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P6: Detection of pathologic and polymorphic cryptic chromosomal imbalances by array-CGH using a BAC-array targeting regions between segmental duplications
E. González et al. / European Journal of Medical Genetics 48 (2005) 443–476
451
Subtelomeric and interstitial microdeletions and microduplications have been shown to be a major cause of mental retardation with multiple congenital anomalies (MR/MCA). With the advent of array based CGH, genome-wide high-resolution screening for such imbalances in mentally retarded patients is now possible. 52 well-characterized patients with MR (mostly MR/MCA) of unknown aetiology were screened for genomic imbalances by array-CGH using two BAC/PAC-arrays (“6k array”: 16 patients; “8k array”: 36 patients). The arrays contained the Sanger Centre 1Mb clone set in addition to more than 3000 or 5000 region- and gene-specific clones, respectively. Nine of the patients were previously analysed using a 2.4k BAC array. Diagnostic thresholds used were ± 5 standard deviations (SD) for the 6k array and ± 7SD for the 8k array. All outliers are being verified by metaphase and/or interphase FISH. On average, 5 clones were verified per patient analysed with the 6k array and 1.4 clones per patient analysed with the 8k array. Parental analyses are performed for all confirmed imbalances. Four copy number changes thought to be causative were detected so far (del 1q24, del 6q11.1-q13, del Xq21.3 and dup 22q11.2). De novo occurrence was demonstrated by parental FISH experiments in the first two cases. FISH analyses are being performed to more precisely define the imbalance breakpoints. Array-CGH revealed a de novo interstitial deletion of 3.7Mb in 1q24 in a six-year-old girl with severely delayed language development, dysmorphic signs, hyperextensibility of joints, muscular hypotonia, seizures and decreased pain sensitivity. This aberration was detected using the 2.4k chip and then re-analysed using the 8k chip. A de novo interstitial deletion of 8.2-10.6 Mb in 6q11.1-q13 (6k chip) was detected in a twelve year old girl with mild MR, several dysmorphic signs, hyperopia and frequent infections. Array-CGH revealed a 2.4–8.6 Mb microdeletion of Xq21.3 (8k chip) in a six year old boy with MR, hypospadia, inguinal and umbilical hernia and several dysmorphic signs. FISH experiments in the patient’s mother revealed the same deletion in one of her X chromosomes. Thus, the patient’s phenotype is most likely caused by the nullisomic form of the deletion as opposed to the heterozygous deletion in the mother. The mother will be evaluated clinically. A 0.6-4.0 Mb duplication within the DiGeorge syndrome critical region / DGCR in 22q11.2 (8k chip) was detected in a seven year old girl with MR, microcephaly at birth and dysmorphic signs. Parental FISH analyses revealed a paternal duplication. Two sibs with learning deficiency / MR will also be analysed. Since DGCR duplications have been described to cause MR/MCA and normal carrier parents have been observed, the patient’s phenotype is most likely caused by this duplication. Here, we present a detection rate of approximately 8% for cryptic chromosomal changes in patients with MR/MCA of unknown aetiology with CGH to DNA arrays containing 6000-8000 large insert clones.
P6: Detection of pathologic and polymorphic cryptic chromosomal imbalances by arrayCGH using a BAC-array targeting regions between segmental duplications Eva González a, Ivon Cuscó b, Olaya Villa c, Mireia Vilardell b, Benjamí Rodríguez b, Juanjo Lozano a, Luis Pérez-Jurado a, Xavier Estivill a, Lauro Sumoy a a Center for Genomic Regulation b Universitat Pompeu Fabra c Hospital del Mar-IMAS The sequencing of the human genome has uncovered the existence of large (5–400 kb), almost identical regions of the genome present in more than one copy, known as segmental duplications (SDs) or low copy repeats. These regions encompass > 5% of our genome and often
452
K. Kok et al. / European Journal of Medical Genetics 48 (2005) 443–476
contain genes, pseudogenes or regulatory elements. Due to their high degree of sequence identity (> 90–95%), SDs can mediate non-allelic homologous recombination leading to chromosome rearrangements underlying genomic disease or genomic polymorphisms (deletions, duplications or inversions). Therefore, regions in between SDs are expected to represent hotspots for genomic mutations causing a significant proportion of human disorder as well as human variation due to copy number polymorphisms (CNPs). As SDs have been identified and mapped (Cheung et al. 2003), we have constructed a microarray targetting the hotspot regions. We included 210 BACs covering single-copy regions flanked SDs (in some cases, BACs contained part of a SD) and all the subtelomeric regions (0.1–1 Mb from p and qter). We also included 18 control probes corresponding to X (15) Y [3] chromosome BACs (ratio controls), Drosophila melanogaster BACs (negative controls), and human genomic DNA (positive controls). Using this CGH-array, we have screened a collection of 101 samples of cytogenetically normal patients with a variety of conditions to look for candidate regions of subchromosomal aneuploidy, including 7 control samples with known deletions. BAC variability was analysed after lowess normalization under more restrictive criteria. Accordingly, we assigned a score to each BAC depending on its variability among samples, considering the interquartile range (IQ/1.35) instead of the standard deviation. All BACs for a slide (case) were then represented and ± 3 IQ/1.35 were considered the normal signal limits of each BAC per slide. We found abnormal BAC signals in 48 samples (including the 7 known controls). A patient-specific de novo rearrangement likely responsible for the disease was detected and confirmed by other methods in 6 patients: 1-) 7qter dup ~10 Mb/10qter del ~8 Mb; 2-) 7q14 dup > 1 Mb; 3-) 15q12 dup 3 Mb; 4-) 15q11 del 0,5 Mb; 5-) 10q23 dup ~4 Mb; 6-) 18qter del 0,5Mb/8pter dup ~3 Mb. We also detected 61 abnormal signals present in 2–9% of the samples that might correspond to CNPs. Almost half of these presumed CNPs (28/61) have also been found by others (Iafrate 2004, Sebat 2004, Bejjani 2005, Sharp 2005). With alternative methods (FISH, MLPA, microsatellites), 5 were confirmed but 6 failed to be reproduced suggesting they were false positive results on the array. The remaining 23 variable loci have not been reported and their confirmation by other methods is in process. Out of these 61 variable BACs, 21 had exclusively decreased signals corresponding to theoretical deletions, 8 had only increased signals suggestive of duplications, while the remaining 32 BACs were either deleted or duplicated in different samples. Our results indicate that array-CGH is a powerful tool to detect sub-microscopic pathogenic rearrangements in patients as well as CNPs. However, careful analysis and confirmation by other methods may be required due to the rate of false positive results. The strategy of targeting putative hotspots located between SDs has proven to be highly effective.
P7: The application of a 6 K genome-wide BAC-array in clinical diagnostic Klaas Kok, Trijnie Dijkhuizen, Birgit Sikkema, Pieter van der Vlies, Yolanthe Swart, Hanny Zorgdrager, Klasien Gerssen-Schoorl, Ton van Essen, Charles H. Buys UMCG, Department of Medical Genetics We have constructed a 6 K BAC array to be applied in array-based CGH analyses of clinical samples. The array is a combination of the 1-Mb clone set that was kindly made available by Dr. Nigel Carter (Wellcome Trust Sanger Institute) and the 1-Mb collection that we obtained from Dr. Pieter de Jong (Children’s Hospital Oakland Research Institute), supplemented with an additional selection of BACs to fill in the gaps that exceeded the 1.5 Mb. Arrays are
451
Subtelomeric and interstitial microdeletions and microduplications have been shown to be a major cause of mental retardation with multiple congenital anomalies (MR/MCA). With the advent of array based CGH, genome-wide high-resolution screening for such imbalances in mentally retarded patients is now possible. 52 well-characterized patients with MR (mostly MR/MCA) of unknown aetiology were screened for genomic imbalances by array-CGH using two BAC/PAC-arrays (“6k array”: 16 patients; “8k array”: 36 patients). The arrays contained the Sanger Centre 1Mb clone set in addition to more than 3000 or 5000 region- and gene-specific clones, respectively. Nine of the patients were previously analysed using a 2.4k BAC array. Diagnostic thresholds used were ± 5 standard deviations (SD) for the 6k array and ± 7SD for the 8k array. All outliers are being verified by metaphase and/or interphase FISH. On average, 5 clones were verified per patient analysed with the 6k array and 1.4 clones per patient analysed with the 8k array. Parental analyses are performed for all confirmed imbalances. Four copy number changes thought to be causative were detected so far (del 1q24, del 6q11.1-q13, del Xq21.3 and dup 22q11.2). De novo occurrence was demonstrated by parental FISH experiments in the first two cases. FISH analyses are being performed to more precisely define the imbalance breakpoints. Array-CGH revealed a de novo interstitial deletion of 3.7Mb in 1q24 in a six-year-old girl with severely delayed language development, dysmorphic signs, hyperextensibility of joints, muscular hypotonia, seizures and decreased pain sensitivity. This aberration was detected using the 2.4k chip and then re-analysed using the 8k chip. A de novo interstitial deletion of 8.2-10.6 Mb in 6q11.1-q13 (6k chip) was detected in a twelve year old girl with mild MR, several dysmorphic signs, hyperopia and frequent infections. Array-CGH revealed a 2.4–8.6 Mb microdeletion of Xq21.3 (8k chip) in a six year old boy with MR, hypospadia, inguinal and umbilical hernia and several dysmorphic signs. FISH experiments in the patient’s mother revealed the same deletion in one of her X chromosomes. Thus, the patient’s phenotype is most likely caused by the nullisomic form of the deletion as opposed to the heterozygous deletion in the mother. The mother will be evaluated clinically. A 0.6-4.0 Mb duplication within the DiGeorge syndrome critical region / DGCR in 22q11.2 (8k chip) was detected in a seven year old girl with MR, microcephaly at birth and dysmorphic signs. Parental FISH analyses revealed a paternal duplication. Two sibs with learning deficiency / MR will also be analysed. Since DGCR duplications have been described to cause MR/MCA and normal carrier parents have been observed, the patient’s phenotype is most likely caused by this duplication. Here, we present a detection rate of approximately 8% for cryptic chromosomal changes in patients with MR/MCA of unknown aetiology with CGH to DNA arrays containing 6000-8000 large insert clones.
P6: Detection of pathologic and polymorphic cryptic chromosomal imbalances by arrayCGH using a BAC-array targeting regions between segmental duplications Eva González a, Ivon Cuscó b, Olaya Villa c, Mireia Vilardell b, Benjamí Rodríguez b, Juanjo Lozano a, Luis Pérez-Jurado a, Xavier Estivill a, Lauro Sumoy a a Center for Genomic Regulation b Universitat Pompeu Fabra c Hospital del Mar-IMAS The sequencing of the human genome has uncovered the existence of large (5–400 kb), almost identical regions of the genome present in more than one copy, known as segmental duplications (SDs) or low copy repeats. These regions encompass > 5% of our genome and often
452
K. Kok et al. / European Journal of Medical Genetics 48 (2005) 443–476
contain genes, pseudogenes or regulatory elements. Due to their high degree of sequence identity (> 90–95%), SDs can mediate non-allelic homologous recombination leading to chromosome rearrangements underlying genomic disease or genomic polymorphisms (deletions, duplications or inversions). Therefore, regions in between SDs are expected to represent hotspots for genomic mutations causing a significant proportion of human disorder as well as human variation due to copy number polymorphisms (CNPs). As SDs have been identified and mapped (Cheung et al. 2003), we have constructed a microarray targetting the hotspot regions. We included 210 BACs covering single-copy regions flanked SDs (in some cases, BACs contained part of a SD) and all the subtelomeric regions (0.1–1 Mb from p and qter). We also included 18 control probes corresponding to X (15) Y [3] chromosome BACs (ratio controls), Drosophila melanogaster BACs (negative controls), and human genomic DNA (positive controls). Using this CGH-array, we have screened a collection of 101 samples of cytogenetically normal patients with a variety of conditions to look for candidate regions of subchromosomal aneuploidy, including 7 control samples with known deletions. BAC variability was analysed after lowess normalization under more restrictive criteria. Accordingly, we assigned a score to each BAC depending on its variability among samples, considering the interquartile range (IQ/1.35) instead of the standard deviation. All BACs for a slide (case) were then represented and ± 3 IQ/1.35 were considered the normal signal limits of each BAC per slide. We found abnormal BAC signals in 48 samples (including the 7 known controls). A patient-specific de novo rearrangement likely responsible for the disease was detected and confirmed by other methods in 6 patients: 1-) 7qter dup ~10 Mb/10qter del ~8 Mb; 2-) 7q14 dup > 1 Mb; 3-) 15q12 dup 3 Mb; 4-) 15q11 del 0,5 Mb; 5-) 10q23 dup ~4 Mb; 6-) 18qter del 0,5Mb/8pter dup ~3 Mb. We also detected 61 abnormal signals present in 2–9% of the samples that might correspond to CNPs. Almost half of these presumed CNPs (28/61) have also been found by others (Iafrate 2004, Sebat 2004, Bejjani 2005, Sharp 2005). With alternative methods (FISH, MLPA, microsatellites), 5 were confirmed but 6 failed to be reproduced suggesting they were false positive results on the array. The remaining 23 variable loci have not been reported and their confirmation by other methods is in process. Out of these 61 variable BACs, 21 had exclusively decreased signals corresponding to theoretical deletions, 8 had only increased signals suggestive of duplications, while the remaining 32 BACs were either deleted or duplicated in different samples. Our results indicate that array-CGH is a powerful tool to detect sub-microscopic pathogenic rearrangements in patients as well as CNPs. However, careful analysis and confirmation by other methods may be required due to the rate of false positive results. The strategy of targeting putative hotspots located between SDs has proven to be highly effective.
P7: The application of a 6 K genome-wide BAC-array in clinical diagnostic Klaas Kok, Trijnie Dijkhuizen, Birgit Sikkema, Pieter van der Vlies, Yolanthe Swart, Hanny Zorgdrager, Klasien Gerssen-Schoorl, Ton van Essen, Charles H. Buys UMCG, Department of Medical Genetics We have constructed a 6 K BAC array to be applied in array-based CGH analyses of clinical samples. The array is a combination of the 1-Mb clone set that was kindly made available by Dr. Nigel Carter (Wellcome Trust Sanger Institute) and the 1-Mb collection that we obtained from Dr. Pieter de Jong (Children’s Hospital Oakland Research Institute), supplemented with an additional selection of BACs to fill in the gaps that exceeded the 1.5 Mb. Arrays are