Investigation of the Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease

CL IN ICAL STUDIES

InvestigationoftheLith6 candidate genesAPOBEC1 andPPARGin humangallstone diseaseClemens Schafmayer1,2, Henry Volzke3, Stephan Buch4,5, Jan Egberts1, Annika Spille5,Huberta von Eberstein2, Andre Franke4, Markus Seeger5, Sebastian Hinz1, Abdou ElSharawy4,Dieter Rosskopf6, Mario Brosch5, Michael Krawczak7, Ulrich R. Foelsch5, Anton Schafmayer8,Frank Lammert9, Stefan Schreiber2,4, Fred Faendrich1, Jochen Hampe5, JuergenTepel1

1 Department of General Surgery and Thoracic Surgery, Christian-Albrechts-University Kiel, Kiel, Germany

2 POPGEN Biobank, Christian-Albrechts-University Kiel, Kiel, Germany

3 Institute for Community Medicine, Ernst Moritz Arndt University Greifswald, Greifswald, Germany

4 Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany

5 Department of General Internal Medicine, Christian-Albrechts-University Kiel, Kiel, Germany

6 Institute of Pharmacology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany

7 Institute of Medical Statistics and Biometry, Christian-Albrechts-University Kiel, Kiel, Germany

8 Department of General Surgery, Klinikum Luneburg, Luneburg, Germany

9 Department of Internal Medicine I, University of Bonn, Bonn, Germany

Keywords

APOBEC1 – gallstone disease – Lith genes –

PPARG

Correspondence

PD Dr Juergen Tepel, Department of General

and Thoracic Surgery, Christian-Albrechts-

Universitat Kiel, Arnold-Heller-Strasse 12,

24105 Kiel, Germany

Tel: 149 431 597 4308

Fax:149 431 597 3883

e-mail: [email protected]

Received 21 August 2006

accepted 13 April 2007

DOI:10.1111/j.1478-3231.2007.01536.x

AbstractBackground: Genetic susceptibility contributes to the aetiology of gallbladder

diseases as shown by multiple epidemiological studies. A major gallstone suscept-

ibility locus (Lith6) was identified in 2003 by quantitative trait locus mapping in

mice. Two attractive positional and functional candidate genes in apolipoprotein B

mRNA-editing protein (APOBEC1) and peroxisome proliferator-activated receptor

g (PPARG) are located in this interval. Aims: To investigate APOBEC1 and PPARG

as candidate genes for common symptomatic gallstone disease in humans. Patients

and methods: Eight hundred and ten patients who underwent cholecystectomy for

symptomatic gallstone disease (median age of onset 50) were compared with 718

sex-matched control individuals. An independent additional sample included 368

gallstone patients and 368 controls. Control individuals were sonographically free of

gallstones. Haplotype tagging and all known coding single nucleotide polymorph-

isms were genotyped for PPARG (N = 32) and APOBEC1 (N = 11). Results: The

investigated high-risk patient sample provides a power of greater than 80% for the

detection of odds ratios down to 1.45. No evidence of association of the two genes in

the single-point tagging markers, coding variants and in the sliding window

haplotype analysis was detected (all nominal single point P-values 4 0.04). A

logistic regression analysis including age, sex and BMI as covariates was also negative

(nominal P-values Z0.08). Conclusions: In the investigated German samples, no

evidence of association of APOBEC1 and PPARG with gallstone susceptibility was

detected. Systematic fine mapping of the complete Lith6 region is required to

identify the causative genetic variants for gallstone in mice and humans.

Gallbladder disease represents a major healthcareproblem both in Europe and the USA. Approximately12% of the US population are affected by gallstonesand more then 750 000 cholecystectomies are per-formed each year (1). Age, gender, ethnicity, obesity,diabetes and parity have all been implicated asrisk factors for the development of gallstones (2, 3).A genetic component in the susceptibility to gallstones

has been recognized as early as 1937 (4). The initialstudies have utilized pathological section statistics (4);more recent studies have turned to ultrasoundsurveys to investigate the familial clustering of chole-lithiasis (5). There is both strong evidence forfamilial clustering and an increased concordance ofthe trait in monocygotic twins as compared withdizygotic twins (6).

Liver International (2007)910 c� 2007 The Authors. Journal compilation c� 2007 Blackwell Munksgaard

Liver International ISSN 1478-3223

Systematic, genome-wide scans of gallstone traits inexperimental crosses of inbred mouse strains haveyielded a susceptibility map of ‘Lith’ loci (7–11).Several of these loci have been found in differentmouse crosses, thus strengthening the case for robustsusceptibility loci in the mouse (11). The humanorthologues of these loci are thus natural positionalcandidates for gallstone susceptibility also in humans.Of note, among all mouse crosses specifically aimed atidentifying Lith loci in mice, only one failed to detectlinkage between cholesterol gallstones and Chr 6. Thelocus was named Lith6 when it was detected withsignificant linkage in the D2�CAST intercross (12).Coincident loci with significant or suggestive linkagepeaks were identified in D2�PERA (11) and129S1�CAST intercrosses (12). Positional candidategenes that colocalize with Lith6 and are involved inlipid homeostasis include the peroxisome proliferator-activated receptor gamma (PPARG) and apolipo-protein B mRNA-editing protein (APOBEC1).Although the exact source of the cholesterol destinedfor biliary excretion is not known, increased biliarycholesterol concentrations may be the result of geneticalterations in lipid metabolism in which both candi-date genes are involved (13). The PPARG gene maps tohuman chromosome 3p25.2, which corresponds tomouse chromosome 6E3. The peroxisome prolifera-tor-activated receptors (PPARs) are members of thenuclear hormone receptor subfamily of transcriptionfactors. PPARs form heterodimers with retinoid Xreceptors and these heterodimers regulate the tran-scription of various genes. There are three knownsubtypes of PPARs, PPAR-a, PPAR-d and PPAR-g.PPAR-g has a central role in adipocyte differentiation.Two protein isoforms of PPAR-g have been identifiedthat stem from the same gene because of alternativepromoter usage and alternative mRNA splicing.PPARg2 is specifically expressed in adipose tissue anddiffers from the more ubiquitously expressed PPARg1by the presence of an additional 28 amino acid N-terminal exon (14, 15).

Several genetic variants have been identified in thePPAR-g locus. The most common variation is asubstitution of proline with alanine at position 12 inexon 1 of PPARg2, with a frequency of about 12% inCaucasians (16) that has been implicated in theprotection against type II diabetes (17). A rare gain-of-function mutation (Pro115Gln) has been observedin four of 121 unrelated obese German subjects (18).The PPAR-g has also been implicated in cases ofinsulin resistance and partial lipodystrophy (19–21).

Apolipoprotein B, a major protein component ofcirculating plasma lipoproteins, exists in two forms:

apoB-100 and apoB-48. In humans, apoB-100 issynthesized in the liver and is present in very low-density lipoproteins and their metabolic products. Onthe other hand, apoB-48 is synthesized in the smallintestine and is present in chylomicrons and chylomi-cron remnants in the form of apoB. The APOBEC1 isinvolved in the production of apoB-48 from apoB-100and is located on human chromosome 12p13.3 – itsmouse homologue Apobec1 is also located in themouse Lith6 locus on chromosome 6F1. Comparedwith control mice, Apobec1 knockout mice fed alithogenic diet display increased biliary cholesterolsecretion rates, decreased bile salt secretion rates, andaccelerated cholesterol gallstone formation (14). How-ever, mice exhibiting a similar apoB-100-only pheno-type generated by abolition of the APOBEC1 targetnucleotide in Apob showed reduced gallstone suscept-ibility, indicating that APOBEC1 may exert additionalfunctions other than Apob mRNA processing thataffect cholesterol gallstone formation. Interestingly,no large investigation of mutations in the PPARG orAPOBEC1 genes for human gallstone susceptibility hasbeen published.

Despite the advanced stage of genetic investigationin the mouse system, there has been a relative scarcityof genetic studies in the human system. An associationof pigment gallstones with a promoter variation in theUDP-glucuronosyltransferase 1A1 (UGT1A1) gene hasbeen reported in patients with chronic haemolyticdisorders (22, 23) and cystic fibrosis (24). Investiga-tions of the 7a-hydroxylase gene (18) and apolipopro-teins A and B (18–20) in 105–221 and apolipoproteinE in 37–169 patients (25, 26) did not yield replicatedassociation findings. A recent genome-wide linkagescan in Mexican Americans identified significant link-age (LOD 3.7) of gallstone susceptibility on chromo-some 1p (27). This represents the first genome-wideinvestigation of gallstone susceptibility in humans andis an excellent starting point for further systematicmapping efforts.

In this report, we have investigated two functionalcandidate genes within the mouse Lith6 locus forassociation with symptomatic gall stone disease in alarge sample of patients who underwent cholecystect-omy for early onset of symptomatic gallstone disease.

Methods

Patients and phenotypes

Patients who underwent cholecystectomy for cholecys-tolithiasis from 2001 to 2005 in the surgical depart-ments of the hospitals at Kiel, Luneburg, Rendsburg,Schleswig, Flensburg, Husum, Niebull, Heide, and

Liver International (2007)c� 2007 The Authors. Journal compilation c� 2007 Blackwell Munksgaard 911

Schafmayer et al. Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease

Eckernforde (all in Northern Germany) were contactedthrough mail by the respective hospitals and invitedto participate in this study. For patients who didnot respond, one written reminder was sent. Indivi-duals who agreed to participate were contacted by thePOPGEN (http://www.popgen.de) recruitment project(28): they were interviewed by mail questionnaire anda venous EDTA blood sample was obtained either atthe POPGEN offices or from the patients’ generalpractitioners. For both cases and controls, this studywas restricted to probands of German ethnicity, i.e.only individuals whose parents were born in Germanywere included. The study protocols were approved bythe institutional ethics committee and the public dataprotection agency. Written, informed consent wasobtained from all study participants. The first conse-cutive 810 participants were included in this study.

Gallstone-free control individuals were recruitedfrom consecutive patients in the years 2003–2004 fromthe routine clinical ultrasound of the Department ofGeneral Internal Medicine, Kiel. Patients with malig-nant disorders were excluded. Index patients wereidentified on the basis of ultrasound reports, andN = 277 probands were recruited using the POPGENinfrastructure using the protocols described above.Additional controls, N = 441, were obtained frompopulation-derived control individuals from thePOPGEN project [patients identified on the basis of theinhabitants register (28)], who underwent an additionalphysical examination at the POPGEN facilities thatincluded an abdominal ultrasound by a trained physi-cian. There was no significant difference in allele fre-quencies between the two control samples (P4 0.1).

DNA was prepared using the FlexiGene chemistry(Qiagen, Hilden, Germany) according to the manu-facturer’s protocols from all samples. This patientsample has been used in a previous study of theauthors (29). An overview of the patient samples isgiven in Table 1.

An additional study population (panel B) fromNorthern Germany utilized 368 cases (median age of

onset 53 years) and 368 sex-matched controls re-cruited from the Study of Health in Pomerania(SHIP), a population-based study representative forthe adult population of West Pomerania (30). Casesreported a history of cholecystectomy or showedevidence of cholelithiasis on ultrasound examination,whereas controls were free of gallstone disease asevidenced by ultrasound. An overview of the studypopulation is given in Table 1.

Genotyping

DNA samples were evaluated by gel electrophoresis andadjusted to 20–30 ng/mL DNA content using the Pico-green fluorescent dye (Molecular Probes – Invitrogen,Carlsbad, CA, USA). One microliter of genomic DNAwas amplified by the GenomiPhi (Amersham, Uppsala,Sweden) whole genome amplification kit and fragmen-ted at 99 1C for 3 min. One hundred nanograms of DNAwere dried overnight in TwinTec hardshell 384-wellplates (Eppendorf, Hamburg, Germany) at room tem-perature. Genotyping was performed on these platesusing the SNPlex chemistry (Applied Biosystems, FosterCity, CA, USA) on an automated platform with TecanFreedom EVO and 384-well TEMO liquid handlingrobots (TECAN, Mannedorf, Switzerland). As imple-mented by the proprietary SNPlex chemistry, the pro-ducts of the whole-genome amplification reaction wereincubated with pooled oligos in order to perform anallele-specific oligonucleotide ligation assay. Ligationproducts were subsequently amplified using universalbiotinylated primers. Polymerase chain reaction pro-ducts were bound to streptavidin-coated plates andhybridized to a mixture of fluorescently labelled oligo-nucleotides, which assume a defined position in thesubsequent capillary electrophoresis owing to coupledelectrophoretic mobility modifiers (‘‘ZipChutes’’). Theresultant ZipChute mixture was then separated on ABI3730� L sequencers. Genotypes were reviewed manuallyusing the GENEMAPPER 4.0 (Applied Biosystems) software.All process data were logged and administered through adatabase-driven LIMS system (31). Genotyping forPPAR-g nonsynonymous coding single nucleotide poly-morphisms (SNPs) Pro12Ala (rs1801282), Pro115Gln(rs1800571) and for mutations Phe388Leu, Arg425Cyswas performed using Taqmans (Foster City, CA, USA)allelic discrimination assays, as described before (12).Minor groove binder (MGB) probe and primer se-quences are available upon request from the authors.

SNP selection and data analysis

Single nucleotide polymorphisms were selected fromHapMap (www.hapmap.org) by the automated

Table 1. Overview of the patient sample

Panel

Patients Controls

Male (%)N Age�Age atDiagnosis� N Age�

Panel A 810 55 50 718 64 50Panel B 368 59 53 368 64 39

�Median values reported.

Both patient samples are independent and have no overlap. Patient

panel A was recruited in Kiel and panel B in Greifswald (both in Northern

Germany).


Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease Schafmayer et al.

selection of haplotype tagging SNPs for Caucasiansfrom the CEU data set (settings: Mendelian errors: 0,minor allele frequency: 0.01, HWE cut-off: 0.01). Inaddition, coding SNPs with a frequency 4 0.01 wereselected from dbSNP. Moreover, for PPARG the raremutation Pro115Gln and two mutations located in theligand-binding domain (R425C and F388L) wereselected based on literature reports as described in theintroduction.

This study was performed using a case–controldesign: Sliding window haplotype analysis was per-formed using COCAPHASE through the UNPHASED suiteof programs (http://www.rfcgr.mrc.ac.uk/�fdudbrid/software/unphased/) (32). Single-point genotypic andallelic tests of association on sex-matched genotyperesults were performed using w2 statistics on contin-gency tables. For all tests, nominal P-values are re-ported. Logistic regression analyses were performedusing SPSS version 11.

Results

The expected power of the combined samples A and Bof 1178 cases and 1068 controls was estimated forsingle-point allelic effects over an odds ratio of one totwo at a nominal significance level of 0.05 (33) for(minor) allele frequencies of 0.1, 0.25 and 0.5 of apotential susceptibility marker. As shown in Fig. 1, thepower for the detection of odds ratios greater than1.45 is approximately 80% or greater in all models. Formore frequent susceptibility factors, odds ratios of1.27 would also be detectable at this power level.

For both candidate genes, haplotype-tagging SNPswere generated using the HapMap genotype data(http://www.hapmap.org) for the candidate gene re-gions (34). Twenty-eight SNP markers in the PPARG

gene were selected. These 28 markers provide goodcoverage as determined by the tagging functionality(35) in HAPLOVIEW (36) (settings: see ‘Methods’). Inaddition, all coding SNPs with an allele frequency4 1% in Caucasians as judged by dbSNP or theliterature were included. In addition, the major SNPsthat have been reported to be associated with diabetesor obesity were genotyped (16–18). A selection ofSNPs that have been reported to cause lipodystrophicinsulin resistance was also genotyped (19–21). Figure 2shows the distribution of markers across the gene andthe regional haplotype structure as generated from theHapMap Caucasian genotypes (category CEU) byHAPLOVIEW. As shown in Fig. 2, the markers tag allmajor haplotype blocks of this gene.

The SNP panel was genotyped in both patientpanels as shown in Table 1. No marker showeddeparture from Hardy–Weinberg equilibrium(P4 0.1), indicating robust genotyping performancein this experiment. Single-point allelic and genotypicassociation tests were performed, yielding nominal P-values ranging from 0.046 to 0.98. In order to improvepower – i.e. to detect association with variants on thehaplotypes not directly tagged by one of the SNPs inthe experiments – a sliding window haplotype analysisusing window sizes of two to five markers wasperformed in the high-risk patient sample (panel A).Here, a nominal significance level of 0.04 was obtainedfor the window size 3 for the haplotype spanning fromrs2938395 to rs796290 and from rs4684104 tors709163 for the window size 5 respectively. However,none of the neighbouring haplotypes showed evidenceof association. Coding SNPs P12A showed no signifi-cant difference in allele or genotype frequencies be-tween cases and controls. For variant rs1800571, theannotation of the NCBI-reference sequence P113Qwas used. This SNP is often also referred to as P115Qin the literature. Coding mutations P113Q, P388L andR425C were monomorphic in both cases and controlsin all investigated samples. Table 2 lists all the resultsfor this gene.

Eleven SNPs were selected for APOBEC1 using thesame approach as described above for PPARG. Theknown coding SNP rs2302515, which leads to anamino acid exchange of isoleucine to methionine, wasincluded in the design. This variant showed no evi-dence of association with the gallstone trait (P4 0.1 inboth the genotypic and allelic single-point associationtest). Single-point analyses for genotypic and allelicassociation yielded nominal P-values in the range from0.06 to 0.94. The sliding window-haplotype analyses inthe high-risk patient sample (panel A) resulted innominal significance levels between 0.10 and 0.91.

Fig. 1. The figure shows a graphical representation of a powerestimation in the combined samples A and B at a significancelevel of Po 0.05 for a two-sided test over an odds ratio (OR)range of 1.0–2.2. The graph was generated using PS-power (33)and shows the test power as a function of the OR (x-axis).



The SNP overview and all results of the associationanalysis are presented in Table 3 for APOBEC1 (Fig. 3).

In order to look for a genotype-specific gallstonerisk in the presence of the covariates of age, sex andBMI, a logistic regression analysis over both patientsamples was performed. For each marker, the modelcontained age, sex and BMI and the respective markergenotypes. Age, sex and BMI were consistently signifi-cant in all models (Po 0.01) – the results for theindividual genetic markers are reported in Tables 2 and3 and are consistently negative.

Discussion

The investigation of gallstone susceptibility in mice hasled to a genomic map of gallstone risk loci in thisspecies. The ‘Lith6’ locus was formally reported in 2003as a new quantitative trait locus (QTL) for cholesterolgallstones from an intercross of CAST/Ei and DBA/2J

(37). This region on mouse chromosome 6 had shownsome degrees of linkage evidence in multiple earlierstudies (9, 10, 38) and was also seen in a QTL analysisfrom an intercross of PERA/Ei and I/LnJ inbred mousestrains with an LOD score of 2.0 (11). Thus, Lith6represents one of the best replicated Lith loci in themouse system. The investigation of human homologuesof the functional candidate genes in these intervalsrepresents an intuitive approach to potential risk var-iants for the common gallstone trait in humans (11, 13).

The patient panel A reported here has utilized over800 patients, who have been operated for symptomaticgallstone disease. The population median age of affec-tion in North Germany for gallstones is about 65 years(30), i.e. approximately 50% of the patients developthe disease up to this age. In order to further improvepower, only patients with an age of onset below 65years were included in this study, yielding a medianage of onset of 50 years in cases. In a further attempt to

Fig. 2. Overview of the physical and genetic structure of the peroxisome proliferator-activated receptor gamma (PPARG) gene regionas generated from the Caucasian HapMap genotypes by HAPLOVIEW (36): The upper panel shows the identifiers of the single nucleotidepolymorphisms (SNPs) in relation to their physical position according to the NCBI genome assembly build 34. The coordinates of thegenomic interval are given in this panel. The exon structure of the PPARG transcripts is given below the SNP list also on the physicalsequence scale. Below the exon structure, a translation of the physical positions into the equidistant presentation of linkagedisequilibrium is presented. The lower panel gives an overview of the pairwise linkage disequilibrium structure of the locus (D0),as generated by HAPLOVIEW (36) from the Caucasian HapMap data.



Tab

le2.

Res

ults

of

the

gen

etic

asso

ciat

ion

anal

ysis

of

the

inve

stig

ated

taggin

gan

dco

din

gSN

Ps(b

old

-typ

e)in

the

PPA

RG

gen

e

dbSN

Pid

Posi

tion�

Panel

APa

nel

BA

1B

Panel

A

MA

Fca

seM

AF

contr

ol

OR

Rec

OR

dom

P alle

licP g

eno

MA

Fca

seM

AF

contr

ol

OR

Rec

OR

dom

P alle

licP g

eno

P reg

HA

P2H

AP3

HA

P4H

AP5

rs7632481

50 fl

anki

ng

0.4

50.4

51.0

30.9

70.9

10.8

10.4

50.4

80.8

10.8

50.3

00.5

80.3

80.7

10.2

20.1

40.1

9rs

12635120

50 fl

anki

ng

0.2

00.2

10.7

40.9

40.4

00.5

20.1

90.1

80.8

81.0

80.7

90.7

70.4

00.4

00.2

50.1

40.0

9rs

2960420

50 fl

anki

ng

0.3

50.3

70.8

90.8

60.2

50.3

30.3

80.3

71.0

71.0

10.8

40.9

40.4

40.6

90.5

90.3

00.2

5rs

4279078

50 fl

anki

ng

0.1

20.1

20.6

41.0

40.9

50.4

50.1

10.0

80.7

11.4

0.1

30.1

70.4

80.9

50.4

20.3

30.2

5rs

4684846

50 fl

anki

ng

0.3

0.2

90.9

21.1

40.5

70.1

70.2

90.3

20.7

40.8

80.2

40.4

80.7

60.4

10.4

00.5

20.3

4rs

12493718

50 fl

anki

ng

0.2

90.2

90.9

31.1

0.6

90.3

50.2

90.3

10.8

10.9

20.4

30.7

10.7

00.5

50.6

80.4

10.6

2rs

7638903

50 fl

anki

ng

0.1

40.1

50.8

30.9

40.5

50.8

10.1

60.1

70.9

70.9

10.5

90.8

20.1

90.8

60.3

60.4

80.6

0rs

10510418

50 fl

anki

ng

0.2

90.2

80.8

71.0

90.8

40.2

80.3

00.2

61.1

1.4

0.1

00.0

50.1

10.4

30.5

20.6

60.5

9rs

6802898

50 fl

anki

ng

0.1

40.1

50.9

60.9

40.6

30.8

70.1

70.1

80.8

90.9

10.5

60.8

40.2

30.3

80.5

70.4

60.4

1rs

1801282

ex1

P12A

0.1

40.1

50.9

20.9

20.4

80.7

60.1

60.1

71.0

90.9

10.6

80.8

00.1

70.8

10.5

60.7

00.8

4rs

4135256

intr

on

0.0

20.0

3N

Aw

0.9

10.7

60.5

60.0

30.0

3N

Aw

1.1

0.7

70.7

60.7

80.9

50.9

50.9

40.9

1rs

2120825

intr

on

0.1

30.1

30.8

91.0

30.9

00.8

90.1

40.1

50.9

80.9

30.7

10.9

10.2

40.7

90.9

20.7

10.1

1rs

1800571

ex3

P113Qz

00

NAw

NAw

NAw

NAw

00

NAw

NAw

NAw

NAw

NAw

–‰–‰

–‰–‰

rs10510419

intr

on

0.1

50.1

50.7

51.1

40.5

00.2

20.1

40.1

70.7

20.7

90.1

40.3

40.9

60.7

90.5

50.0

70.1

6rs

2938395

intr

on

0.3

40.3

40.9

90.9

50.7

80.8

50.3

50.3

21.0

41.3

40.2

40.0

60.1

80.3

60.0

40.1

20.1

8rs

4135268

intr

on

0.0

70.0

90.6

10.8

40.1

70.3

90.0

70.0

51.0

31.2

70.3

20.5

80.4

70.5

40.6

00.7

10.3

9rs

796290

intr

on

0.5

00.4

81.0

91.2

20.5

10.1

10.4

90.5

00.9

50.9

30.7

90.9

00.1

60.5

40.6

40.2

70.8

1–

ex6

F388L

00

NAw

NAw

NAw

NAw

00

NAw

NAw

NAw

NAw

NAw

–‰–‰

–‰–‰

rs709157

intr

on

0.2

70.2

70.9

61.0

10.9

80.9

50.2

80.2

70.8

41.1

50.7

20.2

60.5

10.5

90.3

20.8

20.6

2rs

1175540

intr

on

0.3

20.3

11.1

10.9

90.8

20.7

30.3

10.2

91.0

61.1

90.3

90.4

30.1

80.6

60.8

80.5

00.1

0–

ex7

R425C

00

NAw

NAw

NAw

NAw

00

NAw

NAw

NAw

NAw

NAw

–‰–‰

–‰–‰

rs3856806

ex7

H447H

0.1

40.1

41.2

0.9

90.8

80.8

20.1

50.1

50.6

21

0.8

30.6

90.6

00.8

00.4

30.2

30.2

4rs

1152003

30 fl

anki

ng

0.3

40.3

31.1

61.0

60.4

30.7

00.3

70.3

41.0

51.3

0.3

20.1

00.2

50.4

50.1

80.1

80.3

1rs

9833097

30 fl

anki

ng

0.1

20.1

1.7

21.1

70.1

40.3

00.1

30.1

21

1.0

20.9

40.9

80.2

00.1

50.2

20.1

70.1

9rs

1152004

30 fl

anki

ng

0.1

70.1

90.5

90.8

70.1

10.1

80.1

70.1

70.8

31.0

60.8

70.8

10.4

70.2

50.3

00.2

80.3

6rs

4684104

30 fl

anki

ng

0.4

0.4

10.9

30.9

50.6

40.8

90.4

30.4

11.2

21.1

60.3

40.6

10.6

00.8

10.2

90.3

00.0

4rs

1181795

30 fl

anki

ng

0.1

00.1

02.6

60.9

70.9

00.2

70.1

20.1

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maximize the power of this investigation, only gall-stone-negative (as determined by abdominal ultra-sound investigation) control individuals with amoderately higher median age (64 years) were used.Many polygenic disorders are characterized by a strongcorrelation between the age of onset of affectedrelatives, as has been documented for instance inbreast cancer (39) and Alzheimer disease (40). It isindeed plausible that the genetic influence upon thedevelopment of such disorders is reflected by the age atwhich individuals develop the disorder (41). Thegallstone hazard ratio may therefore be higher in theseyounger patients than the background familial risk oftwo to four reported for gallstones (4, 5, 42–46). Thus,the investigated sample represents a ‘high-risk’ groupof patients that should provide an increased experi-mental power as compared with a general populationsample. In addition, nominal P-values were reportedfor all tests, including the haplotype associations tofurther improve power.

The international HapMap project (www.hapmap.org) has generated a wealth of genotype and markerinformation that significantly aids in the design ofcandidate gene studies (34). For this candidate genestudy, a primary haplotype-tagging approach waschosen, i.e. the genetic variation at both loci wascaptured with a set of carefully selected variants. Thistagging approach is able to detect signals from un-known regulatory or functional elements (35, 47, 48).It thus potentially even offers advantages over a directmutation detection of the coding region of a gene only,because disease susceptibility may also be conferred byother variants, for instance in splice sites or intronicenhancers (49, 50). Tagging SNPs were selected fromthe public HapMap (www.hapmap.org) SNP resources(34). The genotype and allele frequencies of the SNPsinvestigated here were not significantly different fromthe ones for the Caucasian HapMap individuals(P4 0.11; allele frequencies in Tables 2 and 3), thussupporting the selection of variants from this resource.Coding SNPs as described in ‘Results’ were alsoincluded in the marker panel.

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mutations were not found in any of the 1178 patientsand 1068 controls screened. These SNPs were includedfor the sake of completeness: for the syndromaticmutations identified in lipodystrophic insulin resis-tance, their presence in the investigated patient samplewas unlikely owing to the very low frequency of theunderlying syndrome. Variant P115Q (P113Q) wasnot identified in additional patient samples apart fromthe original study (18), indicating an exceedingly lowfrequency of the variant in German Caucasians (51).

The coding SNP rs2302515, which leads to anamino-acid exchange at amino acid position 60 in theAPOBEC1 gene, also showed no evidence of associa-tion with the trait under study. This variant is alsolocated outside the functionally most relevant regionsof the protein, which were studied in depth in anearlier mutagenesis experiment (52).

In summary, this association study is negative forboth genes in both patient samples. A logistic regres-sion analysis in the presence of covariates sex, age andBMI was negative. Thus, in the investigated Germansample, these two loci do not represent major riskfactors for common symptomatic gallstone disease.The mouse Lith6 locus spans a large amount ofgenomic sequence in the mouse and – consequently –also in the orthologous human region. Thus, furtherfine mapping of the mouse gallstone loci and identifi-cation of the causative variants in the animal system isneeded to transfer these discoveries successfully to thehuman disease. Potentially, association mapping of therespective human regions may offer advantages owingto the lower level of disequilibrium in human popula-tions and the abundant genomic resources in humans.Ultimately, the mapping efforts in mice and humans

Fig. 3. Overview of the physical and genetic structure of the APOBEC1 gene region. This gene is transcribed from the genomic minusstrand. The panels are otherwise organized as in Figure 2.



may have to be combined to untangle the genetic basisof gallstones.

Acknowledgements

This study was supported by the German National Genome

Research Network (NGFN) through the POPGEN Biobank

(BmBF 01GR0468), the National Genotyping Platform and

the NGFN network for environmental disorders. The co-

operation of all patients, their families and physicians who

participated in this study is gratefully acknowledged. Espe-

cially, the help of Christine Hoell, Huberta von Eberstein

(POPGEN Biobank project), the ‘‘Genotyping TA’s’’ and of

the heads of the surgical departments Ilka Vogel (Stadtisches

Krankenhaus Kiel), Marco Sailer (Hamburg Bergedorf),

Hermann Dittrich (Rendsburg), Jurgen Belz (Husum),

Rainer Quaschling (Eckernforde), Hodjat Shekarriz (Schles-

wig), Volker Mendel (Flensburg), Werner Neugebauer

(Flensburg), Jiri Klima (Niebull), Friedrich Kallinowski

(Heide) and Anton Schafmayer (Luneburg) is gratefully

acknowledged. Helpful discussions and scientific guidance

from Thomas Bosch (Kiel) are especially acknowledged.

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Investigation of the Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease

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Transcript of Investigation of the Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease