untitled


Common variants in the SLCO1B3 locus are
associated with bilirubin levels and unconjugated
hyperbilirubinemia

Serena Sanna1,{, Fabio Busonero1,{, Andrea Maschio1, Patrick F. McArdle2, Gianluca Usala1,

Mariano Dei1, Sandra Lai1, Antonella Mulas1, Maria Grazia Piras1, Lucia Perseu1, Marco Masala1,

Mara Marongiu1, Laura Crisponi1, Silvia Naitza1, Renzo Galanello3, Gonçalo R. Abecasis4,

Alan R. Shuldiner2,5, David Schlessinger6, Antonio Cao1 and Manuela Uda1,�

1Istituto di Neurogenetica e Neurofarmacologia del Consiglio Nazionale delle Ricerche, Monserrato, 09042 Cagliari,

Italy,
2
Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, Baltimore, MD,

USA, 3Clinica Pediatrica, Ospedale Regionale delle Microcitemie, Dipartimento di Scienze Biomediche e

Biotecnologie, Università degli Studi di Cagliari, 09121 Cagliari, Italy, 4Center for Statistical Genetics, Department of

Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA,
5
Veterans Administration Medical Center, Geriatric

Research and Education Clinical Center, Baltimore, MD, USA and
6
Laboratory of Genetics, National Institute on

Aging, Baltimore, MD, USA

Received February 5, 2009; Revised March 24, 2009; Accepted April 28, 2009

Bilirubin, resulting largely from the turnover of hemoglobin, is found in the plasma in two main forms: uncon-
jugated or conjugated with glucuronic acid. Unconjugated bilirubin is transported into hepatocytes. There, it
is glucuronidated by UGT1A1 and secreted into the bile canaliculi. We report a genome wide association scan
in 4300 Sardinian individuals for total serum bilirubin levels. In addition to the two known loci previously
involved in the regulation of bilirubin levels, UGT1A1 (P 5 6.2 3 10262) and G6PD (P 5 2.5 3 1028), we
observed a strong association on chromosome 12 within the SLCO1B3 gene (P 5 3.9 3 1029). Our findings
were replicated in an independent sample of 1860 Sardinians and in 832 subjects from the Old Order
Amish (combined P < 5 3 10214). We also show that SLC01B3 variants contribute to idiopathic mild unconju-
gated hyperbilirubinemia. Thus, SLC01B3 appears to be involved in the regulation of serum bilirubin levels in
healthy individuals and in some bilirubin-related disorders that are only partially explained by other known
gene variants.

INTRODUCTION

Bilirubin results from hemoglobin catabolism (and to a lesser
extent from other heme-containing proteins) within the
reticulo-endothelial system. Bilirubin is transported into
the liver, where specific carrier proteins belonging to the
Organic Anion Protein Transporter family (1) are thought to
mediate its uptake. In hepatocytes, bilirubin is glucuronidated
by (UDP)-glucuronosyltransferase and thereby transformed
into a water soluble conjugated molecule (2). Conjugated

bilirubin is actively secreted into the bile canaliculi by a
membrane ATP-dependent transporter, designated multidrug
resistance-associated protein 2 (MRP2) (3). In plasma, biliru-
bin occurs primarily unconjugated tightly bound to albumin,
or conjugated as a water-soluble glucuronide. High levels of
unconjugated bilirubin in plasma may indicate increased pro-
duction by hemolysis, ineffective erythropoiesis or decreased
conjugation, whereas conjugated hyperbilirubinemia reflects
decreased excretion by damaged biliary duct or hepatic
parenchymal cells.

†
S.S. and F.B. equally contributed to this work.

�
To whom correspondence should be address. Tel: þ39 0706754591; Fax: þ39 0706754652; Email: manuela.uda@inn.cnr.it

# The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org

Human Molecular Genetics, 2009, Vol. 18, No. 14 2711–2718
doi:10.1093/hmg/ddp203
Advance Access published on May 6, 2009



RESULTS

Within the SardiNIA study we conducted a genome-wide
association (GWA) analysis (4,5) in 4300 Sardinians to ident-
ify genetic factors affecting a series of quantitative traits,
including serum bilirubin levels (see Material and Methods).
The strongest associations were observed on chromosome 2,
in the promoter of UGT1A1 (uridine diphosphoglucuronyl-
transferase) gene (rs887829, P ¼ 6.21 � 10

262
, Fig. 1 and

Supplementary Material, Table S1), a major and well-known
modifier of bilirubin levels (6), and on the X chromosome
near the G6PD (glucose-6-phosphatedehydrogenase) gene
(rs766420, P ¼ 9.40 � 10

209
, Fig. 1 and Supplementary

Material, Table S1).
In addition, a third locus on chromosome 12p12.2 reached

genome-wide significance (Fig. 1). The strongest P-value was
observed at SNP rs2117032 (P ¼ 4.74 � 10

208
, Supplementary

Material, Table S1) downstream of the 30-UTR of the SLCO1B3
(solute carrier organic anion transporter family, member 1B3)
gene (7) (Fig. 2); each copy of the minor allele C was respon-
sible, on average, for an increase of 0.048 mg/dl in bilirubin
levels. The second ranked marker in this region was
rs17680137 (P ¼ 2.3 � 10

27
), located in intron 7 of

SLCO1B3, in modest LD with rs2117032 (r
2
¼ 0.29), and

responsible for a slightly larger effect (0.067 mg/dl per copy
of the minor allele G). Our genotype strategy was designed to
take advantage of the relatedness of the sample. In fact we gen-
otyped first a small number of markers (10K GeneChip) in
largest families to identify shared stretches of chromosome
between individuals, and then genotyped with a denser map
(the 500K chip) a subset of individuals, selected to be dispersed
along the known pedigrees with an overlap of 436 individuals
genotyped with both platforms. Next a within-family imputa-

tion method was used to predict missing genotypes (see Material
and Methods). The two markers, rs17680137 and rs2117032,
belong to the 500 K SNP array and 10 K SNP array, respect-
ively, and thus were imputed the first in the majority of the
samples, the second in only one-fourth. To evaluate the effect
of the different number of genotyped and imputed samples,
we directly genotyped both markers in individuals for whom
only imputed genotypes were available. Using all available gen-
otypes, the association test showed SNP rs17680137 as more
representative of total bilirubin variation (P ¼ 3.9 � 10

29
, see

Table 1 and Supplementary Material, Table S1). Interestingly,
when including this marker in the model, SNP rs2117032 still
showed some evidence for association (P ¼ 0.00050). Further-
more, we observed a significant interaction between the two
markers (P ¼ 1.28 � 10

204
). Using all available genotypes,

we were able to infer the complete haplotypes of 3431 individuals
for which the wet-genotype for both markers were available and
we observed that only haplotypes carrying both positive or both
negative alleles were significant (HGC P ¼ 1.8 � 10

207
and HCT

P ¼ 2.1 � 10
207

, see Table 2). Notably, we observed that the
haplotype frequencies were similar to the minor allele frequency
of the two variants (HGC freq ¼ 0.26 and HCT freq ¼ 0.43), and
that each haplotype was highly correlated with one variant (HGC
and rs17680137 cor ¼ 0.97, HCT and rs2117032 cor ¼ 0.98)
(Table 2). Those results were also replicated in the Amish popu-
lation, where the only two haplotypes associated with bilirubin
levels were those carrying both positives or both negatives
alleles. Each haplotype was highly correlated with one of the
two markers (Table 2). Thus the two SNPs, rather than having
independent effects, are likely imperfectly tagging a third
common variant of which the allele responsible for an increase
in bilirubin levels would be probably associated with the HGC
haplotype, and the other allele with the HCT haplotype.

Figure 1. Genome-wide scan of serum total bilirubin. The figure summarizes association P-values (additive test) for all SNPs that passed quality control tests
(N ¼ 362,129). Position of UGT1A, SLCO1B3 and G6PD genes are annotated. Red dotted line marks Bonferroni threshold (1.3 � 10

27
).

2712 Human Molecular Genetics, 2009, Vol. 18, No. 14



We also evaluated the effect of possible confounding factors
related to the particular hematological features of the Sardi-
nian population, and repeated the association tests including
as covariates Mean Corpuscular Hemoglobin, Mean Corpus-
cular Volume, total Hemoglobin, b-thalassemia carrier status
and G6PD levels (8,9). None of those factors affected the
association at the three loci. As expected, adjustment for
G6PD levels reduced the association at the G6PD locus, some-
what strengthening the UGT1A1 signal (Supplementary
Material, Table S2). In addition, we observed that the associ-
ation of UGT1A1 and SLCO1B3 appeared stronger in women
than in men, although we found no significant difference in the
magnitude of the effect between sexes. In contrast, the associ-
ation with the X-linked G6PD locus was stronger in men, most
likely because of the major impact of G6PD deficiency in
males. In fact, there was no difference in association
between G6PD genotype and bilirubin when G6PD level
was taken into account (Supplementary Material, Table S3).
Finally, none of the tests for epistasis between the top SNPs
at the three loci was significant after correction for multiple
testing (Supplementary Material, Table S4). Additional
studies are required to confirm this observation.

Our findings at the SLCO1B3 locus were successfully repli-
cated in an independent sample of 1860 Sardinians, previously
described and reported as SardiNIA stage 2 (10), with an
effect in the same direction as that observed in the GWA
sample (P , 2 � 10

204
, Table 1). Moreover, we also repli-

cated the association in 832 individuals enrolled in the
Amish Heredity and Phenotype Intervention (HAPI) Heart
study (11) (P , 7 � 10

203
, Table 1), suggesting that variants

in the SLCO1B3 influence bilirubin levels not only in Sardi-
nians but also in other populations of Caucasian origin. Com-
bined, the evidence for association at this locus was P , 5 �
10

214
(Table 1).

As shown in Fig. 2, although our results point to the
SLCO1B3 gene, weaker association encompasses a genomic
region of 700 Kb harboring, in addition to SLCO1B3, four
more genes in the same family of membrane transport pro-
teins. Among them, SLCO1B1 (12) has been previously impli-
cated in bilirubin liver uptake (13) and associated with
bilirubin levels in Asian populations. In this gene two
coding variants, namely Val174Ala/rs4149056 and
Asp130Asn/rs2306283, have been described as major determi-
nants of bilirubin variation (14,15), and they weakly correlated
with rs17680137 (r

2
¼ 0.008 and r

2
¼ 0.359, respectively)

and with rs2117032 (r
2
¼ 0.031 and r

2
¼ 0.302, respectively).

To compare their effects with polymorphisms at SLCO1B3 we
used inferred genotypes based on HapMap (16) CEU (Utah
residents with ancestry from northern and western Europe)
haplotypes available from previous studies (17), and tested for
association with total, conjugated and unconjugated bilirubin.
Only nominal association with total bilirubin was detected
at these coding variants (P ¼ 0.079 and P ¼ 3.6 � 10

203
,

Table 3). In addition, when including rs4149056 and

Figure 2. Evidence of (A) association with serum total bilirubin and (B) linkage disequilibrium around the SLCO1B3 locus. (A) All SNPs in the SLCO1B3 region
(700 Kb) are plotted with association P-values (additive test) compared with physical position. Genomic position and gene annotations refer NCBI Build 36 map.
SNPs are colored according to their linkage disequilibrium with the top variant (rs17680137). Blue line represents the recombination rate (in cM/Mb). Black
diamonds represent combined P-values from SardiNIA and SardiNIA stage 2 cohorts. (B) Patterns of linkage disequilibrium (r

2
) for the CEU HapMap popu-

lation (Utah residents with European ancestry (16)) are plotted and colored as indicated in the palette.

Human Molecular Genetics, 2009, Vol. 18, No. 14 2713



rs2306283 as covariates in the model, SNPs at the SLCO1B3
locus still remain highly significant (Supplementary Material,
Table S5), supporting the hypothesis that the association signal
observed in the SLCO1B3 is not modulated by an indirect effect
of these coding variants. Furthermore, since the SNPs tested in
the GWAS covered 66% of the common variants in the
SLCO1B1 at r

2
. 0.5, it is unlikely that other SNPs in this

gene may have a stronger effect than those in the SLCO1B3
locus. The differential contribution of the two transporters
may be owing to population-specific genetic features. In
Asians, for example, SNP rs17680137 is monomorphic.

Interestingly, we observed that SNPs in SLCO1B1 were
mostly associated with conjugated bilirubin (P ¼ 1.7 �
10

204
for rs4149056), whereas no association was observed

with unconjugated bilirubin (P ¼ 0.26, Table 3). In contrast,
SNP rs17680137 in SLCO1B3 showed a stronger association
with unconjugated bilirubin (P ¼ 5.2 � 10

209
) compared

with conjugated bilirubin (P ¼ 0.0004). A similar pattern of

association was observed in the SardiNIA stage 2 sample
(Table 3), where SNP rs4149056 was directly genotyped.
Again, the association with unconjugated bilirubin at SNPs
rs17680137 and rs2117032 remained significant after account-
ing for the two SNPs in the SLCO1B1 gene (Supplementary
Material, Table S5).

Conjugated bilirubin was not available in the Amish cohort,
and thus a parallel analysis could not be performed.

We next tested for any effect of the UGT1A1 gene on the
association of bilirubin levels with the SLCO1B3 gene. Includ-
ing SNP rs887829, the top marker on the UGT1A1 locus in the
model, the association at rs2117032 and rs17680137 did not
disappear but rather increased (data not shown), suggesting
that SLCO1B3 explains part of the variability not accounted
for by UGT1A1 polymorphisms. To explore this possibility
further, we evaluated whether this locus may be responsible
for idiopathic mild hyperbilirubinemia in patients lacking
mutations in the UGT1A1 gene. Hence, we genotyped 1004

Table 1. Summary of association results for SNPs in theSLCO1B3 locus in all cohorts

Marker Study N Allele High/Low Freq Effect (StdErr) Effect (StdErr) mg/dl P-value

rs17680137 Stage 1
SardiNIA 4300 G/C 0.293 0.170 (0.029) 0.067 (0.011) 3.9 � 10

209

Stage 2
SardiNIA stage2 1860 G/C 0.273 0.143 (0.037) 0.044 (0.014) 1.2 � 10

204

Old Older Amish 832 G/C 0.229 0.171 (0.062) 0.040 (0.020) 6.1 � 10
203

Overall 6992 4.41 � 10
214

rs2117032 Stage 1
SardiNIA 4300 C/T 0.466 0.129 (0.023) 0.048 (0.009) 3.8 � 10

208

Stage 2
SardiNIA stage2 1860 C/T 0.485 0.134 (0.033) 0.040 (0.012) 5.0 � 10

205

Old Older Amish
a

838 C/T 0.415 0.170 (0.051) 0.034 (0.016) 5.2 � 10
204

Overall 6998 2.91 � 10
214

The tables summarize the association results in all cohorts. For each marker, frequency and effect estimates are given with respect to the ‘high frequency’
allele. Association parameters (Effect, StdErr, P-value) are relative to the model where the trait is transformed, thus the effect estimated in this model
represents the change in standard deviation units. Effect in mg/dl is also given for each marker. SardiNIA results are relative to the imputed genotypes
inferred from Affymetrix and TaqMan genotypes.
a
Old Older Amish sample refers to SNP rs10770763, r

2
¼ 0.96 with rs2117032 in CEU HapMap population, for which additional 6 genotyped individuals were

available.

Table 2. Association analysis of haplotypes resulting from all possible combinations of alleles at markers rs17680137 and rs2117032

Haplotype rs17680137 rs2117032
a

Freq Effect
b

StdErr P-value Correlation
c

with
rs17680137 rs2117032

a

SardiNIA
HCC C C 0.294 0.022 0.031 0.49 0.38 0.56

HCT C T 0.434 20.143 0.027 2.10 � 10
207

0.59 0.98
HGC G C 0.262 0.164 0.031 1.84 � 10

207
0.97 0.58

HGT G T 0.010 0.018 0.133 0.893 0.17 0.09

Old Older Amish
HCC C C 0.202 0.058 0.067 0.398 0.23 0.60

HCT C T 0.590 20.150 0.055 0.007 0.64 1.00
HGC G C 0.208 0.162 0.068 0.016 1.00 0.64

HGT G T 0.000 – – – – –

a
For the Amish sample, rs10770763 (T/C alleles) was genotyped instead of rs2117032 (C/T alleles).

b
Effect size and Standard Error are given in standard deviation units.

c
Correlation is given in absolute value.

2714 Human Molecular Genetics, 2009, Vol. 18, No. 14



healthy individuals and 261 unrelated patients with hyperbilir-
ubinemia (defined as serum total bilirubin .1.2 mg/dl) (2)
from the SardiNIA sample for the mutation in the TATAA
element of the 50 promoter region of the UGT1A1 gene (2).
We observed a 5% enrichment in the frequency of the G
allele of SNP rs17680137 in the hyperbilirubinemic patients
compared with the healthy subgroup (P ¼ 0.029; Table 4).
Notably, most of the difference was attributable to patients
homozygous for the wild-type allele, A(TA)6TAA at the
UGT1A locus (N ¼ 95, P ¼ 0.031), rather than to patients car-
rying at least one copy of the mutated allele A(TA)7TAA
(N ¼ 166, P ¼ 0.053) (2). Thus one can infer that SLCO1B3
not only acts in the general population, but also affects mild
hyperbilirubinemias.

DISCUSSION

GWA analysis for serum bilirubin levels on 4300 Sardinian
revealed two striking signals in the UGT1A1 and the G6PD
genes. The association with UGT1A1 is well known. In fact,

mutations in this gene are responsible for the Crigler – Najjar
syndrome as well as the more common mild unconjugated
hyperbilirubinemia of Gilbert syndrome (2). The association
of G6PD with bilirubin levels was also expected, given the
high frequency of deficiency in G6PD in the Sardinian popu-
lation; this condition is indeed associated with a shorter life
span of red blood cells, resulting in a modest increase in bilir-
ubin levels (18). Furthermore, the results revealed an
additional locus on chromosome 2p12.2. The strongest associ-
ated marker in this region, an intronic SNP of the SLCO1B3
gene, was successfully replicated in an independent Sardinian
sample and in a cohort of Amish individuals. Notably, this
gene is highly expressed in the basolateral membrane of hep-
atocytes and is a member of the OATP family encoding the
Organic Anion Transporter Polypeptide Na

þ
-independent,

OATP1B3 (7). The OATP proteins are expressed in a
variety of tissues, including gut, brain, kidney and liver
where they play important roles in drug absorption and distri-
bution and excretion, with a wide spectrum of substrates that
include both endogenous and xenobiotic compounds (1).
Thus SLCO1B3 is a plausible candidate gene responsible for

Table 4. Association of SLCO1B3 with hyperbilirubinemia

N C/C C/G G/G C G Genotypic test P-value Allelic test P-value

Healthy 1004 0.526 0.405 0.069 0.729 0.271
Hyper-bilirubinemia 261 0.446 0.466 0.088 0.679 0.321 0.06 0.029

Hyper (TA6/TA6) 95 0.379 0.547 0.074 0.652 0.348 0.019 0.031

Hyper (TA6/TA7, TA7/TA7) 166 0.488 0.416 0.096 0.695 0.305 0.095 0.053

The table describes the allele and genotype frequencies in a data set of unrelated healthy (total bilirubin ,1 mg/dl) and hyperbilirubinemia patients,
screened for the UGT1A1 A(TA)7TAA mutation. Hyperbilirubinemia was defined as total bilirubin .1.2 mg/dl. P-value ,0.05 are highlighted in bold.

Table 3. Association with total, conjugated and unconjugated bilirubin of SNPs in the SLCO1B3 and SLCO1B1 genes

Locus Marker Study Freq Total bilirubin Conjugated bilirubin Unconjugated bilirubin
Effect (StdErr) P-value Effect (StdErr) P-value Effect (StdErr) P-value

SLCO1B1 rs4149056 (C) SardiNIA
a

0.167 0.070 (0.040) 0.079 0.147 (0.039) 1.7 � 10
204

0.045 (0.040) 0.26
SardiNIA stage2 0.184 0.063 (0.043) 0.14 0.104 (0.042) 0.014 0.056 (0.043) 0.19
Old Order Amish

b
0.103 0.255 (0.086) 3.08 � 10

203
– Na – Na

pCombined 0.0015 7.2 � 10
206

0.095

SLCO1B1 rs2306283 (G) SardiNIA
a

0.485 0.090 (0.030) 3.6 � 10
203

0.100 (0.03) 7.6 � 10
204

0.086 (0.031) 0.0056
SardiNIA stage2 – – Na – Na – Na
Old Order Amish

c
0.319 0.031 (0.054) 0.563 – Na – Na
pCombined 0.0039 7.6 � 10

204
0.0056

SLCO1B3 rs17680137 (G) SardiNIA 0.293 0.170 (0.029) 3.9 � 10
209

0.100 (0.028) 0.0004 0.170 (0.029) 5.2 � 10
209

SardiNIA stage2 0.273 0.144 (0.037) 1.2 � 10
204

0.107 (0.037) 0.0037 0.144 (0.037) 1.2 � 10
204

Old Order Amish 0.219 0.171 (0.062) 6.1 � 10
203

– Na – Na
pCombined 4.41 � 10

214
5.3 � 10

206
2.7 � 10

212

The table summarizes association results with total, conjugated and unconjugated bilirubin for SNP rs4149056 and SNP rs2306283, polymorphisms in
the SLCO1B1 gene previously known to be associated with neonatal hyperbilirubinemia. They are coding mutations, with the following aminoacid
changes: Val174Ala and Asp130Asn, respectively. Effect size and Standard Error are given in standard deviation units. Conjugated and Unconjugated
bilirubin were not available in the Amish cohort.
a
SNPs rs4149056 and rs2306283 were imputed in all SardiNIA samples based on HapMap CEU haplotypes (see Material and Methods). Quality of imputation was

r
2
¼ 0.938 and r

2
¼ 0.929, respectively.

b
SNP rs4149056 was not genotyped in the Amish cohort, and rs11045879 (r

2
¼ 0.86) was analyzed as best proxy among genotyped SNPs.

c
SNP rs2306283 was not genotyped in the Amish cohort, and rs2291075 (r

2
¼ 0.86) was analyzed as best proxy among genotyped SNPs.

Human Molecular Genetics, 2009, Vol. 18, No. 14 2715



changes in bilirubin levels. Interestingly, another solute carrier
organic anion transporter, SLCO1B1, previously implicated in
liver bilirubin uptake (13), maps in the same region, but only
weaker association was observed at this locus. In particular,
we found that SNPs at SLCO1B1 were mostly associated
with conjugated bilirubin, although no association was
detected with unconjugated bilirubin. Although we cannot
exclude the possibility of a differential contribution of the
two transporters in a population-specific fashion, the results
produced in this study indicate that although the SLCO1B3
gene regulates unconjugated serum bilirubin, SLCO1B1
appears to act on conjugated bilirubin. SLCO1B1, with an
high affinity for glucuronated bilirubin (19), may contribute
(together with MPR3) to reverse transport from hepatocytes
to plasma in normal conditions, and very likely also in
cholestasis when the major export pump MPR2 is down-
regulated (20).

Further studies are necessary to identify the SLCO1B3-
specific causative variants and then to clarify the functional
role of both OATP genes on conjugated and unconjugated
bilirubin. Finally, the SLCO1B3 SNPs identified here were
also found to be associated with unconjugated hyperbilirubine-
mia of unknown origin. Speculatively, they may also act as
modifiers of the clinical course of some phenotypes, as
already shown for UGT1A1 polymorphisms in hemolytic
anemia, and anemia resulting from ineffective erythropoiesis,
and neonatal jaundice (8,9,21). Thus, our findings not only
reveal an additional regulator of bilirubin levels in the
general population but may also contribute to the identification
of genetic variants necessary to predict and prevent severe
clinical manifestations in hematological and hepatic disorders
linked to hyperbilirubinemia.

MATERIAL AND METHODS

Sample description

We recruited and phenotyped 6148 individuals, males and
females, ages 14 – 102 years, from a cluster of four towns in
the Ogliastra province of Sardinia (4). During physical exam-
ination, a blood sample was collected from each individual,
and divided into two aliquots. One aliquot was used for
genomic DNA extraction and the second (7 ml peripheral
blood without anticoagulant) to characterize several blood
phenotypes, including evaluation of serum total and conju-
gated bilirubin levels, measured with Express Plus (Bayer)
equipment, according to manufacturer’s instruction.

The normality range was 0.2 – 1.0 and 0 – 0.2 mg/dl for total
and conjugated bilirubin, respectively. Hyperbilirubinemia
was defined when total bilirubin serum concentration
exceeded 1.2 mg/dl (2).

GWAS genotyping

We genotyped from the whole sample 4305 individuals
selected to represent the largest available families, regardless
of their phenotypic values (5). Specifically, 1412 were geno-
typed with the 500K Affymetrix Mapping Array Set and
3,329 with the 10K Mapping Array Set, with 436 individuals
genotyped with both arrays. This genotyping strategy allowed

us to examine the majority of our cohort in a cost-effective
manner because genotypes for the SNPs that passed quality-
control checks could be propagated through the pedigree via
imputation (22,23). Bilirubin measurements were available
for 4300 individuals among the 4305 genotyped. A total of
362 129 SNPs passed initial quality-control checks (5) and
were tested for association. Although the statistical analysis
was ongoing, an additional 2 million autosomal markers
from HapMap imputed data became available. Repeating the
GWA analysis using this larger data set of markers detected
no additional loci, and the top markers at chromosomes 2
and 12 loci were confirmed. Further details of the strategy
for imputation and data analysis are given in the Statistical
Analysis section below.

Replication

We designed a ParAllele Custom Chip (from Affymetrix) to
replicate the regions associated with bilirubin levels as well
as other traits studied in the SardiNIA Project. Genotyping
was performed in 1862 Sardinian, selected within the Sardi-
NIA sample and independent from the individuals included
in the GWA scan (kinship coefficient ¼ 0 with those directly
typed with the 500K chip), and indicated here as SardiNIA
stage 2 data set (17). SNPs rs17680137 and rs2117032, were
included in the ParAllele Custom chip, and also genotyped
in additional individuals using TaqMan SNP genotyping
assay (Applied Biosystems) according to the protocol. The
concordance rate between inferred and wet genotypes was
74.7 and 93.4% for rs17680137 and rs2117032 (over N ¼
1460 and N ¼ 202 samples), respectively. SNP rs4149056,
not included in the custom chip was genotyped in the
SardiNIA stage 2 data set using TaqMan technology.
Mutations in the TATAA element of the 50 promoter
region of the UGT1A1 gene were analyzed by direct sequen-
cing (ABI-PRISM 377; Applied Biosystems, USA) of
genomic DNA.

Statistical analysis

For individuals genotyped with a sparse map, we used a modi-
fied version of the Lander – Green algorithm (23) to estimate
IBD sharing at the location of the SNPs being tested and ident-
ify stretches of haplotype shared with close relatives who were
genotyped at higher density and probabilistically infer missing
genotypes (22). Our inference approach allowed us to account
for uncertainty in genotype assignment, estimating, instead of
the most likely genotype, an expected genotype score, repre-
senting the expected number of copies of a reference allele
(a fractional number between 0 and 2). The genotype scores
were then used in the family-based association test for evalu-
ating the additive effect of each marker, as described else-
where (22,24). Owing to computational constraints, we
divided large pedigrees into sub-units with ‘bit-complexity’
of 19 or less (typically, 20 – 25 individuals) before all the ana-
lyses. The association test fits a simple regression model and
uses a variance component approach to account for correlation
between different observed phenotypes within each family. To
evaluate association on the X chromosome, we modeled
a polygenic variance component shared according to an

2716 Human Molecular Genetics, 2009, Vol. 18, No. 14



X-linked kinship coefficient in addition to the usual autosomal
polygenic variance component (4,24). Prior to all analyses,
traits were normalized using quantile normalization to avoid
inflation of type I error rates; gender, age and age squared
were included as covariates.

In the SardiNIA stage 2 the association with bilirubin levels
was tested as in the SardiNIA GWA sample. To infer haplo-
types we used all available genotypes and Merlin software.
We created a variable for each haplotype coded as 0, 1 or 2,
representing the number of copies carried by an individual.
A linear regression model was fitted for each haplotype. Epis-
tasis and haplotype-specific tests were performed in the GWA
sample using the R kinship package.

Old order Amish

The HAPI Heart Study was initiated in 2002. Participants of
the HAPI Heart Study comprised adults from the Old Order
Amish community of Lancaster County, PA, who were
recruited over a 3-year period. The study aims and recruitment
details, including ascertainment criteria, have been described
previously (11). Physical examinations were conducted at
the Amish Research Clinic in Strasburg, PA. A blood
sample was obtained for measurement of bilirubin levels. Bio-
chemical assays were performed by Quest Diagnostics
(Horsham, PA, USA).

Genotypes for SNPs rs17680137, rs10770763 (proxy of
rs2117032; r

2
¼ 0.96), rs11045879 (proxy of rs4149056;

r2 ¼ 0.86) and rs2291075 (proxy of rs2306283; r
2
¼ 0.86),

were obtained from a previous GWAS performed using the
500K Affymetrix Mapping Array Set.

Statistical analysis was performed using in house developed
software. In brief, we performed a measured genotype
approach utilizing a t-test of the beta coefficient for the SNP
variable. We included sex, age and age squared as fixed cov-
ariates in the model and a polygenic component modeled as a
random effect to account for the full 14 generation pedigree of
the Amish. The bilirubin trait was normalized using quantile
normalization to avoid inflation of type I error rates; gender,
age and age squared were included as covariates, as in the Sar-
diNIA GWA sample.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We thank the many individuals who generously participated in
this study, Monsignore Piseddu, Bishop of Ogliastra, the
Mayors and citizens of the Sardinian towns (Lanusei, Ilbono,
Arzana and Elini), and the head of the Public Health Unit
ASL4, the province of Ogliastra for their volunteerism and
cooperation. In addition, we are grateful to the Mayor and
the administration in Lanusei for providing and furnishing
the clinic site. We thank the team of physicians Angelo
Scuteri, Marco Orrù, Maria Grazia Pilia, Maria Cristina
Spada, Danilo Fois, Liana Ferreli, Francesco Loi, and nurses
Paola Loi, Monica Lai and Anna Cau who carried out

participant physical exams, and Monica Balloi who recruited
the volunteers.

Conflict of Interest statement. None declared.

FUNDING

The work was supported by the following National Institutes
of Health grants: HL084729 to G.R.A., HG002651 to
G.R.A., National Institute of Aging contracts
NO1-AG-1-2109 to the SardiNIA (‘ProgeNIA’) team and
263-MA-410953 to the University of Michigan (G.R.A).The
Old Order Amish study was supported by National Institutes
of Health research grant U01 HL72515, the University of
Maryland General Clinical Research Center, grant M01 RR
16500, the Clinical Nutrition Research Unit of Maryland
P30 DK072488, and the Baltimore Veterans Administration
Medical Center Geriatrics Research.

AUTHOR CONTRIBUTIONS

G.R.A, D.S., A.C., A.R.S. and M.U. designed the study; R.G.,
A.S., D.S. and M.U. provided material and reagents; F.B.,
A.M., G.U., M.D., S.L. and A.M. performed the genotyping;
M.G.P., L.P., M.M., M.M., A.R S. performed characterization
of phenotype and data collection; S.S., P.F.M., analyzed the
data; S.N., L.C., F.B., A.R.S., P.F.M. contributed to draft the
manuscript; S.S., D.S., A.C. and M.U. wrote the paper.

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