Array Comparative Hybridization Research Articles

Two infants with mild, atypical clinical features of Kagami-Ogata syndrome caused by epimutation

Kagami-Ogata syndrome (KOS) is an imprinting disorder characterized by polyhydramnios, bell-shaped thorax with coat-hanger appearance (curved ribs), respiratory distress, abdominal wall defects, and distinct facial features, together with intellectual developmental delay with special needs. Abnormal expression of the imprinted genes on chromosome 14q32.2 causes KOS. Epimutation with aberrant hypermethylation of the MEG3/DLK1: intergenic differentially methylated region (MEG3/DLK1:IG-DMR) and the MEG3:TSS-DMR is one of the etiologies of KOS. We report two infants with KOS caused by epimutation presenting with some characteristic clinical features, mild clinical course, and almost normal motor and intellectual development. Methylation analysis for ten DMRs related to major imprinting disorders using pyrosequencing with genomic DNA (gDNA) extracted from leukocytes showed abnormally increased methylation levels of the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR in both patients, but lower than those in patients with paternal uniparental disomy chromosome 14 (upd(14)pat). The methylation levels in the DMRs other than both DMRs were within normal range. We also conducted methylation analysis for the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR with gDNA extracted from nails and buccal cells of both patients. Methylation levels in the MEG3:TSS-DMR, particularly in buccal cells, were closer to normal range compared to those in leukocytes. Microsatellite analysis for chromosome 14 and array comparative hybridization analysis showed no upd(14)pat or microdeletion involving the 14q32.2 imprinted region in either patient. A differential mosaic ratio of cells with aberrant methylation of DMRs at the 14q32.2 imprinted region among tissues (connective tissue, lung, and brain) might have led to their atypical clinical features. Further studies of patients with epimutation should further expand the phenotypic spectrum of KOS.

P-357 Predicting live birth following single euploid frozen blastocyst transfer - does the journey matter?

Abstract Study question Does the number of available euploid embryos affect the success rate of the first single euploid frozen thawed embryo transfer (FTET)? Summary answer The live birth rate (LBR) following the first single euploid FTET is independent of the number of available euploid embryos. What is known already Preimplantation testing for aneuploidy (PGT-A) is a controversial laboratory technique as an embryo selection tool to increase LBR per transfer and reduce time to pregnancy. There have been many efforts to identify factors predictive of LBR following a euploid embryo transfer. The number of euploid embryos available is known to increase the expected cumulative LBR. However, there is lack of evidence whether the number of available euploid embryos is also significantly associated with chances of live birth for the first euploid FTET cycle. This is important for individualized patient counselling. Study design, size, duration A retrospective analysis of the first, single, euploid FTET of 506 women, between January 2015 to March 2020 following PGT-A was carried out. The indication for PGT-A was advanced maternal age, recurrent miscarriage or implantation failure. Embryos were biopsied on day 5 or 6 and tested with array comparative genetic hybridization or next generation sequencing. The FTET outcomes were compared based on the number of euploid embryos available. Participants/materials, setting, methods 198 women had one euploid embryo available (group 1), 120 women had two (group 2) and 188 women had three or more (group 3). Baseline characteristics were analysed for all participants to adjust for confounders. Reproductive outcomes such as pregnancy rate (PR), clinical miscarriage rate and LBR were compared between the 3 groups for the first euploid FTET. Univariate analysis with Kruskal-Wallis H, chi-square and multivariate logistic regression was performed with SPSS. Main results and the role of chance Baseline characteristics were similar between the groups including age at egg collection (EC), body mass index, use of partner or donor sperm, method of insemination, number of ECs, endometrial preparation protocol and endometrial thickness prior to transfer. Women with three or more euploid embryos were younger at the time of EC compared to groups 1 and 2 (mean age 37 vs 39 and 38). The number of oocytes per EC (15 vs 11 and 11, p < 0.001), the total number of oocytes following batching cycles (20 vs 15 and 16, p < 0.001) and the number of blastocysts biopsied (8 vs 4 and 5, p < 0.001) were significantly higher for Group 3 vs Group 1 and Group 2, respectively. Group 1 embryos were less frequently of excellent and very good morphology and less frequently biopsied on day 5 vs 6. Univariate analysis suggests Group 3 had statistically higher PR and LBR compared to Group 1, but similar miscarriage rate. However, when applying a multivariate logistic regression model, the LBR after first euploid FTET was independent of the number of euploid embryos available but was influenced by age at EC (p = 0.006) and biopsy day (Day 6 vs day 5, OR 0.53, 95% CI 0.33-0.85, p = 0.008). Limitations, reasons for caution The main limitation of this study is its retrospective nature performed in a single centre, and therefore vulnerable to bias and confounding factors that may have not been accounted for. Wider implications of the findings Our findings suggest that the success rate of the first euploid FTET does not depend on the number of euploid embryos but is influenced by the age at EC and the embryo stage at biopsy. This could be useful when counselling women with a low number of euploid embryos available. Trial registration number Not Applicable

Preimplantation genetic testing for aneuploidy: A review of published blastocyst reanalysis concordance data.

Preimplantation genetic testing for aneuploidy (PGT‐A) reduces miscarriage risk, increases the success of IVF, shortens time to pregnancy, and reduces multiple gestation rates without compromising outcomes. The progression of PGT‐A has included common application of next‐generation sequencing (NGS) from single nucleotide polymorphism microarray, quantitative real‐time PCR, and array comparative hybridization platforms of analysis. Additional putative advances in PGT‐A capability include classifying embryos as mosaic and predicting the presence of segmental imbalance. A critical component in the process of technical validation of these advancements involves evaluation of concordance between reanalysis results and initial testing results. While many independent studies have investigated the concordance of results obtained from the remaining embryo with the original PGT‐A diagnosis, compilation and systematic analysis of published data has not been performed. Here, we review results from 26 primary research articles describing concordance in 1271 human blastocysts from 2260 pairwise comparisons. Results illustrate significantly higher discordance from PGT‐A methods which utilize NGS and include prediction of mosaicism or segmental imbalance. These results suggest caution when considering new iterations PGT‐A.

Genomic imbalances defining novel intellectual disability associated loci

BackgroundHigh resolution genome-wide copy number analysis, routinely used in clinical diagnosis for several years, retrieves new and extremely rare copy number variations (CNVs) that provide novel candidate genes contributing to disease etiology. The aim of this work was to identify novel genetic causes of neurodevelopmental disease, inferred from CNVs detected by array comparative hybridization (aCGH), in a cohort of 325 Portuguese patients with intellectual disability (ID).ResultsWe have detected CNVs in 30.1% of the patients, of which 5.2% corresponded to novel likely pathogenic CNVs. For these 11 rare CNVs (which encompass novel ID candidate genes), we identified those most likely to be relevant, and established genotype-phenotype correlations based on detailed clinical assessment. In the case of duplications, we performed expression analysis to assess the impact of the rearrangement. Interestingly, these novel candidate genes belong to known ID-related pathways. Within the 8% of patients with CNVs in known pathogenic loci, the majority had a clinical presentation fitting the phenotype(s) described in the literature, with a few interesting exceptions that are discussed.ConclusionsIdentification of such rare CNVs (some of which reported for the first time in ID patients/families) contributes to our understanding of the etiology of ID and for the ever-improving diagnosis of this group of patients.

Day 6 pgs fresh embryo transfer improves pregnancy outcome and reduce miscarriage rate

There are concerns that day 6 PGS fresh embryo transfer may negatively impact pregnancy outcome due to altered embryo-endometrial synchrony. The current study tests the hypothesis that early day 6 fresh embryo transfers after PGS (D6PGS) will result in increased pregnancy outcome and reduced miscarriage rate compared to day 5 fresh embryo transfer without PGS (D5NPGS). The study also investigates the differences in P4 levels on the day of HCG in both groups. Retrospective analysis. Outcome of 348 ICSI cycles performed at a private ART center between 2010 and 2016 were reviewed. Of these, cycles that undergone D6PGS (N=69) were compared to D5NPGS (N=279), which served as controls. D6PGS strategy involved trophoectoderm biopsy of hatching blastocysts on early day 5, and fresh transfer of euploid embryo in the morning of day 6. Aneuploidy screening was done using array Comparative Genetic Hybridization (a-CGH) technique. Peak P4 levels on the day of HCG were analyzed. The primary outcome measure was the live birth rate. The ongoing pregnancy rate and miscarriage rate were the secondary outcomes. There were no differences in the number of retrieved oocytes (11.24 vs 11.40, P=<0.823) nor in the total mature oocytes (8.75 vs 8.44, P=<0.580). There were no differences in the number of fertilized zygotes (7.94 vs 7.29, P=<0.217). There is significantly lower P4 levels on the day of HCG in D6PGS (0.696 vs 0.907, P=<0.003). Although D5NPGS cycles included younger patients (34.80 vs 35.87, P=<0.062), and had a significantly higher number of embryos transferred (1.78 vs 1.26, P=<0.001), there was a significantly higher clinical pregnancy rate in D6PGS cycles (60.8 vs 45.1%, P=<0.022). There was a trend towards lower miscarriage rate in D6PGS cases (7.1% vs 15.8%, P=<0.19). There was also a trend towards higher live birth rate in D6PGS (56.5% vs 44.0%, P=<0.054). In order to determine the effect on the older patients (37 years or above), 30 cycles with D6PGS were compared against 103 aged-matched cycles with D5NPGS. As noted for the whole study population, P4 levels on the day of HCG in D6PGS were significantly lower (0.695 vs 0.927 ng/ml, P=<0.032). However, in the older patient group there was a significantly higher clinical pregnancy rate in D6PGS cycles (64.5% vs 29.1%, P=<0.001) in spite of significantly higher number of embryos transferred in D5NPGS cycles (2.10 vs 1.29, P=<0.001). Miscarriage rate was significantly reduced in the older patient group in D6PGS cycles (5.0% vs 36.6%, P=<0.026). The live birth rate was significantly higher in D6PGS (61.2% vs 27.8, P=<0.001). Day 6 fresh PGS embryo transfer improved the pregnancy outcome and reduced the miscarriage rate, particularly in older women, compared to day 5 NPGS fresh embryo transfer. Although there is a significant shift to do freeze-all PGS cycles, fresh day 6 PGS embryo transfer benefits the older patients by allowing immediate transfer, avoiding the subsequent frozen thawed cycles and saving time and cost.

First live birth using human oocytes reconstituted by spindle nuclear transfer for mitochondrial DNA mutation causing Leigh syndrome

Mutations in mitochondrial (mt) DNA are maternally inherited and can cause fatal or debilitating disorders without effective treatments.1,2 The severity of clinical symptoms is often associated with the mtDNA mutation load in heteroplasmy.3 Experimental nuclear transfer in metaphase II (MII) spindle oocytes or in pronuclear (PN) zygotes, also called mitochondrial replacement therapy, has been shown to be a novel technology in minimizing mutated mtDNA transmission from oocytes to pre-implantation embryos.4,5 Here we report the first live birth of a boy following spindle nuclear transfer (SNT). Translational research. The patient is a 36-year-old woman with 24.5% mtDNA displaying 8993T>G mutation in subunit 6 of the ATPase gene known to cause Leigh syndrome.6 She had 4 pregnancy losses and 2 deceased children at age 8 months and 6 years from Leigh syndrome as confirmed by >95% mutation load. She was seeking conception of a healthy baby and elected to have SNT over PN transfer for religious reasons. Under IRB-approved protocol with proper consent, the patient’s spindle nuclei were isolated and transferred into the perivitelline space of enucleated donor oocytes. The micro-manipulated complexes were then subjected to 1.4 kV/cm DC voltage for cell membrane fusion. The reconstituted oocytes were fertilized by intracytoplasmic sperm injection (ICSI). The developed blastocysts were biopsied for preimplantation genetic screening (PGS) by array comparative genetic hybridization and whole quantitative genomic mtDNA analysis by Next Generation Sequencing. Five MII oocytes with birefringent spindles were subjected to meiotic SNT. The 5 oocytes were successfully reconstituted and fertilized normally by ICSI. Four out of 5 fertilized oocytes developed into blastocysts. PGS showed that one blastocyst was euploid (46XY), while 3 embryos were aneuploid. The average transmission rate of maternal mtDNA in the biopsied euploid blastocyst was 5.10 ± 1.11% and the heteroplasmy level for 8993T>G was 5.73%. Transfer of the euploid embryo resulted in an uneventful pregnancy with delivery of a healthy boy at 37 weeks of gestation. The average level of transmitted mother’s mtDNA in several neonatal tissues including buccal epithelium, hair follicles, circumcised foreskin, urine precipitate, placenta, amnion, umbilical blood, and umbilical cord was less than 1.60 ± 0.92%. The baby is currently 3 months old and doing well. Human oocytes reconstituted by SNT are capable of producing a healthy live birth. SNT may provide a novel treatment option in minimizing pathogenic mtDNA transmission from mothers to their babies.

Application of Array Comparative Genomic Hybridization in Newborns with Multiple Congenital Anomalies.

Major congenital anomalies are detectable in 2-3 % of the newborn population. Some of their genetic causes are attributable to copy number variations identified by array comparative genomic hybridization (aCGH). The value of aCGH screening as a first-tier test in children with multiple congenital anomalies has been studied and consensus adopted. However, array resolution has not been agreed upon, specifically in the newborn or infant population. Moreover, most array studies have been focused on mixed populations of intellectual disability/developmental delay with or without multiple congenital anomalies, making it difficult to assess the value of microarrays in newborns. The aim of the study was to determine the optimal quality and clinical sensitivity of high-resolution array comparative genomic hybridization in neonates with multiple congenital anomalies. We investigated a group of 54 newborns with multiple congenital anomalies defined as two or more birth defects from more than one organ system. Cytogenetic studies were performed using OGT CytoSure 8 × 60K microarray. We found ten rearrangements in ten newborns. Of these, one recurrent syndromic microduplication was observed, whereas all other changes were unique. Six rearrangements were definitely pathogenic, including one submicroscopic and five that could be seen on routine karyotype analysis. Four other copy number variants were likely pathogenic. The candidate genes that may explain the phenotype were discussed. In conclusion, high-resolution array comparative hybridization can be applied successfully in newborns with multiple congenital anomalies as the method detects a significant number of pathogenic changes, resulting in early diagnoses. We hypothesize that small changes previously considered benign or even inherited rearrangements should be classified as potentially pathogenic at least until a subsequent clinical assessment would exclude a developmental delay or dysmorphism.

Genome instability model of metastatic neuroblastoma tumorigenesis by a dictionary learning algorithm.

BackgroundMetastatic neuroblastoma (NB) occurs in pediatric patients as stage 4S or stage 4 and it is characterized by heterogeneous clinical behavior associated with diverse genotypes. Tumors of stage 4 contain several structural copy number aberrations (CNAs) rarely found in stage 4S. To date, the NB tumorigenesis is not still elucidated, although it is evident that genomic instability plays a critical role in the genesis of the tumor. Here we propose a mathematical approach to decipher genomic data and we provide a new model of NB metastatic tumorigenesis.MethodWe elucidate NB tumorigenesis using Enhanced Fused Lasso Latent Feature Model (E-FLLat) modeling the array comparative chromosome hybridization (aCGH) data of 190 metastatic NBs (63 stage 4S and 127 stage 4). This model for aCGH segmentation, based on the minimization of functional dictionary learning (DL), combines several penalties tailored to the specificities of aCGH data. In DL, the original signal is approximated by a linear weighted combination of atoms: the elements of the learned dictionary.ResultsThe hierarchical structures for stage 4S shows at the first level of the oncogenetic tree several whole chromosome gains except to the unbalanced gains of 17q, 2p and 2q. Conversely, the high CNA complexity found in stage 4 tumors, requires two different trees. Both stage 4 oncogenetic trees are marked diverged, up to five sublevels and the 17q gain is the most common event at the first level (2/3 nodes). Moreover the 11q deletion, one of the major unfavorable marker of disease progression, occurs before 3p loss indicating that critical chromosome aberrations appear at early stages of tumorigenesis. Finally, we also observed a significant (p = 0.025) association between patient age and chromosome loss in stage 4 cases.ConclusionThese results led us to propose a genome instability progressive model in which NB cells initiate with a DNA synthesis uncoupled from cell division, that leads to stage 4S tumors, primarily characterized by numerical aberrations, or stage 4 tumors with high levels of genome instability resulting in complex chromosome rearrangements associated with high tumor aggressiveness and rapid disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0132-y) contains supplementary material, which is available to authorized users.

Chronic type adult T-cell leukemia-lymphoma after autolougous stem cell transplantation for ALK-negative anaplastic large cell lymphoma

An adult male patient was admitted to our hospital with systemic lymphadenopathy. He was diagnosed as having ALK-negative anaplastic large cell lymphoma (ALCL) by lymph node biopsy. Immunohistochemical staining revealed positive reaction for CD4, CD30, CD25, TIA-1, and Granzyme B, but negative for ALK and EBER. Although anti-HTLV-1 antibody in his serum was positive, monoclonal integration of HTLV-1 provirus DNA was not detected in his lymph node by Southern blot analysis. He was treated by combination chemotherapy, and complete remission was obtained by 8 courses of CHOP therapy. Subsequently he received autologous stem cell transplantation (auto-SCT). Fifteen months after auto-SCT, abnormal lymphocytes in the peripheral blood gradually increased. Southern blot analysis of abnormal lymphocytes revealed monoclonal integration of HTLV-1 provirus DNA and monoclonal rearrangement of T-cell receptor b. The patient was therefore diagnosed as chronic type of adult T-cell leukemia-lymphoma (ATL), and immediately progressed to acute type with central nervous system involvement. He died of tumor progression regardless of intensive chemotherapy. We analyzed genomic alterations of the ALCL cells and the chronic type ATL cells using high-resolution array comparative hybridization. These results revealed that the genomic alteration pattern of ALCL was different from those of the chronic type. Chronic type ATL cells had the genetic alterations such as 1q gain and CD58 loss that were frequently observed in acute type ATL than the chronic type, suggesting that these genetic events might contribute to the transformation of this case. TCR rearrangement analyses using PCR were performed for the tumor cells of ALCL and ATL to evaluate the origin of these tumor cells. Results showed that tumor cell clone of the ATL might have already proliferated in ALCL lymph node. This finding suggests that lymph nodes can serve as a niche for ATL development.

Hexasomy 13q31.3q34 due to two marker chromosomes with inverted duplication in a fetus with increased nuchal translucency

Small supernumerary marker chromosomes are structurally rearranged chromosomes that can be formed from different chromosomal fragments and cannot be identified using chromosomal banding analysis. Their examination has to be complemented by additional analyses like fluorescent in situ hybridization or array comparative genomic hybridization. We report on partial hexasomy of chromosome 13q in a fetus of a pregnant woman referred to genetic counseling because of increased fetal nuchal translucency and increased risk of trisomy 21 and trisomy 18 in first-trimester combined prenatal screening. Using chromosome banding analysis, in situ hybridization and array comparative hybridization we revealed the presence of two marker chromosomes with inverted duplication resulting in hexasomy of a 22.6 Mbp fragment in chromosomal region 13q31.3-13q34 with the lack of chromosome 13 centromere. The fetus presented dysmorphic facial features, head and body disproportion, wide neck, ambiguous genitalia, incorrect position of the anus, and symmetrical shortening of the long bones were present in our described case. Some of these features were in accordance with other published cases. Other most often described features in tetrasomy were: microphtalmia or other major eye defects, ear abnormalities and deafness, hemangiomata, hypotelorism, severe learning disability and seizures. Despite a low risk of recurrence for small supernumerary marker chromosomes the possibility of germ line mosaicism exists, thus genetic counseling was offered to the examined family. A full characterization of small supernumerary marker chromosomes in fetal karyotype is necessary for pregnancy prognosis and genetic counseling.

Complex genomic rearrangements in the dystrophin gene due to replication-based mechanisms.

Genomic rearrangements such as intragenic deletions and duplications are the most prevalent type of mutations in the dystrophin gene resulting in Duchenne and Becker muscular dystrophy (D/BMD). These copy number variations (CNVs) are nonrecurrent and can result from either nonhomologous end joining (NHEJ) or microhomology-mediated replication-dependent recombination (MMRDR). We characterized five DMD patients with complex genomic rearrangements using a combination of MLPA/mRNA transcript analysis/custom array comparative hybridization arrays (CGH) and breakpoint sequence analysis to investigate the mechanisms for these rearrangements. Two patients had complex rearrangements that involved microhomologies at breakpoints. One patient had a noncontiguous insertion of 89.7 kb chromosome 4 into intron 43 of DMD involving three breakpoints with 2–5 bp microhomology at the junctions. A second patient had an inversion of exon 44 flanked by intronic deletions with two breakpoint junctions each showing 2 bp microhomology. The third patient was a female with an inherited deletion of exon 47 in DMD on the maternal allele and a de novo noncontiguous duplication of exons 45–49 in DMD and MID1 on the paternal allele. The other two patients harbored complex noncontiguous duplications within the dystrophin gene. We propose a replication-based mechanisms for all five complex DMD rearrangements. This study identifies additional underlying mechanisms in DMD, and provides insight into the molecular bases of these genomic rearrangements.

Preimplantation genetic screening (PGS) with vitrification reduces miscarriage and improves cumulative live birth compared to unscreened frozen embryos

Goals of IVF include achieving a viable pregnancy and live birth of a healthy baby as quickly and efficiently as possible. This study analyzes cumulative live-birth outcomes with and without PGS and array comparative hybridization (aCGH) in patients where blastocysts were frozen and then thawed in subsequent frozen embryo transfers (FET) cycles.

Different mutations in PDE4D associated with developmental disorders with mirror phenotypes

BackgroundPoint mutations in PDE4D have been recently linked to acrodysostosis, an autosomal dominant disorder with skeletal dysplasia, severe brachydactyly, midfacial hypoplasia and intellectual disability. The purpose of the present study...

Iterative phenotyping of 15q11.2, 15q13.3 and 16p13.11 microdeletion carriers in pediatric epilepsies

Microdeletions at 15q11.2, 15q13.3 and 16p13.11 are known genetic risk factors for idiopathic generalized epilepsies and other neurodevelopmental disorders. The full phenotypic range of this microdeletion triad in pediatric epilepsies is unknown. We attempted to describe associated phenotypes in a cohort of pediatric epilepsy patients. We screened 570 patients with pediatric epilepsies including idiopathic generalized epilepsies, focal epilepsies and fever-associated epilepsy syndromes for microdeletions at 15q11.2, 15q13.3 and 16p13.11 using quantitative polymerase chain reaction. Identified microdeletions were confirmed using array comparative hybridization. Ten microdeletions in 15q11.2 (n=3), 15q13.3 (n=3) and 16p13.11 (n=4) were identified (1.8%). 9/10 microdeletions were identified in patients with IGE (6/101, 6.0%) or patients with generalized EEG patterns without seizures (3/122, 2.5%). 6/10 microdeletion carriers had various degrees of ID; the frequency of microdeletions in patients with epilepsy and ID was higher (4.6%) compared to patients with normal intellect (0.9%). Iterative phenotyping revealed a wide range of generalized epilepsy phenotypes. In our pediatric cohort, recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 are mainly associated with phenotypes related to idiopathic generalized epilepsies or related EEG patterns. In contrast to previous reports, these recurrent microdeletions are virtually absent in focal epilepsies, FS, FS+ and GEFS+. Microdeletion carriers have a five-fold risk to present with various degrees of ID compared to patients without these risk factors. This microdeletion triad might help delineate a novel spectrum of epilepsy phenotypes classifiable through clinical, electrographic and genetic data.

Molecular Inversion Probe Array for the Genetic Evaluation of Stillbirth Using Formalin-Fixed, Paraffin-Embedded Tissue

Array comparative hybridization has been used successfully to identify genomic alterations in stillbirth material; however, high DNA quantity and quality requirements may limit its utility in some fetal samples. Molecular inversion probe (MIP) array analysis of FFPE stillbirth autopsy samples circumvents the challenges associated with karyotype and short-term fetal cell culture, requires limited DNA input, and allows for retrospective evaluation of fetal loss. We performed MIP analysis on archival FFPE autopsy tissue to identify underlying genetic abnormalities not previously detected using traditional cytogenetic methods. Archival FFPE stillbirth cases (≥20 weeks gestation) were identified with the following characteristics: i) the phenotype suggested underlying genomic alterations; ii) the karyotype was either normal or not available and there were no other known genetic abnormalities; or iii) previous microarray testing was not performed. Genomic DNA (75 ng) was processed onto a 330,000-feature MIP array. Twenty-seven of 29 (93.1%) FFPE samples had passing MIP quality control scores. Abnormalities were seen in 3 of 27 (11%) archival samples (deletion of 17q12, trisomy 18, and a case of 4qter duplication and 13qter deletion arising from an unbalanced 4q;13q translocation), which, if identified at the time of autopsy, may have changed the course of medical management. This study highlights the benefits of using MIP array analysis for identification of genomic alterations in FFPE stillbirth autopsy tissue.

NRXN1 deletions identified by array comparative genome hybridisation in a clinical case series - further understanding of the relevance of NRXN1 to neurodevelopmental disorders.

BackgroundMicrodeletions in the NRXN1 gene have been associated with a range of neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, speech and language delay, epilepsy and hypotonia.ResultsIn the present study we performed array CGH analysis on 10,397 individuals referred for diagnostic cytogenetic analysis, using a custom oligonucleotide array, which included 215 NRXN1 probes (median spacing 4.9 kb). We found 34 NRXN1 deletions (0.33% of referrals) ranging from 9 to 942 kb in size, of which 18 were exonic (0.17%). Three deletions affected exons also in the beta isoform of NRXN1. No duplications were found. Patients had a range of phenotypes including developmental delay, learning difficulties, attention deficit hyperactivity disorder (ADHD), autism, speech delay, social communication difficulties, epilepsy, behaviour problems and microcephaly. Five patients who had deletions in NRXN1 had a second CNV implicated in neurodevelopmental disorder: a CNTNAP2 and CSMD3 deletion in patients with exonic NRXN1 deletions, and a Williams-Beuren syndrome deletion and two 22q11.2 duplications in patients with intronic NRXN1 deletions.ConclusionsExonic deletions in the NRXN1 gene, predominantly affecting the alpha isoform, were found in patients with a range of neurodevelopmental disorders referred for diagnostic cytogenetic analysis. The targeting of dense oligonucleotide probes to the NRXN1 locus on array comparative hybridisation platforms provides detailed characterisation of deletions in this gene, and is likely to add to understanding of the importance of NRXN1 in neural development.

From the front line: the realities and challenges of microarray studies in cancer

Metaphase cytogenetics and fluorescence in situ hybridisation (FISH) play an integral role in diagnosis and clinical decision making algorithms for many malignancies. However, inherent methodological limitations mean it is not always possible to adequately characterise the malignant clone using these techniques. DNA microarrays [array comparative hybridisation (aCGH)/sin-gle nucleotide polymorphism arrays (SNP-A)] overcome some limitations of metaphase cytogenetics and FISH. Microarrays are not dependent on the ability to obtain metaphases and detect copy number changes across the genome at high resolution. SNP-A also detect copy neutral loss of heterozygosity (CN-LOH) which may flag the presence of homozygous oncogenic mutations in the neoplastic clone. We have performed microarray testing on samples from patients with acute lymphoblastic leukaemia (ALL), myelodysplastic syndromes (MDS) and chronic lymphocytic leukaemia (CLL) because these disorders are characterised by recurrent copy number changes and CN-LOH with demonstrated clinical significance. In our experience, microarrays provide additional information about the neoplastic clone that complements the standard testing approach. Karyotypic complexity and the presence of contaminating non-malignant cells can pose problems with QC, interpretation, nomenclature and reporting. The position of microarrays in the hierarchy of cytogenetic testing in cancer is likely to be disease specific and is yet to be clearly defined.

Abstract 4919: Pathway profiling of head and neck cancer cell lines

Abstract Introduction: Cell lines retain key genetic aberrations of the originating tumors, which frequently correlate with sensitivity to specific inhibitors. We performed genetic and pathway based profiling of 40 head and neck cancer (HNC) cell lines using three publicly available methods for pathway profiling based on expression and copy number data. Methods: 40 HNC cell lines were analyzed for expression using expression arrays (Agilent), and copy number using array comparative hybridization (aCGH) and/or Nanostring methodologies. Integrative genomic analysis was performed to determine pathway activity using pathway analysis techniques previously described by Gatza et al. (BFRM), Pe'er et al (CONEXIC), Vaske et al. (PARADIGM). Results: Frequent pathway activation was identified in &amp;gt;10 well established signalling pathways for HNC including TP53, PI3K-AKT, TP63/SOX2, Cyclin D1, MYC etc, as well as several - for HNC previously unknown - signalling pathways. Cell lines could be associated with expression-based biologic subtypes in HNC tissues suggesting that specific cell lines should be used preferentially for modelling and therapeutic evaluation for HNC subtypes (including HPV(+) cell lines with characteristics of HPV(+) tumors). The three pathway analysis platforms identified a large number of pathways with some overlap in particular between BFRM and CONEXIC, whereas PARADIGM profiled a much wider set of pathways. Both CONEXIC and PARADIGM employed copy number data, whereas BFRM relied solely on expression data. Conclusion: Pathway alterations characteristic of head and neck cancers were identified in virtually all HNC cell lines. Pathway based classification of cell lines was achieved and correlated with pathway activity in HNC tumor tissues. Three pathway-profiling approaches provided a large set of pathway activity with some significant overlap that may have translational relevance. Validation studies are currently on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4919. doi:1538-7445.AM2012-4919

Further clinical and molecular delineation of the 15q24 microdeletion syndrome

BackgroundChromosome 15q24 microdeletion syndrome is a rare genomic disorder characterised by intellectual disability, growth retardation, unusual facial morphology and other anomalies. To date, 20 patients have been reported; 18 have...

A novel t(8;13)(q21;q22) translocation in a pediatric lipoma

Pediatric lipomas are rare and their etiology is unknown. As in adult lipomas, a segment of 12q13-q15 has been documented as a common chromosomal rearrangement region. Here, we report a pediatric lipoma case without rearrangement of 12q13-q15 but with an apparently balanced translocation involving chromosomes 8 and 13 [t(8;13)(q21;q22)] detected by routine cytogenetic analysis, and small deletions on 5q21.1 and 8q21.11 detected by array comparative genetic hybridization. The small deletion on 8q21.11 is possibly due to chromosomal instability at the breakpoint of the translocation.