Oligonucleotide Array Comparative Genomic Hybridization Research Articles

Lissencephaly in an epilepsy cohort: Molecular, radiological and clinical aspects.

Lissencephaly is a rare malformation of cortical development due to abnormal transmantle migration resulting in absent or reduced gyration. The lissencephaly spectrum consists of agyria, pachygyria and subcortical band heterotopia. In this study we compared genetic aetiology, neuroradiology, clinical phenotype and response to antiepileptic drugs in patients with epilepsy and lissencephaly spectrum malformations. The study group consisted of 20 patients - 13 males and 7 females, aged 18 months to 21yearsat the time of data collection. Genetic testing was performed by oligonucleotide array comparative genomic hybridization (microarray), multiplex ligation-dependent probe amplification (MLPA), targeted gene panels and whole exome/genome sequencing. All neuroradiological investigations were re-evaluated and the malformations were classified by the same neuroradiologist. Clinical features and response to anti-epileptic drugs (AEDs) were evaluated by retrospective review of medical records. In eleven patients (55%) mutations in PAFAH1B1 (LIS1) or variable microdeletions of 17p13.3 including the PAFAH1B1 gene were detected. Four patients (20%) had tubulin encoding gene mutations (TUBA1A, TUBG1 and TUBGCP6). Mutations in DCX, DYNC1H1, ADGRG1 and WDR62 were identified in single patients. In one patient, a possibly pathogenic intragenic deletion in TRIO was detected. A clear radiologic distinction could be made between tubulinopathies and PAFAH1B1 related lissencephaly. The majority of the patients had therapy resistant epilepsy and epileptic spasms was the most prominent seizure type. The best therapeutic response to seizure control in our cohort was obtained by the ketogenic diet, vigabatrin, clobazam, phenobarbital and valproate. The most common genetic aetiologies in our cohort of 20 individuals with epilepsy and lissencephaly spectrum were intragenic deletions or single nucleotide mutations in PAFAH1B1 or larger deletions in 17p13.3, encompassing PAFAH1B1, followed by mutations in tubulin encoding genes. Radiological findings could reliably predict molecular results only in agyria with a posterior to anterior gradient. Radiological and molecular findings did not correlate consistently with severity of clinical outcome or therapeutic response.

Identification of novel pathogenic copy number variations in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is a hereditary sensory-motor neuropathy characterized by a strong clinical and genetic heterogeneity. Over the past few years, with the occurrence of whole-exome sequencing (WES) or whole-genome sequencing (WGS), the molecular diagnosis rate has been improved by allowing the screening of more than 80 genes at one time. In CMT, except the recurrent PMP22 duplication accounting for about 60% of pathogenic variations, pathogenic copy number variations (CNVs) are rarely reported and only a few studies screening specifically CNVs have been performed. The aim of the present study was to screen for CNVs in the most prevalent genes associated with CMT in a cohort of 200 patients negative for the PMP22 duplication. CNVs were screened using the Exome Depth software on next generation sequencing (NGS) data obtained by targeted capture and sequencing of a panel of 81 CMT associated genes. Deleterious CNVs were identified in four patients (2%), in four genes: GDAP1, LRSAM1, GAN, and FGD4. All CNVs were confirmed by high-resolution oligonucleotide array Comparative Genomic Hybridization (aCGH) and/or quantitative PCR. By identifying four new CNVs in four different genes, we demonstrate that, although they are rare mutational events in CMT, CNVs might contribute significantly to mutational spectrum of Charcot-Marie-Tooth disease and should be searched in routine NGS diagnosis. This strategy increases the molecular diagnosis rate of patients with neuropathy.

Cytogenetic characterization of the malignant primitive neuroectodermal SK-PN-DW tumor cell line

BackgroundThe SK-PN-DW cell line was established in 1979 and is commercially available. Despite the use of this cell line as an in vitro model for functional and therapeutic studies of malignant primitive neuroectodermal tumor (PNET), there is a lack of complete information about the genetic alterations that are present at the cytogenetic level. Thus, the current study aimed to characterize the cytogenetic profile of this cell line.MethodsRoutine G-banded chromosome analysis, fluorescence in situ hybridization, and oligonucleotide array comparative genomic hybridization assays were performed to characterize the chromosomal changes in this cell line.ResultsThe G-banded karyotype analysis showed that the number of chromosomes in this cell line ranged between 36 and 41. Importantly, all cells displayed a loss of chromosomes Y, 11, 13, and 18. However, some cells showed an additional loss of chromosome 10. Additionally, the observed structural changes indicated: a) unbalanced translocation between chromosomes 1 and 7; b) translocation between chromosomes 11 and 22 at breakpoints 11q24 and 22q12, which is a classical translocation that is associated with Ewing sarcoma; c) a derivative chromosome due to a whole arm translocation between chromosomes 16 and 17 at likely breakpoints 16p10 and 17q10; and d) possible rearrangement in the short arm of chromosome 18. Moreover, a variable number of double minutes were also observed in each metaphase cell. Furthermore, the microarray assay results not only demonstrated genomic-wide chromosomal imbalance in this cell line and precisely placed chromosomal breakpoints on unbalanced, rearranged chromosomes, but also revealed information about subtle chromosomal changes and the chromosomal origin of double minutes. Finally, the fluorescence in situ hybridization assay confirmed the findings of the routine cytogenetic analysis and microarrays.ConclusionThe accurate determination of the cytogenetic profile of the SK-PN-DW cell line is helpful in enabling the research community to utilize this cell line for future identity and comparability studies, in addition to demonstrating the utility of the complete cytogenetic profile, as a public resource.

Analytical validation and chromosomal distribution of regions of homozygosity by oligonucleotide array comparative genomic hybridization from normal prenatal and postnatal case series

BackgroundRegions of homozygosity (ROH) are continuous homozygous segments commonly seen in the human genome. The integration of single nucleotide polymorphism (SNP) probes into current array comparative genomic hybridization (aCGH) analysis has enabled the detection of the ROH. However, for detecting and reporting biologically relevant ROH in a clinical setting, it is necessary to assess the analytical validity of SNP calling and the chromosomal distribution of ROH in normal populations.MethodsThe analytical validity was evaluated by correlating the consistency of SNP calling with the quality parameters of aCGH and by accessing the accuracy of SNP calling using PCR based restriction enzyme digestion and Sanger sequencing. The distribution of ROH was evaluated by the numbers, sizes, locations, and frequencies of ROH from the collection of data from parental, postnatal, and prenatal case series that had normal aCGH and chromosome results.ResultsThe SNP calling failure rate was 20–30% with a derivative Log2 ratio (DLR) below 0.2 and increased significantly to 30–40% with DLR of 0.2–0.4. The accuracy of SNP calling is 93%. Of the 958 cases tested, 34% had no ROH, 64% had one to four ROH, and less than 1% had more than five ROH. Of the 1196 ROH detected, 95% were less than 10 Mb. The distribution of numbers and sizes of ROH showed no differences among the parental, pediatric and prenatal case series and test tissues. The chromosomal distribution of ROH was non-random with ROH seen most frequently in chromosome 8, less frequently in chromosomes 2, 6, 10, 12, 11 and 18, and most rarely seen on chromosomes 15, 19, 21 and 22. Recurrent ROH occurring with a frequency greater than 1% were detected in 17 chromosomal loci which locates either in the pericentric or interstitial regions.ConclusionWith a quality control parameter of DLR set at below 0.2, the consistency of SNP calling would be 75%, the accuracy of SNP call could be 93%, and the observed chromosomal distribution of ROH could be used as a reference. This aCGH analysis could be a reliable screening tool to document biologically relevant ROH and recommend further molecular analysis.

Transfer of embryos with segmental mosaicism is associated with a significant reduction in live-birth rate

To evaluate the impact of segmental mosaicism on pregnancy outcomes from the transfer of embryos previously designated as euploid. Retrospective cohort analysis. Single, private, high-volume fertility center. Three hundred and twenty-seven women who underwent 377 frozen single euploid embryo transfers. Trophectoderm biopsy of embryos cultured to the blastocyst stage, where all transferred embryos were designated euploid by high-density oligonucleotide array comparative genomic hybridization (aCGH); after ascertaining all outcomes, revaluation of aCGH results for evidence of segmental mosaicism (defined as mosaicism on a portion of a chromosome). Live-birth rate and spontaneous abortion rate. Of the 377 embryos transferred, 357 were euploid with no mosaicism, and 20 embryos had segmental mosaicism. Segmental mosaics had a statistically significantly lower live-birth rate compared with euploid controls (30.0% vs. 53.8%). When controlling for age and day of Trophectoderm biopsy, the odds for live birth after transfer of segmental mosaics were reduced by 66% compared with euploid controls (0.34; 95% confidence interval, 0.13-0.92). The spontaneous abortion rate was statistically significantly higher after transfer of segmental mosaics compared with euploid controls (40.0% vs. 18.2%). Blastocysts with segmental mosaicism have reduced reproductive potential but retain the ability to result in live birth. These results support reporting segmental mosaicism to optimize selection of a single embryo for transfer that will maximize the chance of life birth.

A Review of Copy Number Variants in Inherited Neuropathies.

The rapid development in the last 10-15 years of microarray technologies, such as oligonucleotide array Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphisms (SNP) genotyping array, has improved the identification of fine chromosomal structural variants, ranging in length from kilobases (kb) to megabases (Mb), as an important cause of genetic differences among healthy individuals and also as disease-susceptibility and/or disease-causing factors. Structural genomic variations due to unbalanced chromosomal rearrangements are known as Copy-Number Variants (CNVs) and these include variably sized deletions, duplications, triplications and translocations. CNVs can significantly contribute to human diseases and rearrangements in several dosage-sensitive genes have been identified as an important causative mechanism in the molecular aetiology of Charcot-Marie-Tooth (CMT) disease and of several CMT-related disorders, a group of inherited neuropathies with a broad range of clinical phenotypes, inheritance patterns and causative genes. Duplications or deletions of the dosage-sensitive gene PMP22 mapped to chromosome 17p12 represent the most frequent causes of CMT type 1A and Hereditary Neuropathy with liability to Pressure Palsies (HNPP), respectively. Additionally, CNVs have been identified in patients with other CMT types (e.g., CMT1X, CMT1B, CMT4D) and different hereditary poly- (e.g., giant axonal neuropathy) and focal- (e.g., hereditary neuralgic amyotrophy) neuropathies, supporting the notion of hereditary peripheral nerve diseases as possible genomic disorders and making crucial the identification of fine chromosomal rearrangements in the molecular assessment of such patients. Notably, the application of advanced computational tools in the analysis of Next-Generation Sequencing (NGS) data has emerged in recent years as a powerful technique for identifying a genome-wide scale complex structural variants (e.g., as the ones resulted from balanced rearrangements) and also smaller pathogenic (intragenic) CNVs that often remain beyond the detection limit of most conventional genomic microarray analyses; in the context of inherited neuropathies where more than 70 disease-causing genes have been identified to date, NGS and particularly Whole-Genome Sequencing (WGS) hold the potential to reduce the number of genomic assays required per patient to reach a diagnosis, analyzing with a single test all the Single Nucleotide Variants (SNVs) and CNVs in the genes possibly implicated in this heterogeneous group of disorders.

Prenatal diagnosis of low-level mosaicism for trisomy 13 at amniocentesis associated with a favorable outcome

ObjectiveWe present prenatal diagnosis of low-level mosaicism for trisomy 13 at amniocentesis associated with a favorable outcome. Case reportA 35-year-old woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+13[5]/46,XY[20]. Oligonucleotide array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed arr [GRCh37] (13)×3 [0.10], (X,Y)×1 compatible with trisomy 13 mosaicism. Prenatal ultrasound was unremarkable. Repeat amniocentesis was performed at 21 weeks of gestation. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes revealed a mosaic trisomy 13 level of 10% (10/100 cells). aCGH analysis on uncultured amniocytes revealed a result of arr 13q12.11q34 (20,407,323–115,092,619)×2.1 with a log2 ratio of 0.06 compatible with a 10% level of mosaicism. Polymorphic DNA marker analysis excluded uniparental disomy 13. The parental karyotypes were normal. Conventional cytogenetic analysis using cultured amniocytes at repeat amniocentesis revealed a karyotype of 46,XY in 23/23 colonies. The pregnancy was carried to 37 weeks of gestation, and a 3600-g phenotypically normal male baby was delivered. When examined at 8 months of age, the infant was doing well and was normal in psychomotor and growth development. The peripheral blood had a karyotype of 46,XY, and interphase FISH analysis on uncultured urinary cells revealed a mosaic trisomy 13 level of 4.4% (2/45 cells). ConclusionLow-level true mosaicism for trisomy 13 at amniocentesis without ultrasound abnormalities can be associated with a favorable fetal outcome.

Prenatal diagnosis of a 1.6-Mb 4p16.3 interstitial microdeletion encompassing FGFRL1 and TACC3 associated with bilateral cleft lip and palate of Wolf-Hirschhorn syndrome facial dysmorphism and short long bones

ObjectiveWe present prenatal diagnosis of a 4p16.3 interstitial microdeletion associated with bilateral cleft lip and palate and short long bones on prenatal ultrasound, and we discuss the genotype–phenotype correlation. Materials and methodsA 32-year-old woman underwent amniocentesis at 22 weeks of gestation because of bilateral cleft lip and palate and short limbs on prenatal ultrasound. Conventional cytogenetic analysis was performed on cultured amniocytes and parental bloods. Oligonucleotide array comparative genomic hybridization (aCGH) was performed on the DNAs extracted from uncultured amniocytes, parental bloods and umbilical cord. Metaphase fluorescence in situ hybridization (FISH) was performed on cultured amniocytes. ResultsAmniocentesis revealed a karyotype of 46,XY. The parental karyotypes were normal. aCGH analysis on uncultured amniocytes revealed a 1.66-Mb interstitial microdeletion at 4p16.3 encompassing 23 Online Mendelian Inheritance of in Man (OMIM) genes including FGFRL1 and TACC3. The parents did not have such a deletion. The pregnancy was subsequently terminated, and a malformed fetus was delivered with typical Wolf-Hirschhorn syndrome (WHS) facial appearance and bilateral cleft lip and palate. aCGH analysis of the umbilical cord confirmed the prenatal diagnosis with a result of arr 4p16.3 (72,447–1,742,649) × 1.0 [GRCh37 (hg19)]. Metaphase FISH analysis of cultured amniocytes confirmed a 4p16.3 microdeletion. ConclusionHaploinsufficiency of FGFRL1 and TACC3 at 4p16.3 can be associated with bilateral cleft lip and palate of WHS facial dysmorphism and short long bones. Prenatal diagnosis of facial cleft with short long bones should raise a suspicion of chromosome microdeletion syndromes.

Transfer of embryos with segmental mosaicism is associated with a significant reduction in live birth rate compared to euploid controls

Segmental mosaicism is a complex issue with minimal to no data on established thresholds for labeling an embryo euploid versus aneuploid, or on pregnancy outcomes on the transfer of embryos with segmental mosaicism. We aim to evaluate the impact of segmental mosaicism on pregnancy and live birth rates (LBR) from the transfer of embryos previously designated as euploid. Retrospective cohort analysis. All PGS-screened frozen single embryo transfers from January 2015 - December 2015 from a high-volume fertility center were included. Transferred embryos were all designated euploid by high-density oligonucleotide array comparative genomic hybridization (aCGH) PGS by the reporting laboratory. Embryos with segmental mosaicism, designated as euploid, were classified by the reporting laboratory based on the size, location, and percentage of mosaicism of the duplication or deletion. The detailed aCGH results from all transferred euploid embryos were subsequently unmasked to determine the impact of segmental mosaicism on pregnancy outcomes. Chi-square analyses were used to detect differences between euploid versus segmental mosaic embryos. Logistic regression was performed to control for age and day of blastocyst biopsy. A total of 327 women underwent 377 frozen single euploid embryo transfers. After unmasking PGS results, 357 embryos were euploid with no mosaicism and 20 embryos had segmental mosaicism. Average patient age was similar between groups. LBR was significantly lower in the segmental mosaic group (30.0% vs 53.8%, p= 0.04). The odds of a live birth after transfer of embryos with segmental mosaicism was reduced by 66% compared to euploid embryos, controlled for age and day of biopsy (0.34 [95% CI, 0.13- 0.92]) (Table 1). Spontaneous miscarriage, defined as a failed pregnancy following a positive pregnancy test, was significantly higher in the segmental mosaic group compared to euploid controls (40.0% vs. 18.2%, p= 0.04). Blastocysts with segmental mosaicism result in significantly decreased LBR and higher SAB rates compared to euploid controls. These results support reporting all segmental mosaicism, regardless of degree, to optimize embryo selection and LBR.Tabled 1Transfer outcomes of embryos with segmental mosaicism compared to euploid controlsEuploid Embryos N=357Segmental Mosaicism Detected N=20Adjusted Odds Ratio (95% CI)Live Birth N (%)192 (53.8%)6 (30.0%)0.34 (0.13-0.92)Spontaneous Miscarriage N (%)65 (18.2%)8 (40.0%)3.02 (1.18-7.76) Open table in a new tab

Malignant canine mammary tumours: Preliminary genomic insights using oligonucleotide array comparative genomic hybridisation analysis

Neoplastic mammary disease in female dogs represents a major health concern for dog owners and veterinarians, but the genomic basis of the disease is poorly understood. In this study, we performed high resolution oligonucleotide array comparative genomic hybridisation (oaCGH) to assess genome wide DNA copy number changes in 10 malignant canine mammary tumours from seven female dogs, including multiple tumours collected at one time from each of three female dogs. In all but two tumours, genomic imbalances were detected, with losses being more common than gains. Canine chromosomes 9, 22, 26, 27, 34 and X were most frequently affected. Dissimilar oaCGH ratio profiles were observed in multiple tumours from the same dogs, providing preliminary evidence for probable independent pathogenesis. Analysis of adjacent samples of one tumour revealed regional differences in the number of genomic imbalances, suggesting heterogeneity within tumours.

Abstract P1-05-19: Homologous recombination deficiency in PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma

Abstract Introduction: Homologous recombination (HR) is essential for repairing double-stranded DNA breaks. Tumors deficient in DNA HR display high levels of copy number aberrations including amplifications, deletions, and breakpoints. Studies in BRCAmut cancers have shown that HR deficiency (HRD) renders triple negative breast carcinoma (TNBC) sensitive to carboplatin or Poly(ADP-ribose) polymerase (PARP) inhibitors via a synthetic lethal interaction, as previously described. Clinical observations in breast and other solid tumors suggest that the HRD phenotype may arise independently of BRCA1/2 mutation. The implications of HRD enrichment in PD-L1, PD-L2, Jak2 (PDJ) 9p24.1 amplified TNBC are not known. Our goal was to determine the HRD status in PDJ enriched tumors, and the potential association of the PDJ amplicon with a BRCA like HRD phenotype. Methods: Archival fresh frozen and formalin-fixed paraffin embedded (FFPE) tumor samples from 41 patients with TNBC were obtained from 1998-2015. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted using DNA content flow cytometry and interrogated with oligonucleotide array comparative genomic hybridization (aCGH). Tumor homologous recombination deficiency (HRD) scores were calculated by number of interstitial aberrations (IA) in their genomes (HRD-IA). A cut-off of < 40 (low), > 40 (high) or > 50 (very high) was assigned based on our studies of known BRCAmut tumors. Data were compared between HRD groups using chi-square, ANOVA F-test or log-rank test. Results: 14/41 (34%) TNBC patients had high HRD-IA score and 6/41 (14.6%) had very high HRD-IA score. The median age was 61.8 years (SD = 12.8), the average tumor size was 3.1cm (SD = 2.6), 42% were node positive, 24% were pre-menopausal, 19% were Black, Hispanic, or Asian/Pacific Islander. Genetic testing was negative for BRCA1/2 mutations in 9/41 patients, including 3 high and very high HRD-IA score patients. There was no difference in the OS at 5-years [74% versus 59% (p = 0.907)] in the high HRD-AI group. 12/41 (29%) patients were PDJ+. In the PDJ+ patients, 4/12 (33.3%) had HRD > 40 and 1/12 (8.3%) had HRD > 50 compared to the PDJ- patients where 10/29 (34%) had high HRD-IA score and 5/29 (17%) had a very high HRD-IA score, p=0.463. Patients with inflammatory TNBC were significantly more likely to have high HRD-IA score 4/6 (66%) compared to non-inflammatory TNBC 8/31 (26%), p=0.05. Conclusions: We describe a unique group of TNBC patients with similar patient characteristics, tumor size, nodal status, and stage when compared by low, high and very high HRD-IA score. For inflammatory TNBC, there was statistically significant percentage of patients with a high HRD-IA score. We did not see a statistically significant correlation with 9p24.1 amplification based on HRD-IA score which has been suggested in other data sets. The HRD-IA score did not statistically impact OS but there was a suggestion that those with a higher score may have an improved OS potentially due to chemotherapy sensitivity. Further study with a greater number of patients to adequately power the study is needed to determine whether our initial findings can be confirmed and whether the HRD-IA score can be used to define a specific subset of TNBC patients. Citation Format: Gawryletz CD, Anderson KS, Northfelt DW, Kosiorek HE, Linnaus ME, Ocal IT, McCullough AE, Pockaj BA, Barrett MT. Homologous recombination deficiency in PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-19.

Genomic profiling of canine mast cell tumors identifies DNA copy number aberrations associated with KIT mutations and high histological grade.

Mast cell tumor (MCT) is the most common skin malignancy of domestic dogs and presents with a widely variable clinical behavior. Although activating KIT mutations are present in approximately 20% of canine MCTs, molecular etiology is largely unknown for the majority of this cancer. Characterization of genomic alterations in canine MCTs may identify genomic regions and/or genes responsible for their development and progression, facilitating the discovery of new therapeutic targets and improved clinical management of this heterogeneous cancer. We performed genome-wide DNA copy number analysis of 109 primary MCTs derived from three popular canine breeds (the Boxer, Labrador Retriever, and Pug) as well as nontarget breeds using oligonucleotide array comparative genomic hybridization (oaCGH). We demonstrated a stepwise accumulation of numerical DNA copy number aberrations (CNAs) as tumor grade increases. DNA sequencing analysis revealed that KIT mutations were found less frequently in the Pug tumors and were strongly associated with high histological grade. Tumors with KIT mutations showed genome-wide aberrant copy number profiles, with frequent CNAs involving genes in the p53 and RB pathways, whereas CNAs were very limited in tumors with wild-type KIT. We evaluated the presence of four CNAs to predict aggressive tumor phenotypes. This approach predicted aggressive tumors with a sensitivity of 78-94% and specificity of 88-93%, when using oaCGH and droplet digital PCR platforms. Further investigation of genome regions identified in this study may lead to the development of a molecular tool for classification and prognosis, as well as identification of therapeutic target molecules.

Familial Case of Pelizaeus-Merzbacher Disorder Detected by Oligoarray Comparative Genomic Hybridization: Genotype-to-Phenotype Diagnosis

Introduction. Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive hypomyelinating leukodystrophy characterized by nystagmus, spastic quadriplegia, ataxia, and developmental delay. It is caused by mutation in the PLP1 gene. Case Description. We report a 9-year-old boy referred for oligoarray comparative genomic hybridization (OA-CGH) because of intellectual delay, seizures, microcephaly, nystagmus, and spastic paraplegia. Similar clinical findings were reported in his older brother and maternal uncle. Both parents had normal phenotypes. OA-CGH was performed and a 436 Kb duplication was detected and the diagnosis of PMD was made. The mother was carrier of this 436 Kb duplication. Conclusion. Clinical presentation has been accepted as being the mainstay of diagnosis for most conditions. However, recent developments in genetic diagnosis have shown that, in many congenital and sporadic disorders lacking specific phenotypic manifestations, a genotype-to-phenotype approach can be conclusive. In this case, a diagnosis was reached by universal genomic testing, namely, whole genomic array.

Comprehensive genomic characterization of five canine lymphoid tumor cell lines.

BackgroundLeukemia/lymphoma cell lines have been critical in the investigation of the pathogenesis and therapy of hematological malignancies. While human LL cell lines have generally been found to recapitulate the primary tumors from which they were derived, appropriate characterization including cytogenetic and transcriptional assessment is crucial for assessing their clinical predictive value.ResultsIn the following study, five canine LL cell lines, CLBL-1, Ema, TL-1 (Nody-1), UL-1, and 3132, were characterized using extensive immunophenotyping, karyotypic analysis, oligonucleotide array comparative genomic hybridization (oaCGH), and gene expression profiling. Genome-wide DNA copy number data from the cell lines were also directly compared with 299 primary canine round cell tumors to determine whether the cell lines represent primary tumors, and, if so, what subtype each most closely resembled.ConclusionsBased on integrated analyses, CLBL-1 was classified as B-cell lymphoma, Ema and TL-1 as T-cell lymphoma, and UL-1 as T-cell acute lymphoblastic leukemia. 3132, originally classified as a B-cell lymphoma, was reclassified as a histiocytic sarcoma based on characteristic cytogenomic properties. In combination, these data begin to elucidate the clinical predictive value of these cell lines which will enhance the appropriate selection of in vitro models for future studies of canine hematological malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0836-z) contains supplementary material, which is available to authorized users.

Esophageal atresia with tracheoesophageal fistula in a patient with 7q35–36.3 deletion including SHH gene

Terminal 7q deletion is rarely reported in the literature. Holoprosencephaly and sacral dysgenesis are found in association with this deletion, due to haploinsufficiency of SHH and HLBX9 genes respectively. We report on a 2-year-old boy with 7q35–36.3 deletion encompassing SHH identified by oligonucleotide array comparative genomic hybridization. In addition to other frequent features, the patient presented with esophageal atresia and tracheoeosophageal fistula diagnosed at birth. This case, together with two others previously described, one presenting with esophageal atresia, the other with congenital esophageal stenosis, confirms the possible association between congenital esophageal malformations and 7q terminal deletion including SHH.

Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media

To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of <0.85 (a quality control metric of oligonucleotide array comparative genomic hybridization). The fluid sample and trophectoderm results were identical demonstrating (45XY, -13). Three samples were reamplified 1hour later and tested showing improving DLRSD. One of the three samples with a DLRSD of 0.85 demonstrated (46XY), consistent with the biopsy. Overnight DNA amplification showed DNA in all samples. We demonstrate two novel findings: the presence of free embryonic DNA in spent media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future.

Familial GPC3 and GPC4-TFDP3 deletions at Xq26 associated with Simpson-Golabi-Behmel syndrome

We report a familial case of Simpson-Golabi-Behmel syndrome type 1 (SGBS1) with prenatal ultrasound findings of bilateral ventriculomegaly and polyhydramnios and postnatal findings of macroglossia, macrosomia and other dysmorphic features. Oligonucleotide array comparative genomic hybridization (aCGH) analysis and further polymerase chain reaction (PCR) sequencing identified a 167.071kb hemizygous deletion including the entire TFDP3 gene and exons 3–9 of the GPC4 (Glypican 4) gene, and a 86.943kb deletion including exon 3 of the GPC3 (Glypican 3) gene. Joined sequences at the breakpoints indicate that the deletion of TFDP3 and GPC4 is likely caused by microhomology-mediated end joining while the deletion within the GPC3 gene is due to non-homology end joining. Both deletions are considered loss of function due to the loss of large portions of coding sequences. The prenatal ultrasound findings of the affected male fetuses in this family are comparable with two previously reported cases with large deletions of the GPC3 and GPC4 genes. Further investigation of more cases with genetic defects of both GPC3 and GPC4 genes is needed for assessing the genotype-phenotype correlation.

Copy-Number Variation Contributes to the Mutational Load of Bardet-Biedl Syndrome

Bardet-Biedl syndrome (BBS) is a defining ciliopathy, notable for extensive allelic and genetic heterogeneity, almost all of which has been identified through sequencing. Recent data have suggested that copy-number variants (CNVs) also contribute to BBS. We used a custom oligonucleotide array comparative genomic hybridization (aCGH) covering 20 genes that encode intraflagellar transport (IFT) components and 74 ciliopathy loci to screen 92 unrelated individuals with BBS, irrespective of their known mutational burden. We identified 17 individuals with exon-disruptive CNVs (18.5%), including 13 different deletions in eight BBS genes (BBS1, BBS2, ARL6/BBS3, BBS4, BBS5, BBS7, BBS9, and NPHP1) and a deletion and a duplication in other ciliopathy-associated genes (ALMS1 and NPHP4, respectively). By contrast, we found a single heterozygous exon-disruptive event in a BBS-associated gene (BBS9) in 229 control subjects. Superimposing these data with resequencing revealed CNVs to (1) be sufficient to cause disease, (2) Mendelize heterozygous deleterious alleles, and (3) contribute oligogenic alleles by combining point mutations and exonic CNVs in multiple genes. Finally, we report a deletion and a splice site mutation in IFT74, inherited under a recessive paradigm, defining a candidate BBS locus. Our data suggest that CNVs contribute pathogenic alleles to a substantial fraction of BBS-affected individuals and highlight how either deletions or point mutations in discrete splice isoforms can induce hypomorphic mutations in genes otherwise intolerant to deleterious variation. Our data also suggest that CNV analyses and resequencing studies unbiased for previous mutational burden is necessary to delineate the complexity of disease architecture.

Abstract P6-03-13: The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma

Abstract Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-13.

Rapid Increase in frequency of gene copy-number variants during experimental evolution in Caenorhabditis elegans.

BackgroundGene copy-number variation (CNVs), which provides the raw material for the evolution of novel genes, is widespread in natural populations. We investigated whether CNVs constitute a common mechanism of genetic change during adaptation in experimental Caenorhabditis elegans populations. Outcrossing C. elegans populations with low fitness were evolved for >200 generations. The frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, PCR, DNA sequencing across breakpoints, and single-worm PCR.ResultsMultiple duplications and deletions rose to intermediate or high frequencies in independent populations. Several lines of evidence suggest that these changes were adaptive: (i) copy-number changes reached high frequency or were fixed in a short time, (ii) many independent populations harbored CNVs spanning the same genes, and (iii) larger average size of CNVs in adapting populations relative to spontaneous CNVs. The latter is expected if larger CNVs are more likely to encompass genes under selection for a change in gene dosage. Several convergent CNVs originated in populations descended from different low fitness ancestors as well as high fitness controls.ConclusionsWe show that gene copy-number changes are a common class of adaptive genetic change. Due to the high rates of origin of spontaneous duplications and deletions, copy-number changes containing the same genes arose readily in independent populations. Duplications that reached high frequencies in these adapting populations were significantly larger in span. Many convergent CNVs may be general adaptations to laboratory conditions. These results demonstrate the great potential borne by CNVs for evolutionary adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2253-2) contains supplementary material, which is available to authorized users.