Common and rare variants associating with serum levels of creatine kinase and lactate dehydrogenase

Abstract

Creatine kinase (CK) and lactate dehydrogenase (LDH) are widely used markers of tissue damage. To search for sequence variants influencing serum levels of CK and LDH, 28.3 million sequence variants identified through whole-genome sequencing of 2,636 Icelanders were imputed into 63,159 and 98,585 people with CK and LDH measurements, respectively. Here we describe 13 variants associating with serum CK and 16 with LDH levels, including four that associate with both. Among those, 15 are non-synonymous variants and 12 have a minor allele frequency below 5%. We report sequence variants in genes encoding the enzymes being measured (CKM and LDHA), as well as in genes linked to muscular (ANO5) and immune/inflammatory function (CD163/CD163L1, CSF1, CFH, HLA-DQB1, LILRB5, NINJ1 and STAB1). A number of the genes are linked to the mononuclear/phagocyte system and clearance of enzymes from the serum. This highlights the variety in the sources of normal diversity in serum levels of enzymes.

ARTICLE

Received 15 Jun 2015 | Accepted 30 Dec 2015 | Published 3 Feb 2016

Ragnar P. Kristjansson1, Asmundur Oddsson1, Hannes Helgason1,2, Gardar Sveinbjornsson1,Gudny A. Arnadottir1, Brynjar O. Jensson1, Aslaug Jonasdottir1, Adalbjorg Jonasdottir1, G. Bragi Walters1, Gerald Sulem1, Arna Oskarsdottir1, Stefania Benonisdottir1, Olafur B. Davidsson1, Gisli Masson1,Olafur Th Magnusson1, Hilma Holm1,3, Olof Sigurdardottir4, Ingileif Jonsdottir1, Gudmundur I. Eyjolfsson5, Isleifur Olafsson6, Daniel F. Gudbjartsson1,2, Unnur Thorsteinsdottir1,7, Patrick Sulem1 & Kari Stefansson1,7

Creatine kinase (CK) and lactate dehydrogenase (LDH) are widely used markers of tissue damage. To search for sequence variants inuencing serum levels of CK and LDH, 28.3 million sequence variants identied through whole-genome sequencing of 2,636 Icelanders were imputed into 63,159 and 98,585 people with CK and LDH measurements, respectively. Here we describe 13 variants associating with serum CK and 16 with LDH levels, including four that associate with both. Among those, 15 are non-synonymous variants and 12 have a minor allele frequency below 5%. We report sequence variants in genes encoding the enzymes being measured (CKM and LDHA), as well as in genes linked to muscular (ANO5) and immune/inammatory function (CD163/CD163L1, CSF1, CFH, HLA-DQB1, LILRB5, NINJ1 and STAB1). A number of the genes are linked to the mononuclear/phagocyte system and clearance of enzymes from the serum. This highlights the variety in the sources of normal diversity in serum levels of enzymes.

1 deCODE genetics/Amgen, Inc., 101 Reykjavik, Iceland. 2 School of Engineering and Natural Sciences, University of Iceland, 101 Reykjavik, Iceland.

3 Department of Internal Medicine, Landspitali The National University Hospital of Iceland, 101 Reykjavik, Iceland. 4 Department of Clinical Biochemistry, Akureyri Hospital, 600 Akureyri, Iceland. 5 Icelandic Medical Center (Laeknasetrid), Laboratory in Mjodd (RAM), 109 Reykjavik, Iceland. 6 Department of Clinical Biochemistry, Landspitali University Hospital, 101 Reykjavik, Iceland. 7 Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. Correspondence and requests for materials should be addressed to P.S. (email: mailto:[email protected]

Web End [email protected] ) or to K.S. (email: mailto:[email protected]

Web End [email protected] ).

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Common and rare variants associating with serum levels of creatine kinase and lactate dehydrogenase

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10572

The release of intracellular enzymes into the serum is an indicator of tissue damage and physiological cell turnover. Thus, measurements of intracellular enzymes in serum are

widely used to diagnose tissue damage, monitor its course and severity and gauge the effect of therapy.

Creatine kinase (CK) is an enzyme, catalyzing the ATP-dependent phosphorylation of creatine that is important for energy buffering in tissues with variable energy demands, most notably skeletal and cardiac muscle1. Elevated serum CK levels can indicate tissue damage, and are observed in a number of pathological conditions, including statin-induced myopathy2. Monitoring changes in serum CK levels is therefore important in statin-treated patients who display muscle pain or weakness3, and in patients deemed at risk of rhabdomyolysis for various reasons4.

Lactate dehydrogenase (LDH) is an enzyme with a ubiquitous expression5. It is responsible for catalyzing the anaerobic, nicotinamide adenine dinucleotide phosphate-dependent conversion of pyruvate to lactate, which is important during times of high muscular activity6. Serum CK and LDH levels were previously used as biomarkers to diagnose myocardial infarction. Because of low specicity, however, they have been replaced by troponin T and troponin I7, measured through high-sensitivity assays8.

The heritability of LDH levels has been estimated between 40 and 50% (refs 9,10) and upwards of 38% for CK (ref. 11). Our own data indicates a 19.33 and 19.36% heritability for CK and LDH, respectively. A recently published genome-wide association study (GWAS) of 3,232,779 imputed variants in 3,412 statin users found two missense variants affecting serum CK levels, one in CKM (rs11559024) and one in LILRB5 (rs12975366) (ref. 12). An association of a variant in CD163 (rs7136716) with serum CK levels has also been reported13. To date, no systematic GWAS has been carried out to search for sequence variants inuencing serum LDH levels.

To search for common and rare variants that associate with CK or LDH levels, we tested variants detected in a large sequencing study in Iceland for association with these traits14. This unbiased approach has the potential to uncover previously unexpected molecular mechanisms regulating levels of this enzyme in serum. A thorough understanding of sequence variants inuencing these biomarkers is important to improve the usefulness of their measurements, and in the assessment of tissue damage15.

ResultsSummary of ndings. To search for sequence variants associating with mean serum levels of CK and/or LDH, we analysed28.3 million variants, initially identied through whole-genome sequencing of 2,636 Icelanders and subsequently imputed into chip-typed individuals through long-range haplotype phasing16. Genotype probabilities were calculated for rst and second-degree relatives of chip-typed individuals17. We tested for association between sequence variants and serum CK in 63,159 individuals (35,623 chip-typed and 27,536 with chip-typed rst or second-degree relatives), and serum LDH in 98,585 individuals (52,581 chip-typed and 46,004 with chip-typed rst- or second-degree relatives).

We used methodology outlined by Sveinbjornsson et al.18 to determine weighted genome-wide signicance thresholds for different variant classes based on the total number of variants tested per class: loss-of-function (N 6,476;

P 2.6 10 6); missense (N 100,502; P 1.7 10 7); and

other (N 23,854,999; P 7.0 10 10).

We found a total of 13 variants associated with CK, and 16 associated with LDH. Of those, four were associated with both

enzymes (Table 1, Figs 1 and 2). In total, all of the reported sequence variants explain 1.9% of CK variance, and 1.8% of LDH variance. Our results include replication of variants in CKM, LILRB5 and CD163 reported in GWASs of serum CK levels12,13. None of the variants showed more signicant associations when alternate inheritance models were tested (Supplementary Tables 2 and 3), and no additional variants were detected using these models.

Eleven of the variants are common (minor allele frequency (MAF)45%), eight are of low frequency (MAF 0.5-5%) and

ve are rare (MAFo0.5%). Five loci contain several independent signals, associating with either one or both of the enzymes measured (Fig. 3). For loci with multiple signals, we present P values and effects before and after adjusting for other signicant markers at that locus (Table 1). All variants correlated (r240.6)

with the variants we report are shown in the Supplementary Data 1 and Supplementary Figs 533.

Most of the suspected genes are implicated in immune/ inammatory response, enzyme clearance or muscular function, but two encode subunits of the enzymes measured (cis signals) and one encodes a protein that affects rates of CK clearance from plasma.

Cis signals. We observed variants in CKM and LDHA, genes that encode subunits of CK and LDH, respectively, with minor alleles that associate with a lower level of their corresponding serum enzyme levels. Enzymatic levels were measured by assessing quantity through enzymatic activity. Our results, therefore, indicate that these sequence variants act to either decrease the amount of the enzyme produced, or their catalytic ability.

CKM. Through a stepwise conditional analysis at 19q13.3, we found that four independent missense variants in CKM associate with serum CK levels. With one of these, rs11559024 [C] (MAF 2.15%, Glu83Gly), we replicated a reported association12.

The associations of the remaining three low-frequency and rare missense variants are novel. Minor alleles of all four CKM variants have a large lowering effect on CK levels. We never observed more than one of the minor alleles on the same chromosome (all r2r2.2 10 4; Supplementary Table 4).

CKM encodes CK-M, one of two subunits of the CK dimer. Three isoforms of the enzyme exist, consisting of different combinations of CK-M and/or CK-B. Each isozyme has a unique expression prole; CK-MM is expressed in skeletal muscle, CKMB in cardiac muscle and CK-BB in smooth muscle and the brain19. CK-MM typically accounts for the majority of serum CK (ref. 20).

LDHA. The missense variant rs116841148 [T] (MAF 0.652%,

Ala147Ser) at 11p15.1 in LDHA associates with serum LDH levels (Table 1). LDHA encodes the M subunit of the LDH enzyme, expressed in all but one of the ve isozymes of LDH (ref. 21).

CPN1. At 10q24.2, the low-frequency missense variant rs61751507 [T] (MAF 4.06%, Gly178Asp) in CPN1 associates

with lower CK levels (Table 1). CPN1 encodes carboxypeptidaseN. This protein is expressed in blood, hydrolyzes CK-MM10s

C-terminal lysine and converts CK-MM1, the enzymes unaltered form as expressed in tissue, into either CK-MM2 or CK-MM3. This hydrolyzation alters CK-MMs isoelectric point and half-life without affecting properties such as enzymatic activity22,23. It is, therefore, plausible that if the variant in CPN1 induces a change in activity of carboxypeptidase N, it would affect CK-MM clearance rather than having a direct effect on enzymatic activity.

Immune system genes associating with CK and/or LDH levels. Seven loci harbouring variants associating with serum CK and/or LDH levels have genes that are likely to be responsible for the

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Table 1 | Summary of single marker associations for CK (N 63,159) and LDH (N 98,585) in Iceland.

Variant Allele (min/maj)

MAF

(%)

Gene* CK LDH

Unadjusted Adjustedw Unadjusted Adjustedw P value Effect

(Amin)

P value Effect

(Amin)

P value Effect

(Amin)

P value Effect

(Amin)

rs333947 A/G 14.92 CSF1 6.8 10 3 0.021 2.8 10 10 0.042

rs2274700 A/G 38.62 CFH 0.58 0.003 4.1 10 12 0.034

rs150956780z C/G 0.078 STAB1 0.60 0.05 1.3 10 61 1.526

rs17412833z T/A 13.76 HLA-DQB1 1.5 10 4 0.032 1.5 10 22 0.07

rs12342201 A/G 49.45 NINJ1 0.70 0.002 1.1 10 12 0.034

rs61751507z T/C 4.06 CPN1 5.1 10 11 0.091 0.34 0.011

rs116841148z T/G 0.65 LDHA 0.17 0.049 2.9 10 11 0.198

rs7481951z A/T 37.45 ANO5 6.4 10 16 0.047 7.0 10 15 0.045 2.5 10 3 0.015

rs137854526z C/T 0.24 ANO5 4.5 10 4 0.204 1.1 10 3 0.190 0.25 0.058

chr11:22241070z A/T 0.27 ANO5 3.9 10 6 0.245 1.7 10 5 0.229 0.05 0.09

rs2930191 A/G 37.22 2.3 10 4 0.021 4.7 10 13 0.035

chr12:7282745 TA/!TA 21.13 0.49 0.005 1.2 10 25 0.064 2.3 10 12 0.043

rs145411783z A/C 0.66 CD163L1 0.90 0.005 8.7 10 12 0.203 8.9 10 10 0.184

rs4072797z T/C 4.21 CD163L1 0.63 0.007 9.9 10 89 0.236 5.5 10 85 0.239

rs117692263 C/T 9.31 CD163 1.2 10 17 0.083 1.2 10 11 0.067 6.1 10 28 0.09 1.5 10 13 0.062

rs4883263z T/C 3.73 CD163 5.7 10 5 0.059 1.8 10 19 0.114 1.4 10 6 0.063

rs7305678 T/G 16.16 3.0 10 21 0.072 2.8 10 15 0.061 2.0 10 18 0.057 1.2 10 23 0.069

chr12:110830276 C/T 2.09 2.9 10 4 0.071 2.1 10 12 0.119

rs7318906 A/G 47.08 2.7 10 13 0.041 2.9 10 3 0.014

rs149354459z G/C 0.21 CKM 6.0 10 20 0.55 2.8 10 21 0.567 0.54 0.031

rs145987658z A/G 0.086 CKM 8.9 10 9 0.59 2.1 10 9 0.611 0.58 0.047

rs17357122z A/G 0.99 CKM 4.3 10 9 0.165 4.3 10 9 0.175 0.45 0.018

rs11559024z C/T 2.15 CKM 1.8 10 115 0.446 1.1 10 117 0.45 0.16 0.023

rs393600 G/A 25.17 LILRB5 1.4 10 33 0.079 1.3 10 9 0.046 1.6 10 28 0.062 1.7 10 5 0.028

rs12975366z C/T 41.62 LILRB5 6.5 10 44 0.08 4.7 10 20 0.061 7.0 10 51 0.074 4.1 10 28 0.062 CK, creatine kinase; LDH, lactate dehydrogenase; MAF, minor allele frequency.

Variants showing signicant association are in bold, based on the 25 variants tested (Po0.05/25 2 10

3). Further information can be found in Supplementary Table 1.

*Genes given for intronic/exonic variants.

wAll variants are adjusted for all other signicant variants in the same megabase. zNon-synonymous coding variants.

Serum CK

CKM

LILRB5

25

20

15

10

5

0

CD163/CD163L1

ANO5

CPN1

log 10(p)

13q22.1

CACNG1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 23

21

Chromosome

Figure 1 | Manhattan plot showing the seven loci harbouring genome-wide signicant signals inuencing serum CK levels in the Icelandic population. Variants are plotted by chromosomal position (x axis) and log10 P values (y axis). P values above 1 10 25 are represented. Two loci (CKM and LILRB5)

harbour variants with P values below this cutoff (rs11559024: P 1.8 10 115; rs12975366: P 6.5 10 44 and rs393600: P 1.4 10 33). The red line

indicates the threshold for genome-wide signicance, determined by the number of tests performed (P 0.05/28.3 million 1.8 10 9).

associations that are involved in immune or inammatory response; CSF1, CD163/CD163L1, STAB1, CFH, LILRB5, HLA-DQB1 and NINJ1. Three of these genes (CSF1, CD163 and STAB1) have direct links to the clearance of products of cell lysis from the serum through the mononuclear phagocytic system (MPS) (refs 2426). The remaining four implicated

genes do not have a known link to enzyme clearance, although most are preferentially expressed in cells of the myeloid lineage.

CSF1. At 1p13.3, the intronic variant rs333947 [A] (MAF 14.92%) in CSF1 associates with lower LDH levels

(Table 1). We observed no correlated coding variants (r240.6)

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Serum LDH

CD163/CD163L1

60

50

40

30

20

10

0

STAB1

LILRB5

log 10(p)

HLA-DQB1

11p11.2

NINJ1 LDHA

13q22.1

CFH CSF1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 23

21

Chromosome

Figure 2 | Manhattan plot showing the 10 loci harbouring genome-wide signicant signals inuencing serum LDH levels in the Icelandic population. Variants are plotted by chromosomal position (x axis) and log10 P values (y axis). P values above 1 10 60 are represented. One locus (CD163L1)

harbours a variant with P value below this cutoff (rs4072797: P 9.9 10 89). The red line indicates the threshold for genome-wide signicance,

determined by the number of tests performed (P 0.05/28.3 million 1.8 10 9).

40

40

a

100 80 60 40 20 0

100 80 60 40 20 0

100

b

chr19:59451173

chr19:59451173

r

r

Recombination rate (cM/Mb)Recombination rate (cM/Mb)

0.80.60.40.2

0.80.60.40.2

80 60 40 20 0

100

80

60 40 20 0

Recombination rate (cM/Mb)

chr19:59450914

chr19:59450914

30

30

log 10(P value)

20

20

log 10(P value)

log 10(P value)

40 30 20 10

0

10

10

0

0

LILRA6 LILRB3 LILRB2 MIR4752

LILRA3 LILRA5

LILRB3

LILRB3 LILRA6 LILRB5 LILRB2 MIR4752

LILRA3 LILRA5

59.4

59.42 59.44 59.46 59.48 59.5 59.52 59.4 59.42 59.44 59.46 59.48 59.5 59.52

Position on chr19 (Mb)

Position on chr19 (Mb)

59.4 59.42 59.44 59.46 59.48 59.5 59.52

Position on chr19 (Mb)

c

d

r

r

50

0.8

0.80.60.40.2

log 10(P value)

Recombination rate (cM/Mb)

50

30 20 10

0

0.60.40.2

40

LILRB3 LILRA6 LILRB5 LILRB2

MIR4752

LILRA3 LILRA5

LILRB3 LILRA6 LILRB5 LILRB2

MIR4752

LILRA3 LILRA5

59.4 59.42 59.44 59.46 59.48 59.5 59.52

Position on chr19 (Mb)

Figure 3 | Locus plots depicting variants at the LILRB5 locus associating with serum enzyme levels. Leading variants are labelled and shown in purple, other variants are coloured according to correlation (r2) with the leading marker (legend at top-right). log10 P values are shown along the left y

axis and correspond to the variants depicted in the plot. The right y axis shows calculated recombination rates at the chromosomal location, plotted as a solid blue line. (a) Association between the signal represented by rs393600 and serum CK levels. (b) Association between the signal represented by rs12975366 and serum CK levels. (c) Association between the signal represented by rs393600 and serum LDH levels. (d) Association between the signal represented by rs12975366 and serum LDH levels.

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that could explain the association (Supplementary Data 1). The position shows a high degree of evolutionary conservation (GERP (ref. 27) 4.83; Supplementary Table 5). CSF1 encodes

human macrophage-specic colony stimulating factor, a cytokine necessary for the differentiation of monocyte lineage cells, including hepatic Kupffer cells (KCs) (ref. 24). In animal models, reducing KC numbers affects serum enzyme levels, including CK, LDH and aspartate transaminase (AST), without evidence of skeletal or liver damage24. Rs333947 [A] also associates with lower AST levels (Supplementary Table 6). We also observed a suggestive association with CK levels. The associations we observed are consistent with CSF1 function and may indicate an increased macrophage activity, promoting faster clearance of serum enzymes.

We assessed all sequence variants discussed in this paper for associations with AST levels (Supplementary Table 6). Seven variants, including rs333947, associate with AST based on the number of tests performed (Po0.05/25 2.0 10 3). The

direction of effect was always consistent; each allele either had an increasing or decreasing effect on the levels of all enzymes it associates with.

CD163/CD163L1. At 12p13.31, the region containing CD163 and its paralog CD163L1, we observed associations with CK and LDH levels (Table 1). Two variants associated with both CK and LDH levels and four only with LDH levels.

Two common variants associate with higher serum levels of CK and LDH; the intergenic variant rs7305678 [T] (MAF 16.16), and the intronic rs117692263 [C]

(MAF 9.31%). Three low-frequency missense variants in

the region associate solely with serum LDH levels; rs4072797[T] (MAF 4.21%, Asp588Asn) and rs145411783 [A]

(MAF 0.66%) in CD163L1, and rs4883263 [T] (MAF 3.73%,

Ile342Val) in CD163. Finally, a common intergenic indel (chr12:7282745:0:TA MAF 21.13%) associates with LDH levels.

CD163 encodes a scavenger receptor expressed on macrophages and monocytes, including KCs, that is responsible for uptake of the haemoglobinhaptoglobin complex from the bloodstream25. The CD163L1 gene, encoding the M160 receptor, is closely related to CD163. The paralog is expressed by many of the same cells and has a sequence that is highly similar to that of CD163 (ref. 28), but does not have afnity for the same ligands as CD163 (ref. 25).

Association of the intergenic variant rs7305678 with serum CK levels replicates a previously published association between a single marker, rs7136716 at the CD163 locus and serum CK levels in the Japanese13. Rs7136716 correlates with rs7305678 in both the Chinese/Japanese (r2 0.8529) and Icelandic (r2 0.75)

populations, but is independent of other signals we report in the region.

STAB1. At 3p21.1, the very rare missense variant rs150956780[C] (MAF 0.078%, Val1522Leu) at a highly conserved site

within STAB1 (GERP (ref. 27) 4.56) associates with drastically

lower serum LDH levels (effect 1.526SD) (Table 1). An

association signal was observed across a large region and rs150956780 is more frequent outside of Iceland (Tuscany (TSI) MAF 1.0%, CEPH Utah (CEU) MAF 0.6% (ref. 30);

Supplementary Table 5). This could reect a recent introduction of the sequence variant into the Icelandic population. STAB1 encodes stabilin-1, a transmembrane scavenger receptor expressed in a number of tissues including activated macrophages26.

CFH. At 1q31.3, in a locus containing the CFH gene, the synonymous variant rs2274700 [A] (MAF 38.62%) associates

with lower LDH levels (Table 1). Rs2274700 is fully correlated (r2 1.00) with rs1410996 [A] (MAF 38.58%). Rs1410996 is

one of a large number of sequence variants within genes of the complement system reported to associate with age-related

macular degeneration31,32. In our data, the [A] allele associates with lower risk of associate with age-related macular degeneration. Mutations in CFH have also been shown to cause both atypical haemolytic uraemic syndrome33. CFH encodes complement factor H (FH), a key regulator of the alternative pathway of the complement system, produced and secreted in abundance by KCs34. FH also binds to long pentraxin 3 (PTX3) through two of FHs SCR domains (SCR7 and SCR19-20)35. PTX3 inuences the regulation of the complement system36 and plays a non-redundant role in the orchestration of tissue repair and remodelling37. FHs specic functions and its associations suggest that alterations of enzyme levels occur through modulation of complement activation or tissue repair and remodelling38.

LILRB5. At 19q13.42, a locus containing LILRB5, we observed associations of two common and modestly correlated (r2 0.24)

sequence variants with CK and LDH levels. The strongest signal is a missense variant rs12975366 [C] (MAF 41.62%, Asp247Gly,

Asp147Gly) that associates with lower levels of both CK and LDH. A second marker, the intronic variant rs393600 [G] (MAF 25.17%) in LILRB5, similarly associates with both CK

and LDH levels. LILRB5, encoding leukocyte immunoglobulin-like receptor subfamily B member 5, belongs to the LILR class of genes expressed on cells of myeloid lineage, and is involved in inhibition of inammatory responses39,40. Showing association of the marker rs12975366 with CK at LILRB5 replicates previous ndings12.

HLA-DQB1. In the human leukocyte antigen (HLA) region at 6p21.3, the best association signal with LDH levels is represented by the missense variant rs17412833 [T] (MAF 13.76%,

Phe119Tyr) in HLA-DQB1. The alleles of the different HLA genes are strongly correlated to each other and often discussed as long haplotypes41. The HLA genes control the adaptive immune response through presentation of antigens to T cells42. We tested imputed HLA alleles of six of the classical HLA genes: HLA-A; HLA-B; HLA-C; HLA-DQA1; HLA-DQB1; and HLA-DRB1 for association43. The HLA molecular type associating most strongly with serum LDH is HLA-DQB1*06:04 (r2 0.53). We note that

the [T] allele of rs17412833 is present in the following imputed HLA molecular types: DQB1*05:01; 05:02; 05:03; 06:04; and 06:09. HLA-DQB1 forms a part of the dimeric HLA-DQ molecule44. HLA-DQB1*06:04 has previously been implicated in myasthenia gravis in the Chinese45 as well as cervical dystonia46. Conditional analysis shows that the HLADQB1*06:04 signal is fully explained by the missense variant rs17412833, but indicates that the association of rs17412833 is not explained by the HLA-DQB1*06:04 signal.

NINJ1. At 9q22.31, the common intron variant rs12342201 [A] (MAF 49.45%) in NINJ1 associates with lower LDH levels

(Table 1). NINJ1 encodes the ninjurin-1 protein, an adhesion molecule reported to be upregulated in myeloid cells during inammation and important in immune cell migration following neuronal injury47. NINJ1 expression is ubiquitous, and it has been implicated in liver function and hepatocellular senescence48.

Muscle-linked genes associating with CK and/or LDH levels. Variants at two loci are coding variants in genes that are preferentially expressed in muscle and play a role in its function.

ANO5. At 11p14.3, we observed three variants in ANO5 that associate with serum CK levels. The common missense variant rs7481951 [A] (MAF 37.45%, Leu322Phe) associates

with lower serum CK levels. The remaining two rare variants, the missense variant rs137854526 (MAF 0.24%, Phe578Ser),

and the nonsense variant chr11:22241070:S (MAF 0.27%,

Cys601X), associates with higher CK levels (Po0.05/15 non-

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synonymous 3.3 10 3). Rs137854526 occurs at a highly

conserved site (GERP (ref. 27) 5.69). ANO5 encodes the

protein anoctamin-5, a chloride channel in the endoplasmic reticulum membrane49, expressed mainly in skeletal and cardiac muscle. Variants in the gene, including these two rare mutations, have been reported to cause limb-girdle muscular dystrophy, which primarily affects skeletal muscle but often presents with cardiac pathologies50,51. Chr11:22241070:S shows suggestive association with heart failure (effect 1.629, P 1.0 10 3,

N 11,374 cases).

Two cases of compound heterozygotes for ANO5 mutations presenting with skeletal muscle dysfunction and cardiac abnormalities have been described in the literature49,52. We support this with a further two homozygotes for rare ANO5 mutations presenting with cardiovascular phenotypes. Firstly, an individual homozygous for rs137854526 and, secondly, an individual homozygous for chr11:22241070, both presenting with several cardiovascular phenotypes (Supplementary Table 7). These ndings provide further evidence for the effect of ANO5 variants on dysfunctions of the heart.

We note another coding variant in a gene specically expressed in muscle has a P value just above the signicance threshold. The common missense variant in CACNG1 rs1799938 [A] (MAF 10.64%, Gly196Ser) suggestively associates with CK

levels (P 3.2 10 7; effect 0.046). CACNG1 encodes the g

subunit of a 1,4-dihydropyridine (DHPR) -sensitive calcium channel, preferentially expressed in skeletal muscle53.

Other signals associating with CK and/or LDH Levels. The three remaining association signals are at loci without any obvious causative gene.

We observed a signal at 11p11.2 associating with lower serum LDH levels, represented by a large group of 276 correlated markers (r240.6; Supplementary Data 1). The marker showing the strongest association is the common intergenic variant rs2930191 [A] (MAF 37.22%).

At 12q24.13, the low-frequency variant chr12:110830276:S (MAF 2.09%) associates signicantly with lower LDH levels,

and suggestively with CK levels.

Finally, at 13q22.1, the common variant rs7318906 [A] (MAF 47.08%) associates with decreased CK levels.

We used GTEx Portals eQTL Browser to assess whether any of our reported variants affected gene expression. None of the 25 markers we discussed in the current study was directly reported or correlated (r240.2) to any GTEX cis-eqtl (Po1 105).

Signals observed specically in statin takers. We examined statin usage, stratifying our CK and LDH measurements by statin intake. Prescription data for statins were available for a period of time ranging from 2003 to 2009 and the stratied analysis therefore only included blood measurements from this interval. No additional signals were detected (Supplementary Figs 14). Our data showed no indication of the overall results being driven by individuals treated by statins, or any genetic susceptibility to statin side effects, for any of the loci in question (Supplementary Tables 8 and 9). Furthermore, the reported variant rs4363657 in SLCO1B1 showed no effect on any of the tested phenotypes (Supplementary Table 10).

Advantages of whole-genome sequencing. For the 25 reported variants, twenty were present in the 1000G data set (Supplementary Table 11). Of these, three could not be imputed, and a further three showed very low correlation (r2o0.64)

between the directly typed and the 1000G imputed phenotypes. Fourteen variants showed high correlation between directly typed

and imputed genotypes (r2Z0.8) and could therefore plausibly have been discovered using only 1000G imputation.

Five variants were not present in 1000G, but had correlates (r240.6) within the data set (Supplementary Table 12). Of these, four showed good correlation (r240.6) between imputed and correlated genotypes, and could, therefore, have shown association with CK or LDH levels in a study relying purely on a 1000G imputation data set.

We therefore report seven variants that could not have been discovered using a 1000G imputation set.

DiscussionElevation of serum enzyme levels can be a result of two separate processes; increased leakage from tissue into the serum, or reduced clearance54. Furthermore, observations of altered serum enzyme levels can result from a change in enzymatic activity. We report variants affecting serum enzyme levels through all three mechanisms.

Our discovery of a high number of genes linked to immune response is consistent with the MPSs role in clearing debris and foreign material from the bloodstream, including through receptor-mediated endocytosis of short-lived cellular enzymes55,56. Although the MPS has been implicated, the specic receptors responsible for uptake of serum enzymes have not yet been identied24. Nonspecic receptors, responsible for uptake of several enzymes, have been postulated24,57. We report a number of variants in genes expressed preferentially in phagocytic cells, notably hepatic KCs, which affect levels of serum enzymes. The protein products encoded by CD163 and STAB1, and LILRB5 are all scavenger receptors expressed by macrophages58,59, and could be the unidentied receptors responsible for endocytosis of serum enzymes by cells of the MPS.

Non-synonymous variants in ANO5 associates with serum CK levels, consistent with the fact that serum CK levels are largely inuenced by leakage of CK from damaged myocytes60.

The results of this study underscore the diversity in sources of variation of serum enzyme levels, each of which are inuenced by sequence variants, and must be kept in mind when interpreting the measurements.

Methods

Population. Measurements of serum CK levels were available for a total of 63,159 Icelanders. Of these, 35,623 were genotyped using Illumina chips and imputed using long-range-phased haplotypes. Genotype probabilities were calculated for 27,536 individuals based on genetic information available for rst and second-degree relatives. Measurements of serum LDH levels were available for a total of 98,585 individuals, 52,581 of whom were chip-typed using Illumina chips, and 46,004 of whom had genotype probabilities calculated. All individuals had provided consent, and the study was approved by the Data Protection Commission of Iceland and the Icelandic National Bioethics Committee.

Stratication by statin intake. CK and LDH measurements were stratied by statin intake. CK measurements were available for 8,900 statin takers (CKon statin 5,207; CKoff

statin

6,877), and LDH measurements were available for 9,851 statin takers (LDHon statin 6,266; LDHoff

statin

6,877), respectively, with an overlap of 7,534 individuals. These cohorts were used to test sequence variants for association with serum enzyme levels during and outside of times of statin use.

Serum enzyme measurements. Serum enzyme measurements were obtained from three laboratories in Iceland: The Laboratory of the Icelandic National University Hospital; The Icelandic Medical Center Laboratory in Mjodd; and Akureyri Hospital. We used measurements of serum CK (N 63,159, geometric

mean 211.1), and LDH (N 98,585, geometric mean 113.1). Additional

measurement characteristics can be found in Supplementary Table 13. Serum levels were adjusted for sex, age and laboratory of origin. When multiple measurements were available for an individual, the mean adjusted value was used.

Whole-genome sequencing and Illumina single-nucleotide polymorphism chip genotyping. The process used to whole-genome sequence the 2,636 Icelanders,

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and the subsequent imputation from which the data for this analysis were generated has been extensively described in a recent publication14.

Association analysis. All serum enzyme measurements were corrected for age, sexand laboratory of origin, and were subsequently standardized to have a normal distribution. To test for association between quantitative traits and sequence variants, a generalized form of linear regression was used (see Supplementary Note 1). The quantitative trait was used as the response variable and the expected allele count (gene dosage) as the covariate.

In the regression analysis of CK/LDH, we used genealogy information and assume a variance covariance matrix proportional to the kinship matrix (see Supplementary Note 1). To account for relatedness and stratication within the case and control sample sets, we applied the method of genomic control61. The ination factor lg of the w2 statistics was estimated on the basis of a set of about 300,000 common variants distributed across the genome, and P values were adjusted by dividing the corresponding w2 values by this factor. The genomic control was calculated to be l 1.16 for CK and 1.22 for LDH (Supplementary

Figs 34 and 35).

Signicance thresholds. Sequence variants were weighted according to their prior probability of affecting gene function. Thresholds for genome-wide signicance were applied, depending on variant class, as described by Sveinbjornsson et al.18. The type I error rate of 0.05 was allocated equally between three classes of variants; loss-of-function (N 6,476), missense (N 100,502) and other variants

(N 23,854,999). This yielded class-specic Bonferroni genome-wide signicance

thresholds of 2.6 10 6, 1.7 10 7 and 7.0 10 10, respectively.

Sanger sequencing and reimputation. Two sequence variants were poorly imputed due to a low number of sequenced carriers in the original Icelandic data set. A group of suspected carriers and non-carriers of the missese variants rs145987658 in CKM (info 0.63) and rs150956780 in STAB1 (info 0.58) were

Sanger sequenced, and reimputation was subsequently carried out. Imputation information following reimputation increased for both variants.

Fraction of variance explained. Fractions of CK and LDH variance explained by the reported variants were calculated using the formula 2f (1 f) a2, where

f MAF and a effect.

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Acknowledgements

We thank the individuals who participated in this study, and all colleagues who

contributed to collection of data, sample handling and genotyping.

Author contributions

R.P.K., P.S., U.T. and K.S. designed the study and interpreted the results. R.P.K., Hi.H.,

O.S., G.I.E., I.O., D.F.G. and P.S. carried out subject ascertainment and recruitment. As.J.,

Ad.J. and O.T.M. performed genotyping of samples. R.P.K., As.O., Ha.H., Ga.S., B.W.,

Ar.O., O.B.D., G.M., D.F.G. and P.S. performed statistical and bioinformatics analyses.

R.P.K., As.O., Ga.S., G.A.A., Ge.S., S.B., I.J., U.T., P.S. and K.S. drafted the manuscript.

All authors contributed to the nal version of the paper.

Additional information

Accession codes: European Variant Archive: PRJEB8636.

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