Oligonucleotide Array Comparative Genomic Hybridization Research Articles

Prenatal Diagnosis and Postnatal Followup of Partial Trisomy 13q and Partial Monosomy 10p: A Case Report and Review of the Literature

We report prenatal diagnosis and postnatal findings of a fetus with partial trisomy of 13q21.33-qter and partial monosomy of 10p15.3-pter. The mother is a known carrier of a balanced translocation, t(10;13)(p15.3;q21.33), ascertained by history of one miscarriage and two neonatal deaths. The fetal karyotyping on cultured amniocytes showed 46,XX,der(10)t(10;13)(p15.3;q21.33). Oligonucleotide array comparative genomic hybridization (aCGH) defined a 2.339 Mb distal deletion at 10p15.3 (chr10:126,161–2,465,089) and a 46.344 Mb duplication of 13q21.33–q34 (chr13:67,779,708–114,123,540). Ultrasound examination showed polydactyly and polyhydramnios in the fetus. After genetic counseling, the mother decided to continue the pregnancy, and follow-up ultrasound monitoring found no further abnormalities. A girl was delivered at 37+6 weeks of gestation and was transferred to the intensive care unit for intermittent convulsions within 26 hours. She was diagnosed with neonatal hypoxic ischemic encephalopathy and experienced several episodes of apnea in the following month. Her birth weight was 2900 g (10–25th centile) and at five months was 5500 g (5–10th centile). She had dysmorphic features and mild psychomotor retardation. A review of the literature found three previously reported cases with similar compound 10p/13q abnormalities. We discuss a two-step approach to assess fetal viability and phenotype using genomic information from partial trisomy and monosomy.

Deletion of the AP1S2 gene in a child with psychomotor delay and hypotonia

We identified a 495 Kb interstitial deletion of chromosome Xp22.2, centered on the AP1S2 gene, by means of oligonucleotide array comparative genomic hybridisation (array-CGH) in a child with marked hypotonia in the first months of life, psychomotor retardation, severely delayed walking and speech development, and unspecific dysmorphic facial features. The deletion was inherited from the healthy mother. Point mutations of the AP1S2 gene have been identified in patients with X-linked mental retardation (XLMR). The clinical features of our patient are quite similar to those reported in male patients carrying point mutations, thus suggesting that point mutations and deletions of the AP1S2 gene lead to a recognisable XLMR phenotype in males.

A de novo 3.54 Mb deletion of 17q22‐q23.1 associated with hydrocephalus: A case report and review of literature

We describe a female newborn with a de novo 3.54 megabase (Mb) deletion of 17q22-q23.1 (chr17:53,072,536-56,612,662, hg18) including genes from MSI2 to BCAS3 detected by oligonucleotide array comparative genomic hybridization (aCGH). Prenatal ultrasound examination noted oligohydramnios and ventriculomegaly in the fetus. Postnatal examination found hypotonia, macrocephaly, arachnodactyly of fingers and toes, dysmorphic features, bilateral hearing loss and heart defect. Review of reported cases with genomic findings noted one case with proximal deletion involving the NOG gene and a case series with distal recurrent microdeletions involving the TBX2 and TBX4 genes. Our case presented a unique deletion partially overlapped with the above deletions but not including the NOG, TBX2, and TBX4 genes. A genomic map for deletions in this 17q22-q23.1 region was constructed to further define the common deletion intervals for potential haplo-insufficient genes.

Microdeletion and microduplication 17q21.31 plus an additional CNV, in patients with intellectual disability, identified by array-CGH

The recognition of the 17q21.31 microdeletion and microduplication syndrome has been facilitated by high resolution oligonucleotide array comparative genome hybridization technology (aCGH). Molecular analysis of the 17q21.31 microdeletion/duplication syndrome demonstrated a critical region involving at least six genes, including STH and MAPT. The 17q21.31 microdeletion syndrome has an incidence of 1 in 16,000 births, while the microduplication 17q21.31 has been reported so far in only five patients. In general, phenotypes associated with 17q21.31 microduplication seem to be milder than those associated with the microdeletion. Here, we present four patients who have been referred for genetic evaluation by clinical geneticists due to developmental delay and minor congenital abnormalities. Previous standard karyotypes were negative, while aCGH analysis revealed three patients with 17q21.31 microdeletion and one with the respective microduplication, being the sixth reported case so far. Most importantly one of the microdeletion cases involves only partial MAPT gene deletion while leaving the STH gene intact. Two of our patients, one with the 17q21.31 microdeletion and another with the respective microduplication, carried additional clinically relevant microdeletions (del Xq21.31 and del 15q11.2, respectively), possibly modifying their phenotype.

Association between deletion size and important phenotypes expands the genomic region of interest in Phelan–McDermid syndrome (22q13 deletion syndrome)

BackgroundThe clinical features of Phelan–McDermid syndrome (also known as 22q13 deletion syndrome) are highly variable and include hypotonia, speech and other developmental delays, autistic traits and mildly dysmorphic features. Patient...

Identification of FOXO3 and PRDM1 as tumor-suppressor gene candidates in NK-cell neoplasms by genomic and functional analyses

AbstractOligo-array comparative genomic hybridization (CGH) and gene-expression profiling of natural killer (NK)–cell neoplasms were used in an effort to delineate the molecular pathogenesis involved. Oligo-array CGH identified two 6q21 regions that were most frequently deleted (14 of 39 or 36%). One of these regions included POPDC3, PREP, PRDM1, ATG5, and AIM1, whereas the other included LACE1 and FOXO3. All genes located in these regions, except for POPDC3 and AIM1, were down-regulated in neoplastic samples, as determined by gene-expression analysis, and were therefore considered to be candidate tumor-suppressor genes. A20 and HACE1, the well-known tumor-suppressor genes located on 6q21-23, were included as candidate genes because they also demonstrated frequent genomic deletions and down-regulated expression. The Tet-Off NK cell line NKL was subsequently established for functional analyses. Seven candidate genes were transduced into Tet-Off NKL and forced re-expression was induced. Re-expression of FOXO3 and PRDM1 suppressed NKL proliferation, but this was not the case after re-expression of the other genes. This effect was confirmed using another NK cell line, SNK10. Furthermore, genomic analyses detected nonsense mutations of PRDM1 that led to functional inactivation in one cell line and one clinical sample. PRDM1 and FOXO3 are considered to play an important role in the pathogenesis of NK-cell neoplasms.

Proximal Promoter of the Cytochrome P450 Oxidoreductase Gene: Identification of Microdeletions Involving the Untranslated Exon 1 and Critical Function of the SP1 Binding Sites

POR (cytochrome P450 oxidoreductase) is a ubiquitously expressed gene encoding an electron donor to all microsomal P450 enzymes and several non-P450 enzymes. POR mutations cause an autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, and disorders of sex development. Although recent studies have indicated the presence of a CpG-rich region characteristic of housekeeping genes around the untranslated exon 1 (exon 1U) and a tropic effect of thyroid hormone on POR expression via thyroid hormone receptor-β, detailed regulatory mechanisms for the POR expression remain to be clarified. Our objective was to report a pivotal element of the proximal promoter of POR. We first studied three patients (cases 1-3) with POR deficiency due to compound heterozygosity with an p.R457H mutation and transcription failure of an apparently normal allele, by oligoarray comparative genomic hybridization and serial direct sequencing of the deletion fusion points. Consequently, a 2,487-bp microdeletion involving exon 1U was identified in case 1 and an identical 49,604-bp deletion involving exon 1U and exon 1 was found in cases 2 and 3. We next analyzed the 2,487-bp region commonly deleted in cases 1-3 by in silico analysis, DNA binding analysis, luciferase assays, and methylation analysis. The results showed a critical function of the evolutionally conserved SP1 binding sites just upstream of exon 1U, especially the binding site at the position -26/-17, in the transcription of POR. The results suggest that the SP1 binding sites constitute an essential element of the POR proximal promoter.

Mitochondrial DNA polymerase γ mutations: an ever expanding molecular and clinical spectrum

Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial diseases in children and adults. This study sequenced the exons and flanking intronic...

Disruption of the SCN2A and SCN3A genes in a patient with mental retardation, neurobehavioral and psychiatric abnormalities, and a history of infantile seizures

Mutations in genes encoding voltage-gated sodium channels are significant factors in the etiology of neurological diseases and psychiatric disorders, including various types of idiopathic epilepsy. Using a clinical exon-targeted oligonucleotide array comparative genomic hybridization (aCGH), we have identified a de novo ~110-kb deletion involving exons 1-2 of SCN2A and non-coding exon 1a of SCN3A in a 25-year-old female with mental retardation, neurobehavioral and psychiatric abnormalities, and a history of infantile seizures with abnormal EEG. We propose that haploinsufficiency of SCN2A may play an important role in the genetic basis of neurodevelopmental and neurobehavioral disorders and emphasize the efficacy of detecting exonic copy-number variation (CNV) by exon-targeted oligo aCGH.

Evidence for disease penetrance relating to CNV size: Pelizaeus–Merzbacher disease and manifesting carriers with a familial 11 Mb duplication at Xq22

The potential causes for the incomplete penetrance of Pelizaeus-Merzbacher disease (PMD) in female carriers of PLP1 mutations are not well understood. We present a family with a boy having PMD in association with PLP1 duplication and three females who are apparent manifesting carriers. Custom high-resolution oligonucleotide array comparative genomic hybridization (aCGH) and breakpoint junction sequencing were performed and revealed a familial complex duplication consisting of a small duplicated genomic interval (∼56 kb) and a large segmental duplication (∼11 Mb) that resulted in a PLP1 copy number variation gain. Breakpoint junction analysis implicates a replication-based mechanism underlying the rearrangement formation. X-inactivation studies (XCI) showed a random to moderate advantageous skewing pattern in peripheral blood cells but a moderate to extremely skewed (≥90%) pattern in buccal cells. In conclusion, our data show that complex duplications involving PLP1 are not uncommon, can be detected at the level of genome resolution afforded by clinical aCGH and duplication and inversion can be produced in the same event. Furthermore, the observation of three manifesting carriers with a large genomic rearrangement supports the contention that duplication size along with genomic content can be an important factor for penetrance of the PMD phenotype in females.

Genomic characterization of prenatally detected chromosomal structural abnormalities using oligonucleotide array comparative genomic hybridization

Detection of chromosomal structural abnormalities using conventional cytogenetic methods poses a challenge for prenatal genetic counseling due to unpredictable clinical outcomes and risk of recurrence. Of the 1,726 prenatal cases in a 3-year period, we performed oligonucleotide array comparative genomic hybridization (aCGH) analysis on 11 cases detected with various structural chromosomal abnormalities. In nine cases, genomic aberrations and gene contents involving a 3p distal deletion, a marker chromosome from chromosome 4, a derivative chromosome 5 from a 5p/7q translocation, a de novo distal 6q deletion, a recombinant chromosome 8 comprised of an 8p duplication and an 8q deletion, an extra derivative chromosome 9 from an 8p/9q translocation, mosaicism for chromosome 12q with added material of initially unknown origin, an unbalanced 13q/15q rearrangement, and a distal 18q duplication and deletion were delineated. An absence of pathogenic copy number changes was noted in one case with a de novo 11q/14q translocation and in another with a familial insertion of 21q into a 19q. Genomic characterization of the structural abnormalities aided in the prediction of clinical outcomes. These results demonstrated the value of aCGH analysis in prenatal cases with subtle or complex chromosomal rearrangements. Furthermore, a retrospective analysis of clinical indications of our prenatal cases showed that approximately 20% of them had abnormal ultrasound findings and should be considered as high risk pregnancies for a combined chromosome and aCGH analysis.

Identification of a complex 17q rearrangement in a metanephric stromal tumor

Metanephric stromal tumor is a rare benign entity belonging to the group of metanephric renal tumors in children. Although metanephric stromal tumors can be cured by simple nephrectomy, differential diagnosis based on histopathologic criteria with other pediatric renal tumors requiring aggressive chemotherapy can be difficult. To our knowledge, cytogenetic characterization of metanephric stromal tumor has never been reported. We describe conventional ("R-bands" karyotyping) and molecular [fluorescence in situ hybridization (FISH), multicolor FISH, oligo array-comparative genomic hybridization] cytogenetic examinations of a metanephric stromal tumor in a 3-year-old boy. Cytogenetic analysis revealed a complex homogeneous gain between bands 17q22 and 17q25.3, resulting in partial triplication of the segment between bands 17q22 and 17q24.3, and duplication of the segment between bands 17q24.3 and 17q25.3. Cytogenetic confirmatory studies in metanephric stromal tumors are currently needed to assess 17q22q25.3 gain as a recurring cytogenetic abnormality of metanephric stromal tumors.

Homozygous deletion of ADAM3A revealed by genome-wide analysis of early-stage NSCLC in China showed to be correlated with poor prognosis.

e21177 Background: As a highly heterogenous disease, individual variation in non-small cell lung cancer (NSCLC) survival outcome may in part be explained by genomic copy number variations. Here we conducted a study to identify novel genes whose focal copy number variations may correlate with the survival of patients after complete resection of early-stage NSCLC in China. Methods: High-resolution 244 K oligo array comparative genomic hybridization (CGH) was used to analyze DNA from formalin-fixed paraffin-embedded early-stage (IA-IIIA) NSCLC samples. Data of 117 samples passed quality control. Segment cluster value of 0.8 and -1.0 were used as the cut-off value for amplification group and homozygous deletion group according to the user manual of Nexus Copy Number™. Probe distributions of ADAM3A high copy variation samples were checked individually to verify the focal variations. Log rank test was performed to do survival analysis between groups. Kaplan-Meier curve was generated to show survival characteristics. Results: Focal amplification of ADAM3A was detected in 52.1% (61/117) of cases. While homozygous deletion was detected in 6.8% (8/117) of cases. Prognosis of patients with focal amplification of ADAM3A showed no statistical significant difference when compared with normal cohort (median OS 38.8 months versus 40.8 months; p = 0.864). Homozygous deletion of ADAM3A was associated with significantly poorer outcomes than normal cohort (median OS 12.4 months versus 40.8 months; p = 0.000). Prognosis of homozygous deletion group also showed poorer outcomes when compared with focal amplification cohort (median OS 12.4 months versus 38.8 months; p = 0.000). Conclusions: This genome-wide array analysis revealed that focal amplification or homozygous loss of ADAM3A are frequent genetic alterations in early-stage NSCLC. While group of focal amplification and normal group indicated no significant difference in prognosis, homozygous loss of showed to be correlated with poor prognosis in early-stage NSCLC in China. As little is known about ADAM3A except its role in fertilization, further studies are necessary to explore the mechanism for the prognostic impact of ADAM3A in NSCLC.

Clonal evolution of adult T-cell leukemia/lymphoma takes place in the lymph nodes

AbstractAdult T-cell leukemia/lymphoma (ATLL) is the neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). We performed oligo-array comparative genomic hybridization (CGH) against paired samples comprising peripheral blood (PB) and lymph node (LN) samples from 13 patients with acute ATLL. We found that the genome profiles of the PB frequently differed from those of the LN samples. The results showed that 9 of 13 cases investigated had a log2 ratio imbalance among chromosomes, and that chromosome imbalances were more frequent in LN samples. Detailed analysis revealed that the imbalances were likely caused by the presence of multiple subclones in the LN samples. Five of 13 cases showed homozygous loss regions in PB samples, which were not found in the LN samples, indicating that tumors in the PB were derived from LN subclones in most cases. Southern blot analysis of TCRγ showed that these multiple subclones originated from a common clone. We concluded that in many ATLL cases, multiple subclones in the LNs originate from a common clone, and that a selected subclone among the LN subclones appears in the PB.

TGFBR2 deletion in a 20‐month‐old female with developmental delay and microcephaly

To date, over 70 mutations in the TGFBR2 gene have been reported in patients with Loeys-Dietz syndrome (LDS), Marfan syndrome type 2 (MFS2), or other hereditary thoracic aortic aneurysms and dissections. Whereas almost all of mutations analyzed thus far are predicted to disrupt the constitutively active C-terminal serine/threonine kinase domain of TGFBR2, mounting evidence suggests that the molecular mechanism underlying these diseases is more complex than simple haploinsufficiency. Using exon-targeted oligonucleotide array comparative genomic hybridization, we identified an ∼896 kb deletion of TGFBR2 in a 20-month-old female with microcephaly and global developmental delay, but no stigmata of LDS. FISH analysis showed no evidence of this deletion in the parental peripheral blood samples; however, somatic mosaicism was detected using PCR in the paternal DNA from peripheral blood lymphocytes and lymphoblasts. Our data suggest that TGFBR2 haploinsufficiency may cause a phenotype, which is distinct from LDS. Moreover, we propose that somatic mosaicism below the detection threshold of FISH analysis in asymptomatic parents of children with genomic disorders may be more common than previously recognized.

Complex X chromosome rearrangement delineated by array comparative genome hybridization in a woman with premature ovarian insufficiency

To investigate candidate genes affected by a complex X chromosome rearrangement that may play a role in the diagnosis of spontaneous premature ovarian insufficiency (POI). Prospective cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis and oligonucleotide array comparative genome hybridization (CGH). University medical center. A 36-year-old woman with POI found to have a highly rearrangement X chromosome. FISH analysis and oligonucleotide array CGH. Oligonucleotide microarray analysis to detect duplicated, deleted, or translocated regions of the X chromosome. Complex rearrangement of the X chromosome involving ≥12 breakpoints resulting in two deletions, four duplications, and several intrachromosomal translocations. At least 13 genes with possible relevance to POI may be affected by the rearrangement. Array CGH can reveal candidate genes that may have essential roles in fertility and POI.

Bi-allelic deletions within 13q14 and transient trisomy 21 with absence of GATA1s in pediatric acute megakaryoblastic leukemia

Oligonucleotide array comparative genomic hybridization, karyotype and fluorescence in situ hybridization analyses were employed to delineate the cytogenetic abnormalities in a case of pediatric acute megakaryoblastic leukemia. Here we present a unique genetic profile that includes bi-allelic deletions within 13q14, where the retinoblastoma tumor suppressor gene (RB1) resides, as well as isolated trisomy 21 without a concomitant mutation in the hematopoietic transcription factor GATA1s and translocation (17;22), that does not involve the megakaryoblastic leukemia 1 (MKL1) gene located on chromosome 22. Alteration of the RB1 gene is most likely the critical leukemogenic event in this patient.

Abstract 45: Accurate total and allele-specific copy number measurements in mosaic tumors

Abstract For the characterization of genomic copy number changes that occur in the development and progression of cancer, oligonucleotide array comparative genomic hybridization (aCGH) offers high-resolution and precise determination of chromosomal copy number and genome-wide aberrations. We recently reported a new method for making simultaneous measurements of single nucleotide polymorphisms (SNPs) and copy number alterations in the same microarray assay. The combined assay can detect copy-number neutral events, such as acquired loss of heterozygosity (LOH), as well as allelic imbalance in and around amplified regions. Previously reported algorithms for analyzing Agilent CGH+SNP data were able to genotype primarily diploid samples and to detect regions of constitutional LOH. However, tumor heterogeneity, aneuploidy and variable sample quality create significant challenges for both solid and liquid tumors. Our earlier methods were often unable to cope with the high levels of aneuploidy sometimes found in solid tumors, or with admixtures of normal cells and aberrant cells. We now describe new computational methods which can determine total copy number, aneuploid fraction, and allele-specific copy number in many aneuploid tumor samples, even when mixed with up to 80% normal tissue. We report results from genomic DNA isolated from a cancer cell line, a blood sample, and a solid tumor. Each of these sample types presents novel challenges. The cell line we studied, though largely monoclonal, is highly aneuploid. The blood DNA is of high quality, but the fraction of tumor cells in the sample is low. The solid tumor DNA is of relatively low quality, and composed of multiple aberrant clones. We were able to determine the aneuploid fraction of the tumors, and to measure copy number variation in samples with as little as 20% tumor cell content. In addition to detecting copy-neutral LOH regions, we also measured allele-specific copy number, both of the aberrant clone and of admixed normal cells. The new analysis methods allow the extension of the extra allelic information available from the CGH+SNP assay to cancer samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 45. doi:10.1158/1538-7445.AM2011-45

Abstract 2256: Novel prognostic targets identified using integrated analysis of copy number and expression data in esophageal adenocarcinoma

Abstract Introduction: The incidence of esophageal adenocarcinoma (EAC) has increased 6-fold in the west over the last 30 years, with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. Aims: i) To identify novel molecular targets with clinical relevance using an integrated approach in analyzing genome-wide copy number and expression data of EAC. ii) To correlate reduced expression of molecular targets with promoter hypermethylation in EAC. Methods: 30K oligonucleotide array comparative genomic hybridization (CGH) and gene expression microarray profiling were performed on 56 chemo-naive EAC resection samples with long-term clinical follow-up data (median follow up: 20 months, range 0.5-137) to identify regions of copy number changes leading to differential expression levels. Fluorescence in situ hybridization (FISH) assays were carried out on touch-imprint slides of frozen tumor samples to confirm amplification status. Immunohistochemistry (IHC) assays on tissuemicroarrays (TMAs) on the same (n=56) and an independent (n=371; median follow-up: 17 months, range 0.5-193) cohort of EAC samples were used for subsequent validation at the protein level. Kaplan-Meier survival analyses were performed based on IHC data to determine prognostic significance of novel molecular targets. An Illumina Infinium Array was used to investigate the methylation status of 24 independent EAC tumors, compared with 24 premalignant Barrett's Esophagus (BE) samples. Results: Integrated array CGH data with matched gene expression microarray data highlighted 4 genes with copy number gains (log2 ratio>0.32) and increased expression (fold-change>6.5): EGFR, WT1, NEIL2 and MTMR9. FISH assays confirmed amplifications of these genes (more than 6 copies/centromere) in 11% of EAC samples and IHC assays (n=371 independent cases) demonstrated protein over-expression (IHC scores above 2): EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%), corroborating findings from the integrated analysis. Each of these targets individually had prognostic significance in our cohort (P<0.060). Interestingly, reduced expressions of WT1 (P=0.037) and NEIL2 (P=0.051) were associated with poorer prognosis in EAC. In order to investigate the mechanism of reduced expression of these targets which might have contributed to a worse prognosis, methylation array data was utilized from an independent dataset. This showed increased methylation of WT1 in EAC (63%) compared to BE (55%; t-test P=0.028) and normal controls (50%), but no difference in methylation status of NEIL2 between disease states. Conclusion: Integration of array CGH and gene expression data identified novel molecular targrets in EAC and reduced expression of WT1 may be secondary to methylation. These genes have prognostic potential and require further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2256. doi:10.1158/1538-7445.AM2011-2256

Novel prognostic targets identified using integrated analysis of copy number and expression data in oesophageal adenocarcinoma

IntroductionThe incidence of oesophageal adenocarcinoma (OAC) has increased sixfold in the west over the last 30 years, with a dismal survival rate of less than 20%. Understanding the genomic aberrations...