Complement factor 5 is a quantitative trait gene thatmodifies liver fibrogenesis in mice and humansSonja Hillebrandt1,2, Hermann E Wasmuth1, Ralf Weiskirchen3, Claus Hellerbrand4, Hildegard Keppeler1,2,Alexa Werth1, Ramin Schirin-Sokhan1, Gabriele Wilkens1, Andreas Geier1, Johann Lorenzen5, Jorg Kohl6,Axel M Gressner3, Siegfried Matern1 & Frank Lammert1,2

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative traitlocus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis withrecombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small moleculeinhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human geneC5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to theidentification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal rolein fibrogenesis across species.

Chronic liver diseases, irrespective of etiology, lead to hepatic fibrosisand cirrhosis and are a main cause of morbidity and mortality. Inparticular, chronic hepatitis C virus (HCV) infection affects more than170 million individuals and causes 300,000 deaths annually due tocirrhosis and hepatocellular carcinoma1. Despite antiviral therapies,25% of individuals with chronic HCV infection are likely to developadvanced liver fibrosis within 20 years2. Therefore, current therapeuticstrategies emphasize approaches that reduce fibrogenic liver injury.The marked variability in progression of fibrosis has been attributed toage, gender and environmental factors3. Host genetic factors may alsobe important, but basic research to clarify the role of genetic variantsin liver fibrosis is lacking4. Because such analysis in humans is

hampered by the complex genetics of infectious liver diseases, ourearlier study5 used different genetic susceptibilities for liver fibrosis ininbred mouse strains6 to identify quantitative trait loci (QTLs) forhepatic fibrosis (Hfib loci).

A fibrogenic QTL is a genetic locus whose alleles affect phenotypicvariation in liver fibrosis7. To identify QTLs, a segregating experi-mental cross of inbred mouse strains is phenotyped for fibrosis andthen genotyped for markers that differ between the parental strainsand are distributed evenly throughout the entire genome. In general,QTLs are mapped by statistically locating the genomic regions inwhich the degree of genotypic similarity correlates maximally with thedegree of phenotypic similarity among the progeny7. Here we identify

Table 1 QTL analysis of associations between genetic markers on mouse chromosome 2 and liver fibrosis phenotypes

Genetic marker Position (cM) lod score (collagen)a P (collagen) lod score (stage)a P (stage)

D2Mit6 12.5b 1.9* 0.00310 1.6* 0.00666

Hc 20.9 3.4** 0.00008 2.3* 0.00129

D2Mit238 21.9 3.0* 0.00020 2.0* 0.00221

D2Mit90 37.0 2.2* 0.00143 1.5 0.00755

D2Mit185 55.5 1.8* 0.00378 0.1 0.46505

D2Mit399 65.5 1.7* 0.00448 0.5 0.15128

D2Mit113 93.7 0.3 0.26250 0.1 0.55244

Hepatic collagen concentration or histological stage of fibrosis was examined in 629 (A/J � BALB/cJ) � (A/J � BALB/cJ) progeny.aAccording to permutation testing10, lod scores of Z3.2 and Z1.5 indicate highly significant (**) and significant (*) results, respectively. bThe position of the most proximal marker (D2Mit6) wasobtained from the Mouse Genome Database; all other positions were determined in the experimental cross.

Published online 3 July 2005; doi:10.1038/ng1599

1Department of Medicine III, University Hospital Aachen, Aachen University, Aachen, Germany. 2Department of Internal Medicine I, University Hospital Bonn, BonnerForum Biomedizin, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany. 3Institute of Clinical Chemistry and Pathobiochemistry, University HospitalAachen, Aachen University, Aachen, Germany. 4Department of Medicine I, University Hospital Regensburg, University of Regensburg, Regensburg, Germany. 5Instituteof Pathology, University Hospital Aachen, Aachen University, Aachen, Germany. 6Division of Molecular Immunology, Cincinnati Children’s Hospital ResearchFoundation, Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio, USA. Correspondence should be addressed to F.L. (frank.lammert@ukb.uni-bonn.de).

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complement factor 5 as quantitative trait gene underlying the Hfib2locus on chromosome 2 and confirm the genetic association betweenC5 and progressive liver fibrosis in humans. This study uncovers agene that contributes to liver fibrogenesis in mice and humans anddissects a new pathogenetic pathway with potential relevance forantifibrotic strategies.

RESULTSMapping of the fibrogenic QTL on mouse chromosome 2 (Hfib2 )For this study of mouse QTLs for liver fibrosis, we extended ourprevious QTL analysis5 in an experimental intercross between fibrosis-susceptible BALB/cJ and fibrosis-resistant A/J inbred strains. Wegenotyped 629 intercross progeny for seven polymorphic markerson chromosome 2 and then phenotyped all the progeny that wererecombinant for these markers after inducing liver fibrosis with CCl4.Regression analysis showed that a QTL on proximal chromosome 2substantially affected histological stages of liver fibrosis and hepaticcollagen concentrations (Table 1).

To refine the QTL, we used in silico mapping with a dense SNPmap8 and the known phenotypes of the seven inbred mouse strainsthat we used to identify the critical region on chromosome 2 (ref. 5) asinput quantitative data. Using haplotypes (i.e., unique combinationsof marker alleles present on the same chromosome) better covers thecomplex genomic variation between multiple strains and increases thestatistical power of the analysis8. We inferred haplotypes betweengenetic markers D2Mit6 and D2Mit90 (Table 1) from three-SNPwindows with an average size of 900 kb (ref. 8) and calculated an Fstatistic at each haplotype on the basis of the genotype-phenotypepairings. The chromosomal segment with the most significant asso-ciation between the distribution pattern of haplotypes and hepaticcollagen concentrations (F ¼ 37.2, degrees of freedom (d.f.) ¼ 1,50,P o 0.00001, ANOVA) is defined by SNPs at 30.4 Mb and 35.0 Mb,thus reducing the original 44.7-Mb region between D2Mit6 andD2Mit90 by 90% (Fig. 1). The refined QTL separates into two long-range haplotype blocks, which carry the hemolytic complement gene(Hc) encoding complement factor 5 (C5) at the distal end.

Analysis of liver fibrosis in inbred and congenic miceThe role of Hc as a candidate for underlying the QTL on chromosome2 is substantiated by the fact that some inbred mouse strains (includ-ing A/J) carry a 2-bp deletion in exon 6 that results in C5 deficiency9.We ascertained the Hc genotypes of the seven inbred strains used forin silico mapping (Fig. 1) and verified the association with fibrosisphenotypes. After treatment with CCl4, Hc�/� strains (A/J, AKR/J,DBA/2, FVB/NJ) had significantly (P o 0.01) lower stages of fibrosis(1.40 7 0.15) than did Hc+/+ strains (BALB/c, C3H/HeJ, C57BL/6J;2.43 7 0.24; Fig. 2a). Both at baseline and after CCl4 challenge for6 weeks, Hc�/� strains also had significantly (P o 0.01) lower hepaticcollagen concentrations (322 7 43 mg hydroxyproline per g liver) than

65,597,13065,484,88265,266,11865,057,68764,591,73964,329,35964,051,47963,511,48963,034,35562,584,13862,370,87861,738,71961,307,30961,040,30660,767,08560,697,76360,586,96360,379,75560,121,66559,956,87259,739,39259,528,65459,165,05058,849,65358,449,03258,084,39457,962,72657,740,33557,268,56457,081,63356,428,81956,134,10755,886,12355,687,13855,367,90755,005,562

54,251,95554,397,390

54,069,83453,801,86153,331,57153,143,60052,773,59352,654,66052,535,24452,393,65852,205,28152,097,39351,885,35851,664,38851,505,37450,913,22450,749,85750,149,58649,839,94549,610,69349,495,25049,219,19248,864,65448,618,64448,522,56647,769,86947,124,60346,953,30446,724,44546,031,10845,839,97145,653,21845,289,33745,152,28244,717,79844,578,18344,361,76543,937,98643,642,82143,404,55143,132,63142,084,66141,930,29741,244,14441,071,03440,887,96140,588,36940,417,35439,073,46038,824,43738,486,85838,126,85737,711,24137,702,36537,389,66237,107,10936,893,30436,383,33536,087,17335,357,11435,213,30634,973,62834,583,45234,361,43034,094,76133,986,97233,902,86933,787,72033,587,80633,409,95833,178,32932,946,51032,921,63132,737,19532,574,93332,193,72331,782,68230,645,933

30,147,85030,400,990

29,861,42429,738,88229,460,73229,205,70528,839,55728,429,60228,249,16028,101,33327,291,14426,870,63926,603,67426,442,96426,233,60326,055,54325,831,19925,479,18524,931,77624,436,38724,169,22723,399,36123,028,94322,810,56022,452,07421,675,61321,499,31621,269,09820,904,267

D2Mit90

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16.616.616.616.616.616.616.616.616.616.616.616.616.616.616.616.616.616.616.616.6

8.88.86.17.38.58.5

16.68.58.58.58.58.57.33.03.03.03.03.03.08.58.58.5

16.616.616.616.616.616.616.6

8.58.58.58.57.37.37.37.38.58.5

16.616.616.616.616.616.616.616.616.616.616.616.616.616.6

9.58.1

15.223.237.237.237.237.237.237.237.237.237.237.237.237.237.237.237.237.237.237.2

0.00.00.00.00.00.00.00.1

19.319.319.319.319.319.319.318.318.318.318.318.318.318.318.318.318.318.3

Hap

loty

pes

(F s

tatis

tic)

Figure 1 Fine mapping of the fibrogenic QTL on chromosome 2 by in silico

haplotype analysis using 143 SNPs with an average distance of 313 kb

between microsatellite markers D2Mit6 (20.9 Mb) and D2Mit90 (65.6 Mb).

Hepatic collagen concentrations of 52 mice from seven inbred mouse

strains were used as phenotypes, and genotypes were downloaded from the

Mouse Phenome Database. Haplotypes were inferred from sliding three-SNP

windows8, and an F statistic was calculated for each haplotype on the basis

of the genotype-phenotype pairings. Strain names are positioned above the

alleles, which are indicated by different colors. Mb positions of the SNPs are

listed on the left, and F statistics for each haplotype are given on the right.

The position and size of the refined QTL is indicated by the vertical bar.

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did Hc+/+ mice (584 7 86 mg g�1). Hc genotyping of the intercrossprogeny gave maximum lod scores of 3.4 for hepatic collagenconcentration and 2.3 for histological stage of fibrosis (Table 1).These lod scores fulfill the empirical criteria for highly significant (lodscore Z 3.2) and significant (Z 1.5) QTLs, as determined bypermutation tests10. Hence, our experimental and in silico mappingdata show that the Hfib locus on chromosome 2 contains Hc andidentify a significant genetic association with hepatic fibrosis.

As recommended by the Complex Trait Consortium7, we confirmedthe QTL in congenic mice. A congenic strain is generated throughintrogression of the critical QTL region from a resistant strain into asusceptible background strain (or vice versa) by repeated backcrossing.Here we used a congenic strain that carries Hc�/� alleles and thefibrosis-resistant haplotype at the refined QTL on chromosome 2(30.4–35.0 Mb; Fig. 1) from strain DBA/2J on a susceptible back-ground (C57BL/10SnJ). We hypothesized that this congenic strainshould have phenotypic differences relative to the background strainwith respect to fibrosis susceptibility. At baseline, hepatic collagenconcentrations were similar in both strains. After CCl4 challenge,the Hc–/– congenic strain had significantly (P o 0.05) lower hepaticcollagen levels (399 7 36 mg g�1) than did Hc+/+ backgroundcontrols (598 7 69 mg g�1; Fig. 2b). These results were reflected inliver histology (Fig. 2c); the Hc�/� congenic strain had significantly(P o 0.05) less advanced stages of fibrosis (1.80 7 0.18) than didthe Hc+/+ background strain (2.38 7 0.15). The allele effects areconsistent with liver fibrosis being a quantitative trait that is deter-mined by interacting small effects of multiple genes and environ-mental conditions7,11.

Transgenesis with recombined BACQTL and haplotype analyses did not exclude a causal role for othergenes closely linked to Hc, and the congenic strain carried not only

the Hc�/� alleles from strain DBA/2 but also linked DBA/2 alleles.To prove the identity of Hc and the fibrogenic QTL, we reasoned thatit should be possible to reconstitute the fibrosis-susceptible pheno-type in Hc�/� mice by expression of wild-type Hc. Therefore, weintroduced a 118-kb BAC, which was trimmed by ET recombination12

to contain only Hc from the Hc+/+ strain C57BL/6J together withits flanking regions but no other genes, into the fibrosis-resistantHc�/� strain FVB/NJ. Hc BAC-transgenic mice were healthy andreproduced normally. Hemolytic C5 activity was present in serumand was comparable to that of Hc+/+ BALB/cJ mice in radialimmunodiffusion (75–84%) but was absent in all littermate non-transgenic controls.

After challenge with CCl4 for 6 weeks, BAC-transgenic mice hadsignificantly (P o 0.01) higher hepatic fibrosis stages (2.00 7 0.10)and collagen concentrations (352 7 12 mg g�1) than did nontrans-genic FVB/NJ mice (1.46 7 0.14 and 227 7 10 mg g�1, respectively;Fig. 2d). This in vivo complementation of the fibrosis-resistantphenotype by the Hc transgene confirms that Hc is a quantitativetrait gene for liver fibrogenesis in mice.

Blockade of C5R1 attenuates liver fibrosis in miceN-terminal proteolysis of C5 releases a small peptide consisting of74 amino acids (C5a), which exerts its effector functions throughbinding to a specific G protein–coupled receptor (C5R1)13. Inresponse to liver injury, hepatic C5 levels increase and C5R1 expres-sion occurs in various hepatic cell types14. In line with these findings,we detected C5R1 in cultured hepatic stellate cells (HSCs) by immu-nocytochemistry and confocal laser scanning microscopy (Fig. 3a).HSCs expressed C5r1 mRNA (Fig. 3b), and expression increasedsignificantly during transdifferentiation to myofibroblasts in culture(Fig. 3c). Immunoblots confirmed the presence of the 45-kDareceptor protein in cultured HSCs (Fig. 3d). Because myofibroblasts

+0

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Hc delTAHc–/– strains

Hc+/+ strains(BALB/cJ, C3/HeJ, C57BL/6J)

(A/J, AKR/J, DBA/2J, FVB/NJ)

Hc–/–

Hc+/+

a b c

dFigure 2 Phenotypic characterization of liver fibrosis in inbred mouse strains and

Hc BAC-transgenic mice before and after treatment with CCl4 for 6 weeks.

(a) Histological stages of fibrosis and hepatic collagen contents in different strains,

grouped according to their Hc genotypes. The stage of fibrosis is indicated on the

left axis (F0–F4), hepatic collagen contents are represented by hydroxyproline (Hyp)

concentrations on the right axis, and CCl4 treatment is indicated (+ or �). Dataare given as means of strains (7 s.e.m.; **P o 0.01). The two inserted sequence

chromatograms illustrate the 2-bp deletion in exon 6 of Hc in C5-deficient mice, as

confirmed by DNA sequencing. (b) Histological stages of fibrosis and hepatic collagen

contents in congenic Hc�/� and Hc+/+ mice, using the same layout as in a (n ¼ 8–12;

*P o 0.05). (c) Representative liver sections of congenic Hc�/� and Hc+/+ mice after

CCl4 treatment. The Hc�/� mouse (upper panel) shows minimal fibrotic changes,

whereas the Hc+/+ mouse (lower panel) has periportal fibrosis and prominent fibrotic

scars. Five of twelve Hc�/� mice developed minimal fibrotic changes (F1) and seven mice were staged to have only septal fibrosis (F2). In contrast, four

Hc+/+ mice showed prominent bridging fibrosis (F3) and four other Hc+/+ mice had periportal and septal fibrosis (F2). (d) Histological stages of fibrosis and

hepatic collagen contents in Hc BAC-transgenic (Tg) mice and littermate nontransgenic controls, using the same layout as in a (n ¼ 13–14; **P o 0.01).

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synthesize collagens and other extracellular matrix proteins4,15, theirC5R1 expression is consistent with the concept that C5 is a modifier ofliver fibrogenesis.

Therefore, we hypothesized that inhibition of C5 effects should haveantifibrotic consequences. To show that C5a affects liver fibrogenesis,we carried out in vivo studies using small peptidic C5R1 antagonists,which are derived from the C-terminal segment of C5a (ref. 16).Compared with untreated mice, intravenous administration of theC5R1 antagonist to fibrosis-susceptible Hc+/+ BALB/cJ mice signifi-cantly reduced hepatic collagen levels (256 7 25 versus 445 7 87 mgg�1, respectively; P o 0.05) and stage of fibrosis (1.86 7 0.14 versus2.29 7 0.18, respectively; P o 0.05; Fig. 4a). The common cyclicC5R1 antagonist AcF-[OPdChaWR]17 did not lead to more pro-nounced regression of fibrosis scores (1.73 7 0.16 in untreatedmice versus 2.22 7 0.09 in treated mice; P o 0.05; data notshown). Liver collagen contents and fibrosis stages after C5R1 block-ade in Hc+/+ mice were similar to those in Hc�/� mice (Fig. 1b),suggesting that C5a-C5R1 interactions mediated the pathogenetic

effects in our model. Accordingly, C5R1 antagonist treatment hadno antifibrotic effects in Hc�/� mice (strain A/J; Fig. 4b). To analyzefurther the functional mechanisms of C5R1 antagonists, we deter-mined hepatic mRNA expression of matrix metalloproteinases(MMPs) and their tissue inhibitor TIMP1, because C5a regulatesthe production of MMPs in mouse fibroblasts (J.K., unpublisheddata). C5R1 blockade significantly reduced expression of hepaticcollagen a2(I) (Col1a2; 70 7 10% relative to BALB/cJ mice treatedwith CCl4 only) and Timp1 (59 7 13%; Fig. 4c) but not Mmp2,Mmp3 or Mmp9 (data not shown).

C5 is associated with stage of liver fibrosis in humansOur genomic QTL analysis, the Hc BAC-transgenic mouse model andthe in vivo studies with small-molecule C5R1 inhibitors collectivelyprovide a strong rationale for investigating the role of C5 in liverfibrogenesis in humans. Chronic HCV infection is characterized byrelentless activation of the complement system18. Therefore, we testedthe human gene C5 (the ortholog of mouse Hc) in a hypothesis-driven

Figure 3 Expression of C5R1 in rat HSCs.

HSCs were collected at day 2 (d2) or d3 after

primary culture, and their myofibroblastic

phenotype was obtained after secondary culture.

(a) C5R1 immunocytochemistry of isolated

HSCs using antibodies to C5R1 and fluorescein

isothiocyanate conjugates. HSCs had strongly

positive C5R1 signals when studied by light

microscopy (upper panels, magnification 100�)

and confocal laser scanning microscopy (lower

panels, magnification 800�). The right column

shows negative controls (Co) with preimmune

serum (upper right panel) and normal mouse IgG

(lower panels), respectively. Cell nuclei were

counterstained with propidium iodide. (b) C5r1mRNA expression of isolated rat liver cells. HSCs

or myofibroblasts (MF) cultured for different time

intervals, Kupffer cells (KC), and liver sinusoidal

endothelial cells (LSEC) were analyzed by RT-

PCR (upper panel), with Gapdh as reference

(lower panel). The sizes of selected base-pair

markers (M) are indicated on the right; the size

of the C5r1 amplicon is 409 bp. Co, control sample without template cDNA. (c) Quantification of C5r1 mRNA expression in cultured HSCs and

myofibroblasts (MF) by quantitative RT-PCR. Steady-state mRNA levels are indicated on the left axis (relative units, n ¼ 4, **P o 0.001, ANOVA with

Bonferroni’s post hoc test). (d) Immunoblot analysis of C5R1 protein expression in cultured HSCs and myofibroblasts (MF). The sizes of selected molecular

weight markers (M) are indicated on the right; the size of the C5r1 protein is 45 kDa. Staining for b-actin (ACTB) served as internal loading control.

C5R1 Co

C5r1

Gapdh

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HSC d4 d5 d6 d9 MF M

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kDa

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900409242124

12

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Hc+/+ BALB/cJ

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C5R1A cC5R1A– + – +

C5R1A – + – +

– + – +

cC5R1A – + – +

** * *

a

b

cFigure 4 Phenotypic characterization of CCl4-induced liver fibrosis in

mice treated with C5a receptor antagonists (C5R1A). (a) Histological stages

of fibrosis and hepatic collagen contents in fibrosis-susceptible Hc+/+

BALB/cJ inbred mice. The stage of fibrosis is indicated on the left axis

(F0–F4), hepatic collagen contents are represented by hydroxyproline (Hyp)

concentrations on the right axis (n ¼ 7) and treatment with linear C5R1A

is indicated (+ or � and color-coding). C5R1 blockade significantly (*P o0.05) reduced fibrosis stages and hepatic collagen contents in treated mice

compared with untreated BALB/cJ mice. (b) Histological stages of fibrosis

and hepatic collagen contents in fibrosis-resistant Hc�/� A/J inbred mice,

using the same layout as in a (n ¼ 7). C5R1 blockade had no antifibrotic

effects in Hc�/� controls. (c) Hepatic mRNA expression of Col1a2, Mmp2,

Mmp3 and Timp1 in fibrosis-susceptible BALB/cJ mice. Steady-state mRNAlevels are indicated on the left axis (relative units, n ¼ 5), and treatment

with cyclic cC5R1A is indicated (+ or � and color-coding). C5R1 blockade

reduces Col1a2 (to 70 7 10% of the level observed in mice treated with

CCl4 only; *P ¼ 0.05) and Timp1 expression (59 7 13; *P o 0.05),

whereas mRNA levels of Mmp2 and Mmp3 did not change significantly.

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association study in 277 individuals with chronic HCV infection.Because congenital C5 deficiency is rare in humans19 and suscept-ibility to complex diseases is often due to multiple polymorphismswith modest effects and variable linkage disequilibrium11, we firstdetermined common C5 haplotypes in a large control population. Weidentified five common haplotypes (Table 2), which are tagged by 4SNPs (called haplotype-tagging SNPs, htSNPs). None of the genotypefrequencies deviated from Hardy-Weinberg equilibrium.

The overall distribution of C5 haplotypes did not differ betweenindividuals with chronic HCV infection and uninfected controls(Table 2). But the C5 haplotype distribution did differ significantly(P o 0.05, permutation test) between individuals with HCV infectionwith mild liver fibrosis (stages F0–F1) and those with severe fibrosis(stages F2–F4; Fig. 5a). The most common haplotype (C5_1) wasobserved more often in individuals with advanced fibrosis (46%) thanin those with mild fibrosis (39%), whereas haplotype C5_5 was moreprevalent in individuals with mild fibrosis than in those with pro-gressive disease (15% versus 8%). Individuals homozygous withrespect to the at-risk alleles of the two C5 htSNPs (rs2300929 andrs17611), which distinguish haplotypes C5_1 and C5_5 from theothers (Table 2), had significantly more advanced stages of fibrosisthan did individuals carrying at least one other allele (Fig. 5b).

The haplotype analysis results are reflected by significant differencesin the distributions of alleles and genotypes of the two C5 htSNPsbetween individuals with mild (stages F0–F1) and advanced (stagesF2–F4) fibrosis (Table 3). As shown by contingency table statistics,both C5 htSNPs are significantly (P o 0.05) associated with liverfibrosis stage. To control for potentially confounding effects of genderand age, which are independently associated with stage of liverfibrosis, we carried out logistic regression analysis. This analysisyielded levels of association and statistical significance that weresimilar to the contingency table statistics; therefore, there is noevidence for confounding (Table 3). Our genetic analyses in indivi-duals with chronic HCV infection indicate that common C5 genevariants are associated with liver fibrosis in humans.

High C5 serum levels in individuals with at-risk C5 genotypesTo assess the functional relevance of C5 haplotypes, we determined C5serum concentrations and htSNPs in 100 healthy individuals, becauseprogressive liver fibrosis per se is associated with complement activa-tion and depletion18,20. Serum C5 levels (166 7 5 mg ml�1) variedmarkedly among individuals, ranging from 78 to 344 mg ml�1, but didnot correlate with age or gender. Furthermore, C5 levels weresignificantly elevated in individuals carrying at least one at-risk allele(rs17611 A) of the SNP tagging haplotype C5_1 (174 7 6 versus 1487 8 mg ml�1; P o 0.05) and tended to be lower in individualscarrying the low-risk allele (rs2300929 C) of the less common

haplotype C5_5 (Supplementary Table 1 online). Notably, thesignificant association of high serum C5 levels with the at-risk C5haplotype in humans is consistent with our mouse models, with C5deficiency attenuating (Fig. 2b) and BAC transgenesis aggravating(Fig. 2d) liver fibrogenesis.

DISCUSSIONQTL mapping in experimental crosses of inbred mouse strains hasbeen successfully used to localize genetic loci that control complexdiseases. Two major challenges remain: to positionally clone the

Table 2 Common human C5 haplotypes and htSNPs

C5 htSNPb Haplotype frequency

C5 haplotypea rs2300929 (0.13)c rs1035029 (0.34)c rs17611 (0.45)c rs192408 (0.21)c HCV (2n ¼ 554) Control (2n ¼ 634)

C5_1 T A A T 0.43 0.40

C5_2 T G G A 0.19 0.20

C5_3 T G G T 0.13 0.14

C5_4 T A G T 0.10 0.13

C5_5 C A G T 0.11 0.09

aHaplotypes are designated based on their frequencies (e.g., C5_1 for the most frequent haplotype), as determined by the PHASE algorithm. Only the five most common haplotypes (frequencies 40.05) are listed. bSNP designations are taken from the dbSNP database. cThe minor allele frequency is given in parentheses.

50

Freq

uenc

y (%

)S

tage

of f

ibro

sis

C5 haplotypes

C5 htSNP genotypes

40

30

20

10

2.0

1.5

1.0

0.5

0T/T T/C

rs2300929 rs17611

C/C G/G A/G A/A

0C5_1 C5_1C5_2

*

* *

C5_2C5_3 C5_3C5_4 C5_4C5_5 C5_5

F0–F1

F2–F4

a

b

Figure 5 Association of C5 haplotypes and genotypes with histological

stages of liver fibrosis in 277 individuals with chronic HCV infection.(a) Distribution of common C5 haplotypes in individuals with mild liver

fibrosis (stages F0–F1, light bars) and advanced fibrosis (stages F2–F4, dark

gray bars). Haplotypes (mean frequency 7 s.e.m.) were reconstructed from

genotypes of C5 htSNPs (Table 2). The distribution of C5 haplotypes

differed significantly (*P o 0.05, permutation test) between the two groups:

The most common haplotype C5_1 occurred more often in individuals with

advanced fibrosis than in those with mild fibrosis (46% versus 39%),

whereas haplotype C5_5 was more prevalent in individuals with mild fibrosis

than in those with more progressive disease (15% versus 8%).

(b) Phenotypic effects of different genotypes at the two C5 SNPs that tag

haplotypes C5_1 (red ground) and C5_5 (green ground) on the histological

stage of fibrosis. Dark gray bars indicate individuals who were homozygous

with respect to C5 at-risk alleles, light gray bars designate heterozygous

individuals and light bars represent individuals with protective C5 alleles.

Individuals with homozygous A/A alleles at htSNP rs17611 (tagging C5_1)

and homozygous T/T alleles at htSNP rs2300929 (tagging C5_5) had

significantly (*P o 0.05) higher stages of fibrosis than did individuals

carrying at least one other allele.

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gene(s) underlying the QTLs21 and to transfer the findings fromexperimental models to human diseases. In this study, we dissected amouse QTL for liver fibrosis that has an identifiable quantitative effectfor which Hc (encoding complement factor C5) is a strong candidategene. First, we strengthened and refined the experimental QTLanalysis by in silico haplotype mapping, used for other complex traits8.This combination of methods reduces the likelihood of arriving atfalse-positive computational predictions. In addition, we confirmedthat Hc is a quantitative trait gene for liver fibrosis by generating BAC-transgenic mice. Because the BAC that we used contained severalgenes, we trimmed it by ET recombination12 to carry Hc only. Thein vivo complementation of the fibrosis-resistant phenotype by therecombined BAC supports the causal relationship between the pre-sence of C5 and advanced liver fibrosis.

The Complex Trait Consortium7 suggested several criteria thatshould be fulfilled by a bona fide QTL candidate gene. Hc fulfillsseveral of these criteria, including (i) identification of a polymorphismin the coding region (Fig. 2a), (ii) pathophysiological effect tested inmutant mice (Fig. 2b), (iii) positive in vitro expression studies(Fig. 3), (iv) functional link between gene function and quantitativetrait (Fig. 4) and (v) confirmation in BAC-transgenic mice (Fig. 2d).Next we extended and replicated the genetic findings in mice in ahypothesis-driven association study of the orthologous human geneC5 in individuals with chronic HCV infection. We carried out ahaplotype-based analysis that captures more genetic diversity thansingle-marker statistics and is more powerful statistically22,23. Weidentified significant associations between the histological stages ofliver fibrosis and the distribution of C5 haplotypes and their taggingSNPs. Taken together with the mouse study, these findings provideevidence that C5 has a causative role in human liver fibrosis.

The association of C5 serum concentrations and C5 genotypes inhumans suggests that the genetic variants have functional relevance,but the at-risk alleles and genotypes of htSNPs confer relative risks ofonly 1.4–1.9 (Table 3), and the strength of allelic associations with thepossible functional correlates (i.e., C5 serum levels in healthy indivi-duals) seem moderate. The relative risks, however, are exactly in therange expected for complex traits such as liver fibrosis, which areinfluenced by the combination of multiple genes with small individualeffects on the phenotype7,11. The more factors that determine aquantitative trait, the more difficult it is to map and identify all thecausal quantitative trait genes7. This study shows that the combinationof current genomic technologies can identify unknown small-risk

genotypes, but only the whole ensemble of susceptibility variantsand their combinations24 will provide diagnostic tools for the clinicalmanagement of complex traits such as liver fibrosis. We speculate thatin the future, genetic profiles including complement-related markersmight help to predict rapid progression rates of hepatic fibrosis,such as those present after liver transplantation in individuals withHCV infection25.

C5 can be linked functionally to liver fibrogenesis, as liver sinusendothelial cells, Kupffer cells and activated myofibroblasts (the mostrelevant profibrotic cell type in injured liver15) express the receptorC5R1 (ref. 14; Fig. 3). C5R1 is a member of the G protein–coupledtransmembrane receptor superfamily that specifically binds C5a, thepotent chemoattractive and proinflammatory cleavage fragment of C5,causing leukocyte chemotaxis, release of proinflammatory cytokines,smooth muscle contraction, increased vascular permeability and neu-trophil transmigration13. The link between C5 and liver fibrogenesis isconsistent with more severe inflammation and fibrosis after pulmonaryinjury (e.g., in C5-sufficient mice26,27) and the profibrotic effects of C5activation in tubulointerstitial renal injury28, indicating that C5 mightbe a common modifier of chronic immune-mediated diseases.

To assess the potential therapeutic value of our findings, we testedwhether intervention with small-molecule C5R1 inhibitors couldinhibit liver fibrogenesis in vivo. These hexapeptides are derivativesof the C terminus of C5a, which can bind to the receptor with highaffinity and have inhibitory potency in the nanomolar range16,29.Small C-terminally derived C5a analog peptides block C5a-mediatedeffects specifically in rodent models of inflammatory, ischemic, viraland autoimmune diseases, as well as liver regeneration16,17,30,31. Thetherapeutic effects of anti-C5 strategies have been demonstratedrecently by the effective and safe treatment of complement depletionin paroxysmal nocturnal hemoglobinuria with a humanized mono-clonal antibody against C5 (ref. 32). In our study, C5R1 blockade withsmall molecules attenuated liver fibrosis and reduced hepatic Col1a2and Timp1 expression levels. These findings suggest that the pro-fibrotic effects of C5 might be, in part, mediated by extracellularmatrix remodeling, with TIMP1 favoring the accumulation of fibroticmatrix4,15. Other mechanisms might also pertain, such as a growthfactor–like response through direct stimulation of HSC prolifera-tion30, the release of chemokines from liver sinusoidal endothelialcells (which also express C5R1; Fig. 3b) or the modulation ofC5-mediated signal transduction in bone marrow cells that modifyliver fibrogenesis15. In this respect, we note that C5a downregulated

Table 3 Association of C5 htSNPs with histological stages of liver fibrosis in individuals with chronic HCV infection

Number (frequency) of alleles or genotypes Tests for associationa Logistic regression analysis

C5 htSNP Stages F0–F1 (2n ¼ 278) Stages F2–F4 (2n ¼ 276) OR 95% c.i. P OR s.e.m. z 95% c.i. P

rs17611 A 114 (0.41) 138 (0.50) 1.44 1.03–2.01 0.034

rs17611 G 164 (0.59) 138 (0.50)

rs17611 AA 21 (0.15) 32 (0.23) 1.48 0.028 1.56 0.295 2.35 1.08–2.26 0.019

rs17611 AG 72 (0.52) 74 (0.54)

rs17611 GG 46 (0.33) 32 (0.23)

rs2300929 T 235 (0.85) 250 (0.91) 1.76 1.05–3.01 0.031

rs2300929 C 43 (0.15) 26 (0.09)

rs2300929 TT 102 (0.73) 115 (0.83) 1.59 0.045 1.87 0.523 2.25 1.08–3.24 0.024

rs2300929 TC 31 (0.22) 20 (0.14)

rs2300929 CC 6 (0.04) 3 (0.02)

aOdds ratios (OR), confidence intervals (c.i.) and P values were determined using contingency table statistics and logistic regression analysis with adjustment for age and gender. Odds ratios werecalculated relative to low-risk alleles rs17611 G or rs2300929 C.

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the synthesis of IL-12 family cytokines, thereby inhibitingT helper (Th) 1 cell polarization both in vitro and in vivo33. This isin line with the current paradigm that a shift to Th2 cytokine subsetsstimulates fibrogenesis4,6,15,34.

In conclusion, our combined experimental analyses in mice andhumans provide strong evidence for the identity of Hc and thefibrogenic QTL on mouse chromosome 2. Translation of the mouseQTL analysis to a clinical study identifies a susceptibility gene that isassociated with a complex human disease, confirming the concept thatidentical sets of essential genes have key regulatory roles in complexdiseases across different organisms35. This comparative approach isparticularly successful when the quantitative trait gene has a recogniz-able molecular signature in both species21, such as C5, which is agenetic modifier of liver fibrogenesis in mice and humans. The resultsof this study might be relevant not only for understanding thesequelae of chronic liver diseases that are associated with activationof the complement system36,37 but also for understanding fibrogenesisin other organs and developing new antifibrotic therapies in general.

METHODSInbred mice and experimental crosses. We obtained inbred mouse strains A/J,

AKR/J, BALB/cJ, B10.D2-Hc0/oSnJ, C57BL/6J, C57BL/10SnJ, C3H/HeJ,

DBA/2J and FVB/NJ from The Jackson Laboratory. We intercrossed F1 hybrids

of the fibrosis-susceptible strain BALB/cJ and the fibrosis-resistant strain A/J5

to obtain 629 F2 progeny. The B10.D2-Hc0/oSnJ strain carries the Hc�/�

alleles and closely linked alleles from DBA/2J, after six generations of back-

crossing to C57BL/10SnJ. After weaning them at 3 weeks of age, we housed

mice in groups of five per cage. To induce liver fibrosis, we treated 6- to 8-week-

old mice with CCl4 twice weekly intraperitoneally (1 ml per kg body weight as a

1:1 mixture with mineral oil) for 6 weeks5,6. For congenic and transgenic mice,

we reduced the CCl4 dose by 30% because higher doses caused 450% lethality.

Because no gender differences in susceptibility to CCl4-induced liver fibrosis

were observed in our previous study5, we used male and female mice for

subsequent experiments.

For intervention studies, we treated mice with the small peptidic C5R1

antagonists NmePhe-Lys-Pro-dCha-Trp-dArg (NmeFKPdChaWr) or AcPhe-

[Orn-Pro-dCha-Trp-Arg] (AcF-[OPdChaWR]). These linear and cyclic hexa-

peptides, respectively, were synthesized as described29, and their purity was

490% by HPLC (JPT Peptide Technologies). Mice received sterile saline or the

C5R1 antagonist in saline (3 mg kg�1 intravenously) 30 min before each

intraperitoneal injection of CCl4 (n ¼ 7). The blood clearance of small

molecule C5R1 antagonists after intravenous administration to mice follows

a multiphasic decline over as much as 72 h (ref. 31).

We obtained organs and blood samples 3 d after the last injection; for

expression studies, we collected livers from CCl4-treated mice 24 h after

injection of the C5R1 antagonist. We anesthetized mice with isoflurane and

killed them by cervical dislocation. All experiments were done in compliance

with the relevant laws and institutional guidelines and were approved by the

Animal Care and Use Committee of the District of Cologne (Germany).

Generation of transgenic mice with recombined BAC. We obtained BAC

clone RP23-223C17 from RZPD. Because the 191-kb BAC insert contains the

whole Hc gene from inbred mouse strain C57BL/6J and two additional genes

(encoding centrosomal protein 1 and expressed sequence AI182371), we first

modified it by ET recombination12, eliminating base pairs 117,600–191,329 of

the original BAC. For ET recombination, we purified a PCR amplicon contain-

ing the PGK-gb2-neo template (Gene Bridges) between two 50-bp sequences

homologous to the 5¢ and 3¢ ends of Hc (Supplementary Table 2 online) by gel

electrophoresis (Rapid Gel Extraction System, Marligen Biosciences). We trans-

formed Escherichia coli cells containing RP23-223C17 with expression plasmid

pSC101-BAD-gbaAtet (Gene Bridges) by electroporation. We prepared compe-

tent cells from a single clone containing the BAC and the expression plasmid

and transformed them with the purified PCR product by electroporation after

inducing the ET recombination proteins by adding 0.1% L-arabinose for 1 h

(ref. 12). We isolated BAC DNA using the EndoFree Plasmid Maxi kit (Qiagen).

We verified successful ET recombination of the BAC clone by sequencing the

neo cassette and exon 6 of Hc (Supplementary Table 2 online) using the Big

Dye Termination Cycle Sequencing Ready Reaction kit (Applera) as well as by

XbaI and BamHI restriction digestions with subsequent size-fractionation by

pulsed-field gel electrophoresis (CHEF-DR III system, Biorad).

BAC DNA was linearized with NotI and microinjected into pronuclei of

fertilized eggs from fibrosis-resistant and C5-deficient FVB/NJ inbred mice

(Max Planck Institute of Molecular Cell Biology and Genetics). Eighteen of 125

offspring contained at least one copy of the transgene, and we confirmed the

presence of C5 in sera of these mice by hemolytic complement activity of the

alternative pathway in radial immunodiffusion plates (The Binding Site). We

generated four independent lines of Hc BAC-transgenic mice [FVB/NJ-Tg(Hc)]

by mating transgenic founder mice to FVB/NJ inbred mice.

Human study population. We enrolled 277 individuals of European descent

with a median age of 39.0 years (range 18–75 years; 178 males and 99 females).

All individuals were admitted to the Department of Medicine III at Aachen

University Hospital (n ¼ 161) or the Department of Medicine I at Regensburg

University Hospital (n ¼ 116) owing to chronic HCV infection with elevated

aminotransferase activities. The diagnosis of chronic infection was confirmed

by a positive anti-HCV assay (Abbott) and quantification of HCV RNA by RT-

PCR (Cobas Amplicor, Roche) after nucleic acid purification (QIAamp viral

RNA kit, Qiagen). The approximate duration of infection at the time of liver

biopsy could be determined for 170 individuals with an identifiable time point

of infection (61.4%; mean 7 s.d., 8.0 7 8.6 years). In all individuals, other

chronic liver diseases (chronic hepatitis B, autoimmune hepatitis, alcoholic

liver disease, hemochromatosis, a1-antitrypsin deficiency or Wilson’s disease)

were excluded. We recorded data on estimated daily alcohol consumption, and

individuals who reported a regular high alcohol intake of 440 g d�1 in the year

before liver biopsy were excluded from the study, as described previously38. We

obtained informed consent from all individuals, and the study was approved by

the Research Ethics Committees of the Medical Faculties at Aachen University

and University of Regensburg.

Following the epidemiological criteria recommended by Schulz and

Grimes39, we used 317 DNA samples from unrelated in-hospital individuals

without chronic HCV infection as controls (Table 2), who were comparable to

the individuals with HCV infection with respect to gender distribution, age

range and risk for HCV exposure, as described40. In addition, we determined

C5 genotypes in 100 healthy volunteers (median age 35.0 years, range 21–74

years; 44 males).

Genotyping. We isolated genomic DNA from mouse spleens, tail snips, EDTA-

anticoagulated whole blood or paraffin-embedded liver tissue sections38 using

the QIAamp (Qiagen) protocol. We determined DNA concentrations fluoro-

metrically (Biorad), using the dye PicoGreen (Molecular Probes).

We genotyped mouse microsatellite (Mit) markers as described5. We

visualized PCR products on 4% agarose gels (NuSieve 3:1, FMC BioProducts).

We ascertained the presence or absence of the 2-bp deletion in exon 6 of mouse

Hc9 by direct Big Dye Termination cycle sequencing (Supplementary Table 2

online and Fig. 1a).

We determined mouse Hc genotype and human SNPs using fluorogenic 5¢-nuclease (TaqMan) assays on the ABI PRISM 7000 Sequence Detection System

(Applera). Supplementary Table 2 online provides primer and probe sequences

for Hc allele-specific PCR. PCR reactions contained 5–20 ng of genomic

DNA, 1� TaqMan Universal Master Mix, 900 nM of each primer and 200

nM of VIC-labeled and FAM-labeled probes in 25-ml reactions. Amplification

conditions were 95 1C for 10 min, 40 cycles of 92 1C for 15 s and 60 1C

for 1 min.

QTL and haplotype analysis in mice. We generated a genetic map using the

marker data of the 629 intercross progeny. We calculated genetic distances and

linkage probabilities (lod scores) with Map Manager QTX41. We identified the

associations between phenotypes (stages of fibrosis and collagen concentra-

tions) and individual genetic markers in the intercross mice by regression

analysis41,42. To determine the empirical threshold for statistical significance

that is corrected for multiple statistical comparisons, we carried out permuta-

tion tests with 10,000 permutations10, as implemented in Map Manager41.

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To refine the QTL by haplotype analysis, we retrieved the SNPs that mapped

to the National Center for Biotechnology Information’s Mouse Genome build

33 and had been successfully genotyped in the seven strains A/J, AKR/J, BALB/

cByJ, C3H/HeJ, C57BL/6J, DBA/2J and FVB/NJ from the 10K GNF2 SNP set8

deposited in the Mouse Phenome Database. Examining data from all SNP

projects in the Mouse Phenome Project shows that the haplotypes of inbred

strains BALB/cByJ and BALB/cJ and strains C57BL6/J and C57BL/10SnJ are

identical throughout the critical region.

We then inferred haplotypes from three-SNP windows spanning, on

average, 900 kb, as suggested8, and calculated F statistics by ANOVA with

haplotypes as factor and hepatic collagen concentrations of the inbred mice as

dependent variables.

Haplotype analysis in humans. To determine C5 haplotypes, we retrieved the

nine validated SNPs from dbSNP and the Applied Biosystems database that had

minor allele frequencies Z 0.1 (ref. 22) and covered the whole C5 gene with a

mean spacing of 13.0 kb. We reconstructed haplotypes using the PHASE 2.0

algorithm43 and determined htSNPs tagging common haplotypes (frequencies

4 0.05, coverage 4 0.95) using the BEST algorithm44. All SNPs are located in

introns, except for htSNP rs17611 (c.2434G-A), which leads to the substitu-

tion V802I in the a¢-chain of C5b, and SNP rs25681, which causes a

synonymous substitution at position 544 and does not tag a haplotype. To

test for significant differences in haplotype distributions between groups with

correction for multiple comparisons, we carried out permutation tests with

10,000 permutations, as implemented in PHASE43.

Phenotypic characterization of liver fibrosis. We obtained liver biopsy

samples using the percutaneous Menghini technique under ultrasound

guidance. For histological scoring of liver fibrosis, we stained paraffin-

embedded 2- to 4-mm liver sections with Sirius red5. Hepatic fibrosis

was scored (stages F0–F4) according to Desmet and Scheuer45 by two

pathologists blinded to the study protocol and stratified as mild fibrosis

(F0–F1) or advanced septal (bridging) fibrosis (F2–F4)46. We measured

collagen contents in liver hydrolysates in mice by photometric measurement

of the specific amino acid hydroxyproline5. Regression analysis confirmed a

linear relationship between stages of fibrosis and fibrotic (Sirius red–positive)

areas (n ¼ 29, r ¼ 0.88, P o 0.001, Pearson’s correlation coefficient), as

determined by morphometric analysis using Quantimet software (Biorad)5.

Expression analysis. We isolated hepatic cell subpopulations (HSCs, Kupffer

cells and liver sinusoidal endothelial cells) from in situ–perfused livers of male

Sprague-Dawley rats by the pronase-collagenase method and purified them by

density gradient centrifugation and centrifugal elutriation47. We obtained the

myofibroblastic phenotype by secondary culture of HSCs. The mean HSC

purity was 490%, as estimated by vitamin A autofluorescence, and the yield

ranged from 20 � 106 to 50 � 106 cells per liver47.

We isolated total RNA by guanidinium thiocyanate–phenol–chloroform

extraction. We assessed mRNA expression of C5r1 by RT-PCR, using Super-

Script (Invitrogen) and C5r1 primers and assay conditions as described48. We

carried out quantitative RT-PCR to examine mRNA expression of C5r1,

Col1a2, Mmp2, Mmp3, Mmp9 and Timp1 using Assays-on-Demand (Applera)

on the ABI PRISM 7000 Sequence Detection System. Assays are available from

the Applied Biosystems database. All data were normalized using Gapdh mRNA

or 18S rRNA levels.

For immunoblotting, we separated protein homogenates from HSCs under

nonreducing conditions on 4–12% acrylamide gradient gels in MOPS running

buffer. After blotting, we blocked membranes with 5% (w/v) nonfat milk

powder in Tris-buffered saline containing 0.1% (w/v) Tween 20 and incubated

them with a mouse monoclonal antibody to rat C5R1 (HR63; HyCult

Biotechnology). We then incubated the blots with an antibody to mouse IgG

conjugated to horseradish peroxidase and developed them using SuperSignal

West Dura Extended Substrate (Pierce).

For immunocytochemistry, we washed collagen-coated glass slides with

adherent cells with phosphate-buffered saline, fixed them with 4% (w/v)

paraformaldehyde in phosphate-buffered saline and treated them with acet-

one/methanol (1:1, v/v) for 5 min. We blocked endogenous peroxidase activity

with 0.3% H2O2 in phosphate-buffered saline for 10 min. After incubation with

a polyclonal antibody to C5R1 (1:1,000; DPC Biermann) for 1 h, we applied

the biotinylated secondary antibody to chicken (Vector Laboratories) and

completed staining with Vectastain ABC Reagent (Vector Laboratories). For

immunofluorescence studies, we blocked cells with a biotin-blocking reagent

(X0590; DakoCytomation), incubated them for 16 h with the antibody to C5R1

R63 at a dilution of 1:50 and, after washing, with an antibody to mouse IgG

conjugated to biotin. We then stained cells with a streptavidin–fluorescein

isothiocyanate conjugate (DakoCytomation).

Statistical analysis. We carried out statistical analysis with SPSS 11.0 (SPSS

GmbH). Unless stated otherwise, continuous variables are given as means 7s.e.m., and groups were compared with Student’s t-test (continuous variables)

or Fisher’s exact test (categorical measures).

As tests for association, we compared the distribution of alleles and

genotypes in contingency tables by Pearson’s goodness-of-fit w2 test and

Armitage’s trend test, respectively49, using software by T.F. Wienker (Institute

of Medical Biometry, Informatics and Epidemiology, University of Bonn) and

T.M. Strom (Institute of Human Genetics, GSF-National Research Center for

Environment and Health). We confirmed consistency of genotype frequencies

with Hardy-Weinberg equilibrium using an exact test50. To adjust for poten-

tially confounding effects on stage of fibrosis, we carried out logistic regression

analysis, using Intercooled STATA 7.0 (Stata Corp.) and Genassoc commands

written by D. Clayton (Cambridge Institute for Medical Research).

URLs. Map Manager QTX is available at http://www.mapmanager.org/, PHASE

2.0 is available at http://www.stat.washington.edu/stephens/software.html and

BEST is available at http://genomethods.org/best/. See http://ihg.gsf.de/ihg/snps

for tests for deviation from Hardy-Weinberg equilibrium and tests for

association in case-control studies and http://www-gene.cimr.cam.ac.uk/

for Genassoc commands. For in silico haplotype mapping in mouse strains,

SNP data were retrieved from Mouse Phenome Database at http://aretha.

jax.org/pub-cgi/phenome/mpdcgi?rtn¼snps/door/ (project ID MPD:152).

Data on human SNPs and Assays-on-Demand, assay conditions and reagent

compositions are available from the Applied Biosystems databases at http://

www.appliedbiosystems.com/ (assay IDs C_2783669_1, C_7577339_1,

C_11720402_10, C_11720394_10, Mm00483888_m1, Mm00441724_m1,

Mm00440295_m1 and Rn00586108_m1).

Mouse Genome Database accession numbers. Data on Hc BAC-transgenic

alleles, MGI:3527961, MGI:3527963, MGI:3527964 and MGI:3528826 (http://

www.informatics.jax.org/).

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank H. Matern, M.C. Carey, B. Paigen and T. Sauerbruch for discussionsand comments. This study was supported by grants from DeutscheForschungsgemeinschaft, the German Network of Excellence for Viral Hepatitis(Kompetenznetz Hepatitis), the Ministry of Science and Research of North-Rhine-Westphalia and Aachen University (cooperative project Identification of MolecularMarkers and Gene Therapy for Fibrosis and Wound Healing). This study waspresented in part at the Plenary Session of the Annual Meeting of the AmericanAssociation for the Study of Liver Diseases, Boston, November 2002, andpublished in abstract form in Hepatology (36, 296A; 2002).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 29 March; accepted 19 May 2005

Published online at http://www.nature.com/naturegenetics/

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