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CNV Analysis in Monozygotic Twin Pairs Discordant for Urorectal Malformations

Published online by Cambridge University Press:  09 May 2013

Friederike Baudisch
Affiliation:
Department of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany
Markus Draaken
Affiliation:
Institute of Human Genetics, University of Bonn, Bonn, Germany Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany
Enrika Bartels
Affiliation:
Institute of Human Genetics, University of Bonn, Bonn, Germany
Eberhard Schmiedeke
Affiliation:
Department of Pediatric Surgery and Urology, Center for Child and Adolescent Health, Hospital Bremen-Mitte, Bremen, Germany
Soyhan Bagci
Affiliation:
Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany
Peter Bartmann
Affiliation:
Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany
Markus M. Nöthen
Affiliation:
Institute of Human Genetics, University of Bonn, Bonn, Germany Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany
Michael Ludwig
Affiliation:
Department of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany
Heiko Reutter*
Affiliation:
Institute of Human Genetics, University of Bonn, Bonn, Germany Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany
*
address for correspondence: Heiko Reutter, Department of Neonatology & Institute of Human Genetics, University of Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany. E-mail: reutter@uni-bonn.de

Abstract

Early post-twinning mutational events can account for discordant phenotypes in monozygotic (MZ) twin pairs. Such mutational events may comprise genomic alterations of different sizes, ranging from single nucleotides to large copy-number variations (CNVs). Anorectal malformations (ARM) and the bladder exstrophy-epispadias complex (BEEC) represent the most severe end of the urorectal malformation spectrum. Recently, CNV studies in patients with sporadic ARM and the BEEC have identified de novo events that occur in specific chromosomal regions. We hypothesized that early arising, post-twinning CNVs might contribute to discordance in MZ twin pairs with ARM or the BEEC; knowledge of such CNVs might help to identify additional chromosomal regions involved in the development of these malformations. We investigated four discordant MZ twin pairs (three ARM and one BEEC) using molecular karyotyping arrays comprising 1,140,419 markers with a median marker spacing of 1.5 kb. Filtering the coding regions for possible disease-causing post-twinning de novo CNVs present only in the affected twin, but not in the unaffected twin or the parents, identified a total of 136 CNVs. These 136 CNVs were then filtered against publicly available databases and finally re-evaluated visually. No potentially causative CNV remained after applying these filter criteria. Our results suggest that post-twinning CNV events that affect coding regions of the genome did not contribute to the discordant phenotypes in MZ twin pairs that we investigated. Possible causes for the discordant phenotypes include changes in regulatory elements or smaller genetic changes within coding regions which may be detectable by whole-exome sequencing.

Type
Articles
Copyright
Copyright © The Authors 2013 

The search for differences in genetic constitutions within discordant monozygotic (MZ) twin pairs has been suggested as a promising approach for gene-finding (Zwijnenburg et al., Reference Zwijnenburg, Meijers-Heijboer and Boomsma2010). This suggestion is based on the observation that very early post-twinning mutational events can cause discordance in MZ twin pairs (Helderman-van den Enden et al., Reference Helderman-van den Enden, Maaswinkel-Mooij, Hoogendoorn, Willemsen, Maat-Kievit, Losekoot and Oostra1999; Kondo et al., Reference Kondo, Schutte, Richardson, Bjork, Knight, Watanabe and Murray2002; Kruyer et al., Reference Kruyer, Mila, Glover, Carbonell, Ballesta and Estivill1994; Taylor et al., Reference Taylor, Thum and Abdalla2008; Zwijnenburg et al., Reference Zwijnenburg, Meijers-Heijboer and Boomsma2010). Such mutational events may comprise genomic alterations at different scales, ranging from changes affecting only single nucleotides to larger copy-number variations (CNVs) of varying sizes, resulting in losses or gains of several thousands to millions of base pairs. The investigation of discordant MZ twin pairs may be applicable not only to monogenic disorders but also to multi-factorial disorders, including congenital malformations. In accordance with this, Breckpot et al. (Reference Breckpot, Thienpont, Gewillig, Allegaert, Vermeesch and Devriendt2012) recently identified three possibly disease-causing de novo CNVs in one affected twin out of six MZ twin pairs discordant for congenital heart defects.

In our study, we applied this strategy for the first time to the investigation of urorectal malformations, specifically anorectal malformations (ARM) and the bladder exstrophy-epispadias complex (BEEC). There is substantial evidence that genetic factors contribute to the development of these malformations; in particular, CNV studies in sporadic patients have identified de novo events that occur in specific chromosomal regions (Bartels et al., Reference Bartels, Draaken, Kazmierczak, Spranger, Schramm, Baudisch and Reutter2011; Boocock & Donnai, Reference Boocock and Donnai1987; Boyadjiev et al., Reference Boyadjiev, Dodson, Radford, Ashrafi, Beaty, Mathews and Gearhart2004; Cuschieri & EUROCAT Working Group, Reference Cuschieri2001; Draaken et al., Reference Draaken, Reutter, Schramm, Bartels, Boemers, Ebert and Ludwig2010; Falcone et al., Reference Falcone, Levitt, Peña and Bates2007; Keppler-Noreuil, Reference Keppler-Noreuil2001; Ludwig et al., Reference Ludwig, Ching, Reutter and Boyadjiev2009; Marcelis et al., Reference Marcelis, de Blaauw and Brunner2011; Schramm et al., Reference Schramm, Draaken, Bartels, Boemers, Aretz, Brockschmidt and Reutter2011a, Reference Schramm, Draaken, Bartels, Boemers, Schmiedeke, Grasshoff-Derr and Reutter2011b; Shapiro et al., Reference Shapiro, Lepor and Jeffs1984). We hypothesized that CNVs affecting coding regions that occur after twinning might contribute to discordance in MZ twin pairs with ARM or the BEEC. To test this hypothesis, we investigated four discordant MZ twin pairs (three ARM and one BEEC) using array-based molecular karyotyping.

Materials and Methods

Patients and DNA Isolation

In this study we included four discordant MZ twin pairs who were contacted and recruited through the German self-help organizations for patients with ARM (SoMA e.V.) and BEEC (Selbsthilfegruppe Blasenekstrophie/Epispadie e.V.) as well as through the Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany and the Department of Pediatric Surgery and Urology, Center for Child and Adolescent Health, Hospital Bremen-Mitte, Bremen, Germany. Patients with ARM were classified according to the Krickenbeck Classification System (Holschneider et al., Reference Holschneider, Hutson, Peña, Beket, Chatterjee, Coran and Kunst2005). Patients with the BEEC were classified according to Gearhart (Reference Gearhart, Walsh, Retik, Vaughan and Wein2002). Written informed consent was obtained from all families prior to study entry. The Ethics Committee of the Medical Faculty of the University of Bonn approved the study. For each twin pair, blood or saliva samples were obtained from both twins and their parents for the purpose of DNA extraction and molecular-genetic analyses. Isolation of genomic DNA was carried out using the Chemagic Magnetic Separation Module I (Chemagen, Baesweiler, Germany) or, in the case of saliva samples, the Oragene DNA Kit (DNA Genotek Inc., Kanata, Canada).

Description of Twin Pairs

Twin pair 1

Twin pair 1 was 5-year old at the time of recruitment. These male twins were the second and third of four children of unrelated German parents. The affected twins presented with classic bladder exstrophy (CBE; Table 1). The mother had one intrauterine fetal dismiss due to fetal polycystic kidney disease. The family history was otherwise unremarkable.

TABLE 1 Phenotypes of the Affected Twins Investigated

Twin pair 2

Twin pair 2 was recruited during the newborn period. These male twins were the first children of a Peruvian mother and a German father. The affected twin presented with ARM, dilated lower and upper urinary tract, and distension of the abdominal wall, resembling features of prune belly syndrome (PBS) (Table 1). The family history was unremarkable.

Twin pair 3

Twin pair 3 was 9-year old at the time of recruitment. These male twins were the only children of their unrelated German parents. The affected twin presented with hemifacial microsomia and pre-auricular pits, resembling features of Goldenhar syndrome and ARM (Table 1). The family history was unremarkable.

Twin pair 4

Twin pair 4 was 13-year old at the time of recruitment. These male twins were the first two of four children of their unrelated German parents. The affected twin presented with ARM and fused vertebra of the thoracic spine. Besides his congenital anomalies, he developed an attention-deficit hyperactivity disorder during childhood (Table 1). The third pregnancy of the mother ended in fetal dismiss during the 13th week of gestation, with signs of severe intrauterine growth retardation.

History of pregnancy in all four twin pairs was uneventful for the ARM- and BEEC-associated environmental risk factors, for example, maternal diabetes, uterine vascular pathology, and infertility treatment (Reutter et al., Reference Reutter, Boyadjiev, Gambhir, Ebert, Rösch, Stein and Jenetzky2011; Zwink et al., Reference Zwink, Jenetzky and Brenner2011).

Monozygosity Testing

We tested all twin pairs for their zygosity status before proceeding with molecular karyotyping. Analysis was performed using a PowerPlex® 16 System according to the manufacturer's recommendations (Promega, Madison, WI, USA), allowing co-amplification and three-color detection of 16 loci.

Paternity Testing

The GenomeStudio (version V2011.1, http://www.illumina.com/) genotyping module was used for paternity testing.

Array-Based Molecular Karyotyping

HumanOmni1-Quad_v1 Chip (Illumina, Inc., San Diego, CA, USA), which contains 1,140,419 markers with a median marker spacing of 1.5 kb, was used for molecular karyotyping. All analyses were performed according to the manufacturer's protocol. A DNA sample was considered to have failed if fewer than 95% of loci were generated on the corresponding BeadChip. CNVs were predicted using the program QuantiSNP (v2.2, www.well.ox.ac.uk/QuantiSNP/), which uses an Objective Bayes Hidden-Markov model for the estimation. The log R ratio (LRR) represents a measure of the magnitude of combined fluorescence-intensity signals. The B-allele frequency (BAF) denotes the relative ratio of fluorescence signals from one allelic probe compared with another. Duplications can be identified by an increase of LRR and the occurrence of four clusters in BAF (at 0, ~0.33, ~0.67, and 1). Consequently, a deletion is characterized by a decrease of LRR and lack of heterozygosity (at 0.5) in BAF.

CNV Filtering

Filtering of CNV data was carried out using Cartagenia Bench™ software (released January 2011, Cartagenia, Leuven, Belgium). We excluded (1) CNV calls with a log Bayes factor below 7, (2) all CNV regions with fewer than five aberrant markers, and (3) all CNV calls with a frequency of over 5% in our internal control cohort (n = 531 healthy controls; Table 2). As we included small CNVs comprising ‘5 aberrant’ markers only, a certain number of remaining CNVs comprised less than three rs-markers; for example, 2 rs- and 3 cnvi-markers = ‘intensity only probes’. As cnvi-markers do not carry any genotype information, we excluded CNVs comprising less than three rs-markers later through visual inspection in filter step (7). In filter step (4) the remaining CNVs were filtered for being present only in the affected twin, but not in the unaffected twin or the parents, and in filter step (5) their gene content was filtered using the UCSC genome browser (Dreszer et al., Reference Dreszer, Karolchik, Zweig, Hinrichs, Raney, Kuhn and Kent2012; http://genome.ucsc.edu/) against RefSeqGene (coding regions only, http://www.ncbi.nlm.nih.gov/refseq/rsg/). Next, in step (6) we filtered for the presence of CNVs against the following publicly available databases: Database of Genomic Variants (DGV, v10; Iafrate et al., Reference Iafrate, Feuk, Rivera, Listewnik, Donahoe, Qi and Lee2004), and Database of Chromosomal Imbalance and Phenotype in Humans, using Ensembl Resources (DECIPHER, v5,1; Firth et al., Reference Firth, Richards, Bevan, Clayton, Corpas, Rajan and Carter2009). CNVs determined to exist in either of these databases, with at least 10 full overlapping reports in DGV and at least five full overlapping reports in DECIPHER, were excluded. However, we did not exclude CNV overlapping in at least five reports in DECIPHER if the associated phenotypes included any phenotype investigated here, for example, ARM. In step (7) the remaining CNVs were visually re-evaluated using the GenomeStudio (GS) genotyping module (GS, version V2011.1, http://www.illumina.com/). GS Data Analysis Software visualizes and analyzes data generated by all of Illumina's platforms. GS supports the primary analysis of the microarray-based data generated by the iScan System and BeadXpress Reader. The results of genotyping are displayed in the form of LRR and BAF as described above (see section ‘Array-Based Molecular Karyotyping’). As mentioned above, through visual inspection of the remaining CNVs we excluded all CNVs comprising less than three rs-markers, for example, two rs- and three cnvi-markers.

TABLE 2 Filter Steps for (a) CNVs Present Only in the Affected Twin, but Not in the Unaffected Twin or the Parents, or (b) CNVs Present in Both Twins but Not the Parents

In addition, we used the available data to search for possible disease-causing de novo CNVs with incomplete penetrance. For this analysis, we used identical filter criteria, except that criterion (4) was modified to filter CNVs present in both twins but not in parents.

Quantitative Polymerase Chain Reaction (qPCR)

Reactions for qPCR were performed on an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems, Carslbad, CA, USA) using SYBR Green for detection. Each assay included DNA from four controls (two male and two female samples) and the probands’ DNA at a final concentration of 20 ng/mL; reactions were run in triplicate. Reaction mixtures (10 μL) contained 0.2 μmol of each primer and 5 μL of Power SYBR Green PCR Master Mix (Applied Biosystems), with cycling conditions as follows: Initiation, 50°C for 2 min; denaturation, 95°C for 10 min; followed by 40 cycles at 95°C for 15 s; and a combined annealing and extension step at 60°C for 60 s. The threshold cycle (Ct) values were normalized using the Ct-value of three reference genes (BNC1, CFTR, and RPP38) for each DNA sample. Relative quantification was done using the comparative Ct method (Livak & Schmittgen, Reference Livak and Schmittgen2001).

Results

Screening for CNVs present only in the affected twin, but not in the unaffected twin or the parents (complete penetrance). The numbers of CNVs remaining after various filtering steps are given in Table 2. After applying steps (1), (2), and (3), 125, 142, 174, and 126 CNVs remained in twin pairs 1, 2, 3, and 4 respectively. After applying step (4), 44, 70, 62, and 40 CNVs remained. After applying filtering steps (5), (6), and (7) no potentially causative CNV remained.

Screening for CNVs present in both twins but not the parents (incomplete penetrance). Filtering for possible disease-causing de novo CNVs present in both twins but not in the parents yielded, after step (4), nine CNVs for twin pair 1, eight CNVs for twin pair 2, 31 CNVs for twin pair 3, and 14 CNVs for twin pair 4. After applying filter steps (5), (6), and (7), one CNV remained in twin pair 3 (Table 2, column B). This CNV on chromosome 16p13.3 was suggested to have a size of around 14 kb and overlapped with exons 7–9 of human Rab11 family-interacting protein 3 (RAB11FIP3) gene. However, qPCR performed on the twins and their parents revealed two copies in all individuals and thus did not confirm the presence of a CNV.

Discussion

Our array-based analysis did not identify disease-causing CNVs in any of the four MZ discordant twin pairs that we investigated. This suggests that CNVs affecting coding regions that arise as early post-twinning mutational events are not a frequent cause of discordance among MZ twins with urorectal malformations. It may be the case, however, that we have missed true causative CNVs because our filter criteria excluded CNVs located outside of coding regions and would therefore have missed CNVs in promoter or enhancer regions. It is also possible that the arrays we used, with a median inter-marker spacing of 1.5 kb, missed smaller CNVs, or that mutational events were present involving small DNA changes (e.g., single-nucleotide substitutions) that are not detectable per se using an SNP-based array approach. These changes might well be detectable by systematic sequencing approaches such as exome- or genome-wide sequencing, and these methods therefore represent an important direction for future studies of discordant twin pairs. Furthermore, there could be somatic mosaicism for causal mutation (if any . . .), which remained undetected because we investigated DNA from peripheral lymphocytes. The investigation of this would require investigation of DNA from affected tissue.

In addition to genomic differences, there is also growing evidence that implicates epigenetic events occurring early in embryogenesis in the development of phenotypic discordance in MZ twin pairs (Kaminsky et al., Reference Kaminsky, Tang, Wang, Ptak, Oh, Wong and Petronis2009; Yamazawa et al., Reference Yamazawa, Kagami, Fukami, Matsubara and Ogata2008), and such alterations can be detected in principle with appropriate experimental methods. For ethical reasons, however, investigations of tissue-specific differences in methylation in urorectal malformations are not possible because the unaffected twins are not available for tissue sampling. Retrospective evaluation of pregnancy history in the investigated discordant twin pairs did not identify any possible environmental risk factor. Nevertheless, an environmental contribution cannot be ruled out completely (Reutter et al., Reference Reutter, Boyadjiev, Gambhir, Ebert, Rösch, Stein and Jenetzky2011; Zwink et al., Reference Zwink, Jenetzky and Brenner2011).

Acknowledgments

We thank all patients and their parents for their participation in this study. We thank the German self-help organizations for people with anorectal malformations (SoMA e.V.) and with exstrophy-epispadias complex (SHG Blasenekstrophie/Epispadie e.V.). Grant sponsor: German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF; grant no. 01GM08107). Enrika Bartels, is supported by the University of Bonn, BONFOR (grant no. O-149.0099). Enrika Bartels, Markus M. Nöthen, Michael Ludwig, Heiko Reutter, and Markus Draaken are members of the Network for the Systematic Investigation of the Molecular Causes, Clinical Implications and Psychosocial Outcome of Congenital Urorectal Malformations (CURE-Net), which is supported by a research grant from the BMBF.

References

Bartels, E., Draaken, M., Kazmierczak, B., Spranger, S., Schramm, C., Baudisch, F., . . . Reutter, H. (2011). De novo partial trisomy 18p and partial monosomy 18q in a patient with anorectal malformation. Cytogenetic and Genome Research, 134, 243248.CrossRefGoogle Scholar
Boocock, G. R., & Donnai, D. (1987). Anorectal malformation: Familial aspects and associated anomalies. Archives of Disease in Childhood, 62, 576579.Google Scholar
Boyadjiev, S. A., Dodson, J. L., Radford, C. L., Ashrafi, G. H., Beaty, T. H., Mathews, R. I., . . . Gearhart, J. P. (2004). Clinical and molecular characterization of the bladder exstrophy-epispadias complex: Analysis of 232 families. BJU International, 94, 13371343.Google Scholar
Breckpot, J., Thienpont, B., Gewillig, M., Allegaert, K., Vermeesch, J. R., & Devriendt, K. (2012). Differences in copy number variation between discordant monozygotic twins as a model for exploring chromosomal mosaicism in congenital heart defects. Molecular Syndromology, 2, 8187.Google Scholar
Cuschieri, A., & EUROCAT Working Group. (2001). Descriptive epidemiology of isolated anal anomalies: A survey of 4.6 million births in Europe. American Journal of Medical Genetics, 103, 207215.Google Scholar
Draaken, M., Reutter, H., Schramm, C., Bartels, E., Boemers, T. M., Ebert, A. K., . . . Ludwig, M. (2010). Microduplications at 22q11.21 are associated with non-syndromic classic bladder exstrophy. European Journal of Medical Genetics, 53, 5560.Google Scholar
Dreszer, T. R., Karolchik, D., Zweig, A. S., Hinrichs, A. S., Raney, B. J., Kuhn, R. M., . . . Kent, W. J. (2012). The UCSC Genome Browser database: Extensions and updates 2011. Nucleic Acids Research, 40, 918923.Google Scholar
Falcone, R. A. Jr, Levitt, M. A., Peña, A., & Bates, M. (2007). Increased heritability of certain types of anorectal malformations. Journal of Pediatric Surgery, 42, 124127.Google Scholar
Firth, H. V., Richards, S. M., Bevan, A. P., Clayton, S., Corpas, M., Rajan, D., . . . Carter, N. P. (2009). DECIPHER: Database of chromosomal imbalance and phenotype in humans using Ensembl resources. American Journal of Medical Genetics, 84, 524533.Google Scholar
Gearhart, J. P. (2002). Exstrophy, epispadias, and other bladder anomalies. In Walsh, P. C., Retik, A. B., Vaughan, E. D. & Wein, A. J. (Eds.), Campbell's urology (8th ed., pp. 21362196). Philadelphia, PA: WB Saunders.Google Scholar
Helderman-van den Enden, A. T., Maaswinkel-Mooij, P. D., Hoogendoorn, E., Willemsen, R., Maat-Kievit, J. A., Losekoot, M., & Oostra, B. A. (1999). Monozygotic twin brothers with the fragile X syndrome: Different CGG repeats and different mental capacities. Journal of Medical Genetics, 36, 253257.Google ScholarPubMed
Holschneider, A., Hutson, J., Peña, A., Beket, E., Chatterjee, S., Coran, A., . . . Kunst, M. (2005). Preliminary report on the International Conference for the Development of Standards for the Treatment of Anorectal Malformations. Journal of Pediatric Surgery, 40, 15211526.Google Scholar
Iafrate, A. J., Feuk, L., Rivera, M. N., Listewnik, M. L., Donahoe, P. K., Qi, Y., . . . Lee, C. (2004). Detection of large-scale variation in the human genome. Nature Genetics, 36, 949951.Google Scholar
Kaminsky, Z. A., Tang, T., Wang, S. C., Ptak, C., Oh, G. H., Wong, A. H., . . . Petronis, A. (2009). DNA methylation profiles in monozygotic and dizygotic twins. Nature Genetics, 41, 240245.CrossRefGoogle ScholarPubMed
Keppler-Noreuil, K. M. (2001). OEIS complex (omphalocele-exstrophy-imperforate anus-spinal defects): A review of 14 cases. American Journal of Medical Genetics, 99, 271279.Google Scholar
Kondo, S., Schutte, B. C., Richardson, R. J., Bjork, B. C., Knight, A. S., Watanabe, Y., . . . Murray, J. C. (2002). Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nature Genetics, 32, 285289.CrossRefGoogle Scholar
Kruyer, H., Mila, M., Glover, G., Carbonell, P., Ballesta, F., & Estivill, X. (1994). Fragile X syndrome and the (CGG)n mutation: Two families with discordant MZ twins. American Journal of Medical Genetics, 54, 437442.Google Scholar
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 25, 402408.CrossRefGoogle ScholarPubMed
Ludwig, M., Ching, B., Reutter, H., & Boyadjiev, S. A. (2009). Bladder exstrophy-epispadias complex. Birth Defects Research Part A: Clinical and Molecular Teratology, 85, 509522.Google Scholar
Marcelis, C., de Blaauw, I., & Brunner, H. (2011). Chromosomal anomalies in the etiology of anorectal malformations: A review. American Journal of Medical Genetics Part A, 155A, 26922704.Google Scholar
Reutter, H., Boyadjiev, S. A., Gambhir, L., Ebert, A. K., Rösch, W. H., Stein, R., . . . Jenetzky, E. (2011). Phenotype severity in the bladder exstrophy-epispadias complex: Analysis of genetic and nongenetic contributing factors in 441 families from North America and Europe. Journal of Pediatrics, 159, 825831Google Scholar
Schramm, C., Draaken, M., Bartels, E., Boemers, T. M., Aretz, S., Brockschmidt, F. F., . . . Reutter, H. (2011a). De novo microduplication at 22q11.21 in a patient with VACTERL association. European Journal of Medical Genetics, 54, 913.CrossRefGoogle Scholar
Schramm, C., Draaken, M., Bartels, E., Boemers, T. M., Schmiedeke, E., Grasshoff-Derr, S., . . . Reutter, H. (2011b). De novo duplication of 18p11.21-18q12.1 in a female with anorectal malformation. American Journal of Medical Genetics Part A, 155A, 445449.Google Scholar
Shapiro, E., Lepor, H., & Jeffs, R. D. (1984). The inheritance of the exstrophy-epispadias complex. Journal of Urology, 132, 308310.CrossRefGoogle ScholarPubMed
Taylor, D. M., Thum, M. Y., & Abdalla, H. (2008). Dichorionic triamniotic triplet pregnancy with monozygotic twins discordant for trisomy 13 after preimplantation genetic screening: Case report. Fertility and Sterility, 90, 2017.e5e9.Google Scholar
Yamazawa, K., Kagami, M., Fukami, M., Matsubara, K., & Ogata, T. (2008). Monozygotic female twins discordant for Silver-Russell syndrome and hypomethylation of the H19-DMR. Journal of Human Genetics, 53, 950955.CrossRefGoogle ScholarPubMed
Zwijnenburg, P. J., Meijers-Heijboer, H., & Boomsma, D. I. (2010). Identical but not the same: The value of discordant monozygotic twins in genetic research. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 153B, 11341149.CrossRefGoogle Scholar
Zwink, N., Jenetzky, E., & Brenner, H. (2011). Parental risk factors and anorectal malformations: Systematic review and meta-analysis. Orphanet Journal of Rare Diseases, 6, 25.Google Scholar
Figure 0

TABLE 1 Phenotypes of the Affected Twins Investigated

Figure 1

TABLE 2 Filter Steps for (a) CNVs Present Only in the Affected Twin, but Not in the Unaffected Twin or the Parents, or (b) CNVs Present in Both Twins but Not the Parents