscript. This research was supported (5U54GM069338 and 2P01HL20948) and the Clayton Foundation for Research.

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