Brief Report

Genome-Wide Linkage Scan for BladderExstrophy-Epispadias Complex

Michael Ludwig,1* Franz Ruschendorf,2 Kathrin Saar,2 Norbert Hubner,2 Lothar Siekmann,1

Simeon A. Boyadjiev,3 and Heiko Reutter4,5

1Department of Clinical Biochemistry and Pharmacology, University of Bonn, Bonn, Germany2Max-Delbruck-Centre for Molecular Medicine, Berlin, Germany

3Section of Genetics, Department of Pediatrics, University of California Davis, Sacramento, California4Institute of Human Genetics, University of Bonn, Bonn, Germany

5Department of Neonatology, Children’s Hospital, University of Bonn, Bonn, Germany

Received 1 July 2008; Revised 8 August 2008; Accepted 8 August 2008

BACKGROUND: The bladder exstrophy-epispadias complex represents a spectrum of urogenital anomaliesin which part or all of the distal urinary tract fail to close and are exposed on the outer abdominal wall. Pre-vious studies are suggestive of an underlying multifactorial mode of inheritance. However, no genetic ornongenetic factor has been identified so far. In this study, we sought risk loci by parametric and nonpara-metric linkage analysis, searching for homozygous segments, and more complex inherited loci, respectively.METHODS: Two pedigrees, Spanish and German, each comprising two members affected with classicalbladder exstrophy, were analyzed by genome-wide linkage scan. RESULTS: Evidence for possible risk/modi-fying loci on chromosomes 2p22.1–p21, 2p25.2–p25.1, 4q23–q32.3, 7q21.3–q33, 7q34–q36.1, 14q31.1–q32.2, and19q13.33–q13.43 (LOD scores >1.50) was obtained. CONCLUSIONS: This study was the first positionalapproach to identify chromosomal candidate regions causally related to bladder exstrophy-epispadias com-plex. Our results suggest the presence of susceptibility genes in the regions identified. These regions need tobe confirmed in future studies. Birth Defects Research (Part A) 85:174–178, 2009. � 2008 Wiley-Liss, Inc.

Key words: bladder exstrophy-epispadias complex; linkage analysis; SNP markers; genome-wide linkagescan; exstrophy; epispadias

INTRODUCTION

The bladder exstrophy-epispadias complex (BEEC) is arare birth defect comprising a clinical spectrum rangingfrom isolated epispadias to classic bladder exstrophy(CBE) to its most severe form, cloacal exstrophy (CE), of-ten referred to as OEIS (omphalocele, exstrophy, imperfo-rate anus, and spinal defects) complex (Gearhart andJeffs, 1998; Carey, 2001). The reported prevalence of CBE,the most common form of the BEEC, varies among Euro-pean descendents between 1:20,000 and 1:80,000, withmales being affected more often than females (Shapiroet al., 1984; ICBDMS, 1987; Boyadjiev et al., 2004; Ludwiget al., 2005; Gambhir et al., 2008).

Descriptive epidemiological and clinical data suggestmale gender, race, and advanced parental age (Boyadjievet al., 2004), increased parity even after adjusting for age(Byron-Scott et al., 1998), and in vitro fertilization (Wood

et al., 2003, 2007) as disposing risk factors. Although nogenetic risk factor has been identified so far in humans, acomplex genetic mode of inheritance has been assumed

Parts of this article have been presented at the 57th Annual Meeting of theSuddeutsche Gesellschaft fur Kinderheilkunde und Jugendmedizin, 11.-13.04. 2008, Ulm. PS-24 (abstr.)Grant sponsor: German Federal Ministry of Education and Research (Bundes-ministerium fur Bildung und Forschung, BMBF).Grant sponsor: The German and the Swiss Selfhelp group.Michael Ludwig and Franz Ruschendorf contributed equally.Michael Ludwig and Heiko Reutter are members of the Network forSystematic Investigation of the Molecular Causes, Clinical Implications andPsychosocial Outcome of Congenital Uro-Rectal Malformations (CURE-Net).*Correspondence to: Michael Ludwig, Dept. of Clinical Chemistry and Phar-macology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn.E-mail: mludwig@uni-bonn.dePublished online 11 December 2008 in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/bdra.20512

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� 2008 Wiley-Liss, Inc. Birth Defects Research (Part A) 85:174�178 (2009)

to underlie the BEEC (Boyadjiev et al., 2004). This hasbeen supported by an estimated recurrence risk amongCaucasian siblings of 0.5–2%, a 400-fold (ks 400) increasecompared to the general population (Shapiro et al., 1984;Reutter et al., 2003). Furthermore, pairwise concordancerates among monozygotic and dizygotic BEEC twin pairsof 45% (95%CI: 29–62) for monozygotic and 6% (95%CI:0.1–27) for dizygotic twins are suggestive of genetic influ-ence (Reutter et al., 2007). Familial occurrence is rare,with only 23 families being reported to date (Keppler-Noreuil, 2001; Boyadjiev et al., 2004; Kajbafzadeh et al.,2006). All of these families, except two, have two affectedmembers. In these two families described, there are threeaffected members of both genders, which, however, showvariable defects (Keppler-Noreuil, 2001; Boyadjiev et al.,2004). Most data on these familial cases suggest autoso-mal-dominant inheritance with reduced penetrance(Reutter et al., 2003). To our knowledge, the presentstudy represents the first approach to identify susceptibil-ity genes involved in the etiology of the BEEC by using agenome-wide linkage scan.

MATERIALS AND METHODSSubjects

Blood samples were obtained from all available familymembers with their informed consent and the study wasapproved by the Ethics Committee of the University ofBonn. All family members were recruited by H. R., andbecause patients had undergone most of their surgicalreconstructions, no photographs were taken. However,medical charts of all affected were carefully studied andthe physical examination that had been performed gave aclear picture of the clinical situation.

The first family (no. 33) is a five-generation familyfrom Germany with nine subjects, including two CBEpatients (male and female) who are third-degree cousins.This family has been described before by Reutter et al.(2003). The second family (no. 83), which has not beendescribed previously, is a consanguineous, in regards tothe LOD-calculation applied, five-generation family fromSpain with two CBE males, where 10 individuals couldbe tested. In both these families probands underwent pri-mary bladder closure and bladder neck reconstruction.

Genotyping

Blood DNA was purified and the whole-genome geno-typing scan was performed as described previously (Tim-mann et al., 2007). In brief, a high density SNP genomescan was performed using a whole-genome samplinganalysis approach (Kennedy et al., 2003) with theAffymetrix GeneChip Human Mapping 10K v2 Arraycomprising 10,032 SNP markers with an average hetero-zygosity in Caucasians of 38% and a mean intermarkerdistance of 258 kb/0.36 cM. Mapping order and geneticdistances of markers were obtained from Affymetrix(Affymetrix, NetAffx Annotation files, http://www.affymetrix.com).

Statistical Analysis and Quality Control

Parametric and nonparametric linkage analysis wasperformed with Merlin (Abecasis et al., 2002). Deceased

relatives were entered as unknown, whereas alive mem-bers where no DNA was available (not tested) wereentered as not affected. For a dominant model we usedtrait locus mutant allele frequency of 0.0001 and incom-plete penetrances (0.00, 0.80, 0.80) for homozygous wild-type, heterozygous, and homozygote mutant genotypes,respectively. The recessive model was calculated with atrait locus mutant allele frequency of 0.001 and completepenetrances (0.00, 0.00, 1.00). Allele frequencies from aCaucasian population were used (NetAffx SNPannotationfile).

The graphical user interface ALOHOMORA was usedfor Data Management and Quality Control (Ruschendorfand Nurnberg, 2005). Gender of individuals was esti-mated by counting heterozygote genotypes of X-linkedmarkers for each sample, and compared to the pedigreedefinition. The correct pedigree structure was checked bythe program Graphical Relationship Representation (Abe-casis et al., 2001). All SNPs were analyzed and Mendelianerrors were revealed by PedCheck (O’Connell andWeeks, 1998) and deleted in the family where theyappeared. Non-Mendelian errors or unlikely genotypeswere identified by Merlin (Abecasis et al., 2002) anddeleted in the individual where they appeared. Giventhis and to avoid inflated LOD scores a total of 252markers were excluded from the analysis. To reduce link-age disequilibrium, we also calculated with a depletedset of SNPs: ALOHOMORA allows presetting of a mini-mum spacing (here: 10,000 nucleotides in the case ofmaximum heterogeneity LOD [HLOD] calculation)between the markers that resulted in a dataset compris-ing 6,989 SNPs.

RESULTS

At least three regions yielded suggestive positiveresults in both families investigated (Table 1). In family33 (Fig. 1A), two maximum LODs of 1.916 in regions2p22.1–p21 and 14q31.1–q32.2 were found under a domi-nant model (Fig. 1B,D). These regions were confirmedunder a recessive model with full penetrance (LODs1.906). An additional lower double-peak on chromosome7 (corresponding to 7q21.3–q33 and 7q34–q36.1) wasobserved under the dominant model, but only the firstpeak was also found under the recessive model (Fig. 1C).In family 83, calculations yielded three LOD score peaksof 1.45 in the dominant model in regions 2p25.2–p25,4q23–q32.3, and 19q13.33–q13.43 (Fig. 2). Here, the reces-sive model yielded LODs of 1.504 for the same regions.

Although there was no apparent overlap in the regionsidentified in these families a joint analysis was per-formed. As expected, this analysis gave no significantresults (data not shown).

DISCUSSION

Our genome-wide scan for BEEC yielded seven lociwith a parametric LOD score close to or above 1.5.Unfortunately, as recently summarized (Ludwig et al.,2005), none of the structural aberrations identified inBEEC patients so far affects one of the susceptibility locidetermined here. Aside from seven BEEC cases with nu-merical chromosomal anomalies (47,XXX [observedtwice]; 47,XXY; 47,XYY; 47,XX,121; 47,[no sex re-ported],118; 45,X0/46XX [mosaic]), structural aberrations

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were found in two patients with isolated epispadias(46,XY,dup[9p]; 46,Xydel[4][p?-p?]), in two CBE cases(46,XY,t[8;9][p11.2;q13]; 46,XY,t[2;9][q13;q32]), in two CEpatients (46,X,der[Y]t[Y;9][q11.23;q34.1]-del[Y][q11.2]der[9]t[Y;9]; 46,XY,del[3] [q12.2q13.2]; Kosaki et al., 2005]),

and in one CE patient with Hypomelanosis Ito (diploid/tetraploid/t[1;6] mosaicism), respectively.

On the other hand, possible candidate genes arelocated within the regions identified in the present study,which might contribute to the development of BEEC

Figure 1. (A) Pedigree of family 33 (Germany) with classical bladder exstrophy. Affected individuals are shown with blackenedsymbols, and samples included in the analysis as unaffected but where no DNA was available are indicated by nt (not tested). (B–D)Maximum LOD score values obtained from Merlin parametric analysis predicted under a dominant (red lining) or recessive model (bluelining) for regions on chromosomes 2, 7, and 14.

Table 1Genome-Scan Results, Indicating Suggestive Evidence at Various Chromosomes

Flanking SNP MarkersPosition Parametric

Cytogenetic Physical (bp)* LOD

Family 33 rs1368113 rs1368086 2p22.1–p21 38,585,994 – 43,876,658 1.916y

rs1375671 rs1378647 7q21.3–q33 96,166,612 – 136,090,438 1.906y

rs1986764 rs722933 7q34–q36.1 141,872,311 – 147,601,309 1.906y

rs54566 rs1951094 14q31.1–q32.2 79,705,600 – 98,140,120 1.916y

Family 83 rs771166 rs953071 2p25.2–p25.1 6,911,295 – 11,957,213 1.504{

rs1540053 rs4129776 4q23–q32.3 100,439,332 – 165,639,920 1.504{

rs3810261 rs1368467 19q13.33–q13.43 54,914,726 – 62,128,208 1.504{

*Derived from the NCBI (Build 35.1).yLOD 5 1.916 for regions on chromosome 2 and 14 in a dominant model with complete penetrance and LOD 5 1.906 for regions on

chromosomes 2, 7, and 14 in a recessive model with complete penetrance.{LOD 5 1.45 in a dominant model with 80% penetrance.

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and/or its severity. These include, amongst others,PLEKHH2 on 2p21, which is involved in membrane andjuxtamembrane targeting (DiNitto and Lambright, 2006).Also, the region of interest on chromosome 19 harborsthe serine protease gene cluster with 13 KLK (Kalikrein-related peptidases: KLK) genes. KLKs are involved in theregulation of cell differentiation and tissue regenerationand most of these genes show a tissue-specific expressionprofile (e.g., skin or prostate) (Gan et al., 2000).

From the pedigree structures an autosomal dominant,intermediate, or recessive trait involving each of therisk/modifying loci identified might explain the disease,whereas an X-linked mode seems to be more unlikely, atleast in the German family, where both sexes areaffected. Alternatively, depending on the type of muta-tion, a given mutation in the same gene may be inheritedin a recessive or dominant fashion influenced by muta-tions in (an)other gene(s) (Badano and Katsanis, 2002).Thus, according to a non-Mendelian model, patients maycarry zero, one, or two mutated alleles at one locus,which, together with other risk/modifier alleles, wouldinfluence formation and strength of the BEEC.

Finally, it might be possible—though not very likely—that our data obtained are just chance findings. Because

this study, to our knowledge, represents the first ge-nome-wide scan for BEEC, further studies analyzing in-dependent families need to be performed to replicatethese data and narrow down the suggested regions.

ACKNOWLEDGMENTSWe thank the patients and their family members for

their cooperation.

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