Genomics 85 (2005) 622 – 629 www.elsevier.com/locate/ygeno
A reciprocal translocation 46,XY,t(8;9)(p11.2;q13) in a bladder exstrophy patient disrupts CNTNAP3 and presents evidence of a pericentromeric duplication on chromosome 9 Simeon A. Boyadjieva,T, Sarah T. Southb, Cristi L. Radforda, Ankita Patelc, George Zhanga, David J. Hura, George H. Thomasa,d, John P. Gearharte, Gail Stettena,b a
McKusick–Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, 733 N. Broadway, BRB 469, Baltimore, MD 21205, USA b Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA c Kleberg Cytogenetics Laboratory, Baylor College of Medicine, Houston, TX, USA d Kennedy Krieger Institute, Baltimore, MD, USA e Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Received 5 August 2004; accepted 11 January 2005
Abstract A patient with sporadic bladder exstrophy and de novo apparently balanced chromosomal translocation 46,XY,t(8;9)(p11.2;q13) was analyzed by fluorescence in situ hybridization (FISH) and molecular methods. We were able to map both translocation breakpoints to single genomic clones. The chromosome 8p11.2 breakpoint was mapped to BAC clone RP4-547J18, predicted to contain several hypothetical genes. Characterization of the chromosome 9q13 breakpoint indicated a disruption in the 5Vregion of CNTNAP3 within BAC RP11-292B8. This observation suggests possible involvement of CNTNAP3 in the etiology of bladder exstrophy. Additionally, FISH analysis identified several genomic copies of CNTNAP3 on both sides of the chromosome 9 centromere flanking the polymorphic heterochromatin. Northern blot analysis of lymphoblast and bladder RNA confirmed CNTNAP3 transcripts in these tissues and did not show abnormal CNTNAP3 expression in the proband and two unrelated patients with bladder exstrophy. The identification of multiple copies of three BAC clones in the proband, his parents, and unrelated controls suggests that duplications of CNTNAP3 and the surrounding genomic region have occurred as a result of repeated events of unequal crossing over and pericentric inversions during chromosome 9 evolution. D 2005 Elsevier Inc. All rights reserved. Keywords: Bladder exstrophy–epispadias complex; Contactin-associated protein-like 3 (CNTNAP3); Chromosome 9; Translocation breakpoint mapping
Introduction Classic bladder exstrophy is a congenital anomaly in which the urinary bladder fails to close and is exposed on the outer abdominal wall. It is believed that this condition is a part of a clinical spectrum of the bladder exstrophy–epispadias complex (BEEC), a single developmental disorder lacking clear Mendelian mode of inheritance [1,2]. The phenotypic severity of BEEC ranges from isolated epispadias through classic bladder exstrophy to cloacal exstrophy, which T Corresponding author. Fax: +1 410 502 1853. E-mail address:
[email protected] (S.A. Boyadjiev). 0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2005.01.002
manifests with omphalocele, exstrophy of the bladder, imperforated anus, and spinal defects—OEIS complex [3]. The reported incidence of classic bladder exstrophy varies, but the most commonly cited figure is 1 in 30,000 live births [4]. The majority of BEEC cases are sporadic, with less than 3% of the cases being familial [5]. BEEC has been reported in several monozygotic twin pairs, and in at least two instances the twins were concordant for the defect [6,7]. The recurrence risk for an affected individual is thought to be 1 in 70 live births, which is more than 400-fold greater than the risk in the general population [8]. These observations suggest that genetic factors may be involved in the etiology of BEEC.
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Few chromosomal abnormalities associated with BEEC have been reported. Exstrophy of the cloaca associated with unilateral renal agenesis and mullerian anomalies was reported in a child with 47,XXX [9]. In a series of 86 patients with cloacal exstrophy, 3 were found to have aneuploidy (47,XX,+21; 45,X/46,XX; and 47,XXX) [10]. It is not clear whether these chromosomal defects simply coincide with BEEC or are actually causative. Unlike aneuploidies, structural chromosomal abnormalities such as translocations, deletions, and insertions involving breakpoints may be more helpful in identifying possible candidate loci for BEEC. However, few patients with this birth defect and structural chromosomal abnormalities have been reported [11,12]. Recently, an infant with 9q34.1-qter deletion resulting from a de novo unbalanced translocation between chromosomes 9q and Yq was described in an infant with cloacal exstrophy [13]. In an attempt to identify genetic and nongenetic factors contributing to the risk of BEEC we have initiated the characterization of affected families by epidemiological, clinical, and molecular methods [14]. We identified a male patient with classic bladder exstrophy who has a de novo apparently balanced chromosomal translocation, 46,XY, t(8;9)(p11.2;q13). We were able to demonstrate that the chromosome 9q13 breakpoint maps within BAC clone RP11-292B8, which contains a single gene, CNTNAP3. BAC RP4-547J18 was found to span the chromosome 8p11.2 breakpoint. In the process of mapping the translocation breakpoints we observed that three bacterial artificial chromosome (BAC) clones assigned at that time to chromosome 9q13 are present in multiple copies flanking the centromere of chromosome 9. The duplications of these genomic clones were present in the proband, his parents, and four unrelated controls. Chromosome 9 contains a heterochromatic region just below the centromere that is highly polymorphic in size and orientation [15]. Approximately 8% of the population has increased heterochromatic material, 5% have a very small or absent area, and 3% have a pericentric inversion involving all or part of this region [16]. We suggest that CNTNAP3 and/or other genes on chromosome 9q13 or chromosome 8p11.2 may contribute to the etiology of BEEC. The unexpected observation of multiple BAC copies indicates that segmental duplications of the pericentric region of chromosome 9 and its flanking genes have occurred due to combination of repeated pericentric inversions and unequal crossing over during evolution.
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www.bladderexstrophy.com). The family provided detailed past medical records, including surgical and clinical genetic evaluation, and donated blood samples for molecular analysis. The family history was unremarkable. Review of the available documentation confirmed that the proband has classic bladder exstrophy not associated with other congenital anomalies or developmental delay. An anecdotal report of a chromosomal translocation that was detected soon after birth was independently confirmed at our institution. A reciprocal chromosomal translocation, 46,XY,t(8;9)(p11.2;q13), was identified in the proband (Fig. 1). Both his parents had normal karyotypes. Mapping of the translocation breakpoint We used FISH probes prepared from BAC clones known to map in the vicinity of the breakpoints in an attempt to identify candidate genes for bladder exstrophy. For chromosome 9q13 analysis we used BAC clones RP11-366N18 (GenBank Accession No. AL445584.16), RP11-292B8 (GenBank Accession No. AL162233.14), and RP11-290L7 (GenBank Accession No. AL353729.17). FISH probes prepared from these BACs were hybridized to both metaphase chromosomes and interphase nucleus preparations from the proband, his parents, and four control individuals. The 9q13 copy of the most centromeric BAC RP11366N18 is proximal to the breakpoint, thus resulting in signals on both the p and q arms of the normal chromosome 9 and the derivative chromosome 9 (Fig. 2A). The hybridization patterns on the derivative 9 and derivative 8 chromosomes show that the chromosome 9q13 translocation breakpoint is within the chromosome 9q13 copy of BAC RP11-292B8, resulting in signals on the derivative chromosome 8, the p and q arms of the derivative chromosome 9, and the p and q arms of the normal chromosome 9 (Fig.
Results and discussion Clinical characterization and karyotype analysis The family of an 11-year-old Caucasian boy with corrected classic bladder exstrophy contacted the investigators as a result of an invitation for participation in a study of bladder exstrophy placed on the Internet (http://
Fig. 1. Chromosomal images and ideogram representing de novo reciprocal translocation 46,XY,t(8;9)(p11.2;q13) in a patient with classic bladder exstrophy.
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Fig. 2. (A, D) Partial metaphase and interphase hybridizations with BAC clones RP11-366N18 demonstrate multiple genomic copies of this sequence proximal to the breakpoint. (B, E) BAC RP11-292B8 spans the chromosome 9q13 breakpoint. (C, F) BAC RP11-290L7 is distal to the breakpoint. All three BAC clones are present in multiple copies flanking the pericentric heterochromatic region of chromosome 9.
2B). The 9q13 copy of the most telomeric BAC RP11290L7 is distal to the breakpoint, thereby resulting in signals on the derivative chromosome 8, only the p arm of derivative chromosome 9, and both the p and q arms of the normal chromosome 9 (Fig. 2C). Using a similar strategy we were also able to map the chromosome 8p11.2 breakpoint to BAC clone RP4-547J18 (GenBank Accession No. AC084722, data not shown). At this time no known genes have been mapped to this genomic clone, but several hypothetical genes have been predicted by computational methods (http://genome.ucsc.edu/cgi-bin/ hgTracks?hgsid=38238540 and hgt.out1=1.5x&position= chr8%3A43078551-43255736). We are considering future studies to evaluate if these hypothetical genes are relevant to the bladder exstrophy–epispadias complex. Molecular characterization of the CNTNAP3 gene BAC RP11-292B8, which spans the chromosome 9q13 breakpoint, has been completely sequenced and contains
only one gene, contactin associated protein-like 3 (CNTNAP3), which extends into the overlapping BAC RP11-290L7 (Fig. 3A). The CNTNAP gene subfamily belongs to the larger NCP family (for Neuroxin-IV/ CNTNAP/Paranodin), the biological function of which is thought to be the mediation of neuron–glia interactions [17]. At least four CNTNAP genes with expression restricted primarily to the brain and peripheral nerves have been cloned so far and are presumed to play a role in cell– cell interactions [18–20]. To determine if the translocation in this patient interrupted CNTNAP3, long-range PCR products that spanned various regions of the CNTNAP3 gene were amplified and used as FISH probes. Analysis of the hybridization patterns on the derivative 8 and derivative 9 chromosomes showed that the translocation breakpoint lies within the 23-kb region of the first CNTNAP3 intron (Figs. 3B and C). CNTNAP3 is known to encode several alternatively spliced isoforms, the biological functions of which have not been determined. At least one isoform encodes a transmembrane protein (isoform 1, Accession No. NM_033655) and a shorter isoform 2 (Accession No. NM_024879), is thought to encode a secreted peptide. By BLAST analysis (http://www.ncbi.nlm.nih. gov/BLAST) we identified two additional full-length cDNAs, (GenBank Accession Nos. AK056833 and AKQ 054645), which are likely to represent alternative CNTNAP3 isoforms 3 and 4, respectively (Fig. 3A). All cDNA isoforms map within the two overlapping BAC clones RP11-292B8 and RP11-290L7 that we used for the FISH experiments. We confirmed by RT-PCR that isoforms 1, 3, and 4 are expressed in fetal bladder, normal postnatal bladder, and exstrophic bladder. In addition, using Affymetrix U133 Plus microarrays we demonstrated that CNTNAP3 isoform 1 (Affymetrix probe set 223799_at) is expressed in undifferentiated human embryonic mesenchyme at 8 weeks of gestation and in fetal, normal postnatal, and exstrophic bladders (Boyadjiev et al., unpublished data). In an attempt to determine the effect of the 46,XY,t(8;9)(p11.2;q13) translocation on a molecular level, we analyzed the transcription of CNTNAP3 in the proband and his parents by RT-PCR sequencing analysis. At least two copies of CNTNAP3 were transcribed in the proband and his father based on the presence of polymorphism c.618TNC in both genomic and complementary DNA (Figs. 4A and B). By sequencing, we were unable to determine if the proband with classic bladder exstrophy is hemizygous for CNTNAP3 due to the observed multiple genomic copies of this gene. It is highly likely that some of the transcribed mRNA is nonfunctional as we observed a frameshift due to a 5-bp deletion at the beginning of exon 7 in the proband (Fig. 4A). A larger than expected RT-PCR band was identified in the mother that included a 139-bp unannotated exon inserted between exons 6 and 7 of the CNTNAP3 transcript (Fig. 4C). We were not able to amplify a transcript from her second CNTNAP3 allele. The presence of multiple genomic copies of CNTNAP3 and the observed unusual
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Fig. 3. (A) Schematic organization of CNTNAP3 and its isoforms (not in scale). The locations of the PCR-generated FISH probes and the breakpoint are indicated. (B) FISH analysis with probe A demonstrates that the breakpoint is distal to exon 1 of CNTNAP3. (C) FISH analysis with probe B maps the breakpoint proximal to exon 2.
cDNA forms suggest that more than two transcriptionally active CNTNAP3 alleles may be present in the genome. At this time it remains unclear if the multiple CNTNAP3 isoforms result from an alternative splicing of the CNTNAP3 gene or are transcribed from similar, but divergent copies that have occurred as a result of evolutionary amplification of CNTNAP3. Direct sequencing analysis of blood and bladder DNA from unrelated patients with BEEC identified more than 100 single-nucleotide polymorphisms, of which at least 65 were within protein coding regions. The majority of these DNA variants were also present in control DNA samples.
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To investigate the effect of the translocation on CNTNAP3 expression, we performed northern blot analysis on bladder and lymphoblast RNA, as well as a commercially available Multiple Tissue Northern (MTN) blot. We did not find CNTNAP3 transcripts in bladder tissue on the MTN blot using a probe distal to the translocation breakpoint (exon 1), although a 3.6-kb transcript was detected in the spinal cord (data not shown). However, hybridization of the same probe to a custom northern blot containing lymphoblast RNA of normal controls, the proband with the reported translocation, and another exstrophic patient revealed two CNTNAP3 transcripts (approximately 5.3 and 1.4 kb)(Fig. 5A). A probe proximal to the breakpoint (exons 8 and 9) detected three transcripts of approximately 4.6, 2.6, and 1.4 kb (Fig. 5B). No abnormal CNTNAP3 expression was detected in the affected individuals as compared with controls. As bladder RNA from the proband was not available, we analyzed a northern blot with bladder RNA of two unrelated patients with bladder extrophy and four unaffected controls with the same two probes used for the lymphoblast blot. Transcripts similar in size to those on the lymphoblast blot were observed, and no differences in the expression of CNTNAP3 between affected patients and controls were detected (data not shown). In summary, analysis of these northern blots did not demonstrate aberrant expression of CNTNAP3 in the reported proband nor in other unrelated individuals with bladder exstrophy. We are unable to prove a causal relationship between CNTNAP3 and BEEC. However, subtle dosage changes of CNTNAP3 cannot be excluded by the described methods of analysis. At the same time it is possible that this translocation disrupts a regulatory element for another gene that could be further away on the derivative chromosomes. Such an event has been documented in patients with preaxial polydactyly due to disruption of a cis-acting limb-specific enhancer located approximately 1 Mb away from SHH [21].
Fig. 4. (A) Two CNTNAP3 cDNA species are transcribed in the proband (g, genomic DNA; c, cDNA). A 5-bp deletion (ATCAG, underlined) at the beginning of exon 7 junction leads to a frameshift. (B) Normal biallelic transcription of CNTNAP3 in the proband’s father. (C) Transcription of cDNA from a single CNTNAP3 allele containing previously unannotated exon 6a.
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Fig. 5. Custom lymphoblast northern blot probed with the exon 1 (A) and exon 8–9 (B) probes. Lane 1: Ambion Millenium marker; lane 2: blank control; lanes 3 and 4: RNA from unrelated and unaffected control individuals; lanes 5–7: RNA of the reported proband (*) and his unaffected parents; lanes 8–10: RNA of an unrelated proband with extrophy (A) and his unaffected parents. GAPD was used as a loading control (C). Approximate sizes of the bands are indicated with arrows on the right.
We plan to further investigate whether 8p11.2 and 9q13 chromosomal regions confer increased susceptibility for BEEC by association analysis of case–parent trios using the transmission disequilibrium test [22]. Segmental duplications of the chromosome 9 pericentromeric region In addition to the expected signal on chromosome 9q13, BACs RP11-366N18, RP11-292B8, and RP11-290L7 consistently produced unexpected signals on the proximal short arm of chromosome 9 just above the centromere, suggesting segmental duplication of this genomic region in all samples. Analysis of the hybridization patterns in interphase nuclei showed that there are at least three copies of these BACs on each chromosome 9 in the proband (Figs. 2D–F). The same hybridization pattern was observed in his parents and four unrelated controls and, together with the multiple bands on Southern blot analysis, corroborated the observations of CNTNAP3 duplication (data not shown). Our data suggest that there are between two and five copies of BAC clones RP11-366N18, RP11292B8, and RP11-290L7 above the centromere and a single copy below it on each chromosome 9 among normal individuals. These three clones were initially assigned to chromosome 9q13 but were subsequently reassigned to 9p12 in an opposite order (http://genome.ucsc.edu). In our opinion, the existence of multiple copies of these BACs, which was not detected prior to our experiments, led to these assignment discrepancies. Chromosome 9 is the most polymorphic nonacrocentric chromosome due to variable length of the pericentromeric heterochromatin and a common inv(9)(p11q13) [15]. At
least 12 heteromorphic patterns in amount and position of the chromosome 9 heterochromatin have been documented and reportedly they do not cause problems in reproduction and development. It has been suggested that large-scale variants in the genomes of normal individuals may lead to chromosomal rearrangements that give rise to disease or influence gene expression [23]. DNA rearrangements such as inversions, duplications, deletions, and translocations are thought to occur through nonallelic homologous recombination mediated by low-copy repeats or LCRs [24]. LCRs, also called paralogous segmental duplications, are usually 10–400 kb in size, have greater than 95% similarity, and may constitute more than 5% of the human genome. The pairing in 9p12-9q13 regions may be less stringent due to the LCRs that we have observed. We suggest that these LCRs permit unequal crossing over and small inversions that have led to amplification and redistribution of the heterochromatin and the adjacent genomic regions in which CNTNAP3 and perhaps other genes reside. Such an amplification mechanism may not be unique for chromosome 9. Similar pericentromric heterochromatin regions exist in chromosomes 1 and 16 and may be also implicated in amplification of neighboring genes as suggested by the observation of amplification of the pericentrimeric region on chromosome 1 [25]. Further evidence that the boundaries between euchromatin and heterochromatin frequently acquire sequences as a result of duplication or amplification was provided by the observations of amplification of a pseudogene cassette in the pericentromeric region of chromosome 16 [26]. Although duplications of genes flanking the heterochromatic regions of chromosomes 1 and 16 have not yet been described, we are just beginning to uncover the degree of polymorphic variation in the human genome [23]. We believe that our report is the first to document an amplification of a gene in the euchromatin region bordering a pericientric heterochromatin block as we proved that the CNTNAP3 gene has multiple genomic copies on chromosome 9. Recent sequence analysis data on chromosome 9 has shown intrachromosomal duplications adjacent to both the centromere and the large heterochromatic block, which are consistent with our observations [27]. Further studies are needed to determine if changes in CNTNAP3 and/or in other genes on chromosomes 9q13 and 8p11.2 are risk factors for the occurrence of BEEC.
Materials and methods Subjects This study was approved by the institutional review boards of Johns Hopkins Hospital and was conducted in accordance with the institutional guidelines. Informed consent was obtained from all individuals prior to physical examination and sample collection.
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BAC clone selection and DNA isolation Genomic BAC clones spanning the chromosomal 8p11 and 9q13 regions were selected based on the information from a publicly available genome database (http:// genome.ucsc.edu). Overlapping clones that span these two regions were purchased through BACPAC resources at the Children’s Hospital Oakland Research Institute (http:// bacpac.chori.org). Individual BAC colonies were selected from plates prepared with L-Broth AGAR pellets (BIO 101, Inc.), supplemented with chloramphenicol (25 Ag/ml), and grown overnight in a liquid medium. BAC DNA was isolated using a standard protocol with the Plasmid Midi Kit (Qiagen, Inc). Karyotype analysis and FISH mapping of the breakpoints Chromosomal preparations were made from cultured lymphoblasts according to standard protocols. For FISH mapping with BAC probes, 1 Ag of BAC plasmid DNA was labeled in a nick translation reaction with either Spectrum Green-dUTP or Spectrum Orange-dUTP (Vysis). Two hundred nanograms of labeled probe was then precipitated with a 10-fold excess of human Cot-1 DNA (Gibco) and resuspended in 10 Al of a solution containing 50% formamide, 2 SSC, and 10% dextran sulfate. The probe was then diluted 1:10 in DenHyb solution (Insitus Biotechnologies), denatured for 5 min at 728C, and preannealed at 378C for 30 min. Each BAC probe was hybridized at 378C overnight in a humidified chamber. Following hybridization, slides were washed for 5 min in 2X SSC at 708C followed by a 2-min rinse in 2X SSC at room temperature. Slides were then counterstained with DAPI II (Vysis) and viewed. A minimum of 10 metaphase spreads were scored for each probe, and all images were captured using a Zeiss Axioskop with a SenSys CCD camera (Photometrics) and Quips Smart Capture imaging software (Applied Imaging). Long-range PCR for FISH probes A series of long-range PCRs were performed to generate additional probes for FISH analysis to further refine the breakpoint region on chromosome 9. DNA from BAC clone RP11-292B8 was used as a template for the long-range PCR. The Expand Long Range Template PCR System (Roche Applied Science) was used to amplify PCR fragments between 4 and 8 kb in size with the supplied protocols. The primer sequences and PCR conditions are available on request. The PCR products were purified from gel bands using the GFX PCR Column Purification Kit (Amersham Pharmacia Biotech Inc.) and prepared as probes for FISH analysis. One microgram of PCR product was labeled in a nick translation reaction (Vysis) with digoxigenin-11-dUTP (Roche Applied Science). The labeled probe was hybridized as described above. Following
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hybridization, the slides were washed for 5 min in 2X SSC at 708C followed by a 2-min rinse in 2X SSC at room temperature. For detection of the probe, slides were then incubated in the dark with rhodamine-labeled anti-digoxigenin (Insitus Biotechnologies) for 5 min at 378C, followed by three rounds of 2-min washes with 4X SSC and 0.05% Tween 20 at room temperature. Slides were then counterstained with DAPI II (Vysis) and viewed. A minimum of 10 metaphase spreads were scored for each probe and all images were captured as described above. Sequencing analysis of PCR and RT-PCR products Genomic DNA and total RNA were isolated from lymphoblastoid cell lines of the proband and his parents using standard protocols (Gentra Systems, Inc., and ZR Whole-Blood Total RNA Kit, Zymo Research, respectively). In addition, blood and/or bladder DNA and RNA were extracted from a panel of unrelated BEEC probands for mutation analysis of CNTNAP3. A commercial DNA (Hoffmann–La Roche Ltd.) and fetal bladder RNA samples (Stratagene) were used as control samples. The entire CNTNAP3 gene was sequenced on a panel of lymphoblast genomic DNA and cDNA from BEEC individuals with epispadias, classic bladder exstrophy, or cloacal exstrophy. Considering the possibility of somatic mosaicism, we analyzed paired blood and bladder DNA samples for three of the individuals with classic bladder exstrophy. At least 100 bp of intronic sequence flanking each exon was analyzed for each proband. Different combinations of cDNA primers in the area of exons 9 through 13 were used to detect individual isoforms. The PCR protocols and primer sequences are available on request. PCR products were purified from gel bands or solution using a column purification kit (GFX PCR, Amersham Pharmacia Biotech Inc.). Sequencing was carried out by the dideoxy chain termination method using the Sequenase Version 2.0 DNA Sequencing Kit (U.S. Biochemical) with a relevant primer. The ABI Prism 3700 automated fluorescent DNA analyzer (PE Biosystems) was used. All sequence data were reviewed by at least two independent investigators. Southern blot analysis Southern blot analysis was performed to search for CNTNAP3 rearrangements and/or loss of an allele. Ten micrograms of genomic lymphoblast DNA from the proband, both parents, and four control individuals was digested with either EcoRI or HindIII. The DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond H+ membranes (Amersham Pharmacia Biotech Inc.) by alkaline transfer (1.5 M NaCl and 0.5 M NaOH). Hybridization probes were prepared from cDNA PCR amplicons containing exons 4 through 7 that had been purified and labeled with [32P]-dCTP using standard protocols. Hybridization and wash conditions were per-
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formed according to the manufacturer’s instructions using ExpressHyb solution (Clontech Laboratories Inc.).
and Grants K23 DE00462 (S.A.B.) and NICHD24061 (G.H.T.). This work is dedicated to the memory of Dr. Anton S. Boyadjiev.
Northern blot analysis Total RNA was isolated using a standard TRIzol protocol (Invitrogen) from EBV-transformed lymphoblast cell lines and bladder smooth muscle tissues that were obtained during surgical repair of patients with classic bladder exstrophy. However, no bladder tissue was available from the proband with the reported chromosomal translocation. The tissue samples were immediately preserved in RNAlater solution (Ambion, Inc.) and fat, connective tissue, and urothelium were removed prior to RNA extraction. The integrity of RNA was verified with RNA LabChip kits using the Agilent bioanalyzer (Agilent Technologies), and a custom northern blot with total bladder RNA was prepared. Another blot with 20 Ag of total lymphoblast RNA, including RNA samples from the reported proband and his parents, was also prepared. A commercially available multiple tissue northern blot (BD Biosciences) was used for analysis of CNTNAP3 expression in various tissues. Probes were prepared by PCR of cDNA synthesized from commercially available fetal bladder RNA (Stratagene). To identify aberrant transcription patterns for CNTNAP3 in the reported proband we selected probes that were proximal (ca. 210 to ca.143, covering exon 1 of CNTNAP3) and distal (ca.1081 to ca.1454, covering exons 8 and 9 of CNTNAP3) to the chromosome 9 breakpoint within CNTNAP3 (NM_033655). Primer sequences and PCR conditions are available on request. Probes were labeled with [a-32P] dCTP (3000 Ci/mmol, Perkin–Elmer Life Sciences) using the random prime DNA labeling kit (Roche). Hybridizations were performed overnight at 428C using RapidHyb solution (Ambion, Inc.) with 2 x 106 cpm/ ml of labeled probes. Following hybridization, the membranes were washed twice with 2X SSC, 0.1% SDS, and then twice with 0.1X SSC, 0.1X SDS at 428C, exposed to phosphor Imaging Screen K (Bio-Rad), and developed using the Personal Molecular Imager FX phosphoimager (Bio-Rad). The Quantity One software (Bio-Rad) was used to analyze membranes posthybridization.
Acknowledgments We are grateful to all participating family members for generously donating their time and biological specimens for this study. We thank Nadia Drake, Kamali Carroll, Steve Wowk, and Crystal Lopez for their excellent technical support and the Association for the Bladder Exstrophy Community for hosting an invitation for research participation (http://www.bladderexstrophy.com). These studies were supported in part by Johns Hopkins University School of Medicine General Clinical Research Center Grant M01RR00052, the National Center for Research Resources/NIH,
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