CytoScan HD Array Research Articles

Genome-wide DNA copy number profiling and bioinformatics analysis of ovarian cancer reveals key genes and pathways associated with distinct invasive/migratory capabilities.

Ovarian cancer (OC) metastasis presents major hurdles that must be overcome to improve patient outcomes. Recent studies have demonstrated copy number variations (CNVs) frequently contribute to alterations in oncogenic drivers. The present study used a CytoScan HD Array to analyse CNVs and loss of heterozygosity (LOH) in the entire genomes of 6 OC patients and human OC cell lines to determine the genetic target events leading to the distinct invasive/migratory capacities of OC. The results showed that LOH at Xq11.1 and Xp21.1 and gains at 8q21.13 were novel, specific CNVs. Ovarian cancer-related CNVs were then screened by bioinformatics analysis. In addition, transcription factors-target gene interactions were predicted with information from PASTAA analysis. As a result, six genes (i.e., GAB2, AKT1, EGFR, COL6A3, UGT1A1 and UGT1A8) were identified as strong candidates by integrating the above data with gene expression and clinical outcome data. In the transcriptional regulatory network, 4 known cancer-related transcription factors (TFs) interacted with 6 CNV-driven genes. The protein/DNA arrays revealed 3 of these 4 TFs as potential candidate gene-related transcription factors in OC. We then demonstrated that these six genes can serve as potential biomarkers for OC. Further studies are required to elucidate the pathogenesis of OC.

Single Nucleotide Polymorphism Array-Based Signature of Genetic Ploidy Groups in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is characterised by a number of recurrent chromosomal abnormalities which inform prognosis. Low hypodiploidy (HoTr) and high hyperdiploidy (HeH) are genetic subgroups associated with large non-random ploidy shifts, specifically 30-39 chromosomes and 51-65 chromosomes respectively. HoTr ALL often presents with a near triploid karyotype of 60-78 chromosomes through chromosomal endoreduplication without cytokinesis. This presents a diagnostic challenge in distinguishing this poor risk entity from good risk HeH ALL. To date, classification of such challenging cases has been based on the modal chromosome number, the pattern of specific gains, and identification of loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) arrays where possible. However, loss of cellular context and mixed cell populations (normal diploid, low hypodiploid and near-triploid) when analysing SNP arrays can pose additional analytical difficulty. The aim of this SNP array study was to (1) determine the level of inaccurate genetic subgrouping when cytogenetics only was used to distinguish HeH from doubled up HoTr and (2) develop a diagnostic algorithm to reliably call HeH and HoTr without supporting cytogenetic analysis. SNP arrays were performed on diagnostic ALL samples of 85 patients (46 HoTr, 39 HeH) using Illumina CytoSNP 850k (n=80) or Affymetrix Cytoscan HD (n=5) arrays. Probe level data were uploaded to Nexus Copy Number 10 (BioDiscovery) and manual segments spanning the length of each chromosome were created. No further data pre-processing was carried out and log2 ratio of 0 was automatically assigned to the median log2 ratio of the sample. Chromosomal log2 ratios were normalized within each sample and a variety of machine-learning techniques used to cluster the samples independently of assigned diagnosis. Cases residing in the incorrect cluster based on initial diagnosis were examined in detail using information from cytogenetics and SNP array interpretation. SNP arrays were analysed from 46 HoTr (median age 50.5 years (range 7-87), 43% male) and 39 HeH (median age 7 years (range 1-58), 56% male) patients. Unsupervised clustering of log2 ratios showed a clear distinction between HeH and HoTr patients (figure (A)). Six cases clustered incorrectly based on cytogenetic diagnosis. After detailed interpretation of all cases, including identifying LOH affecting chromosomes 3, 7, 15, 16 and 17, 3/6 cases initially classified as HeH were highly suggestive of HoTr ALL despite having <60 chromosomes. Similarly, 1/6 cases was cytogenetically diagnosed with HoTr but had a SNP array pattern typical of HeH ALL. We identified chromosomes 1, 4, 11, 17, 19, and 21 as those contributing most to the distinction in the HoTr and HeH signatures with the log2 ratio of chromosome 1 the most highly discriminatory in this cohort. Using whole chromosome log2 ratios, HoTr and HeH ALL have distinct profiles. SNP array analysis highlighted at least 4 patients whose ploidy subgroups appeared incorrectly called by cytogenetics, which can affect risk stratification. Crucially, these data call into question the accepted modal chromosome numbers for HeH and HoTr in the near triploid phase and suggest this alone cannot be used to classify patients into these ploidy groups. All samples incorrectly classified as HeH ALL were from adults aged >40 years, suggesting this good risk subgroup is even rarer than previously thought in older adults. After re-classification, our cohort only contained 5/41 adults >40 years with HeH ALL, signifying that HoTr ALL is the commonest genetic ploidy group in older adults with ALL and must still be considered in cases with 50-60 chromosomes. Copy number analysis from SNP arrays is challenging in samples with marked ploidy shift and mixed cell populations. Importantly, a number of our samples had significant contaminating normal DNA and LOH could not be confirmed visually (figure (B)), underlying the need for additional factors to aid classification. Our analysis only takes into account log2 ratio of entire chromosomes, thus permitting a measure of the relative over and under-representation of specific chromosomes within the sample. This method accurately clusters patients even when LOH cannot be clearly visualized from B-allele frequency due to contaminating non-leukemic DNA and supports the development of a diagnostic classifier based on chromosomal log2 ratios. Disclosures Fielding: Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy.

Genetic and Genomic Characterisation of Older Adults with Acute Lymphoblastic Leukemia Treated on the UKALL14 and UKALL60+ Clinical Trials

Genetic and Genomic Characterisation of Older Adults with Acute Lymphoblastic Leukemia Treated on the UKALL14 and UKALL60+ Clinical Trials

CNAs concordance between CRC-PDOs and the original tissue reveals relevant oncogenic pathways alterations

Abstract Background Colorectal cancer (CRC) patients derived organoids (PDOs) are 3D in vitro primary culture representing an increasingly used model for precision medicine. Besides somatic mutations, CRC is characterized by certain levels of copy number alterations (CNAs): somatic gain or loss of DNA sections, which can include oncogenes or tumor suppressors. We aimed to analyze CNAs concordance between CRC-PDOs and the original tumor tissue. Methods Primary or metastatic CRC tissues have been obtained from patients who underwent surgery. Tissue has been washed and incubated with antibiotics. After mechanical and enzymatic digestion, free cells have been seeded in Matrigel with proper medium. Development of organoids has been observed the day after seeding. Organoids have been passaged, expanded and stored in liquid nitrogen to constitute a biobank. After reaching appropriate volume, DNA from PDOs and matched fresh tumor tissue has been extracted. Next generation sequencing (NGS) has been performed with an in-house customized cancer specific 85-gene panel. Cytoscan-HD (Thermo-Scientific) array has been performed according to manufacturer protocol. Data analysis has been performed with Chromosome Analysis Suite (Applied Biosystems v4.0). Results NGS analysis showed high level concordance between PDOs and matched tumor tissue. Cytoscan-HD analysis was feasible and effective with PDOs. Results revealed good concordance between PDOs and original patient tissue. PDOs maintain mosaicism indicating certain level of heterogeneity. Among the CNAs detected we were able to detect alterations of interest, such as loss of regions including SMAD2, SMAD4, MTOR, CASP9, EPHB2, RUNX3, RSPO1, TIE1, JAK1, TGFBR3, VCAM1, PIK3C3 and amplification of regions containing NOTCH2L, CDH1L, MUC1, NTRK1, MAPKAPK2, TGFB2, TP53BP2, PARP1, WNT3A, ARID4B, AKT3, CDH4, AURKA. Conclusions In addition to mutation profile, CRC-PDOs CNAs pattern reflects that of the original patient tissue, allowing to capture genomic alterations other than sequencing. Further validation is needed. Legal entity responsible for the study INCLIVA Biomedical Research Institute. Funding Instituto de Salud Carlos III, Rio Hortega contract, Joan Rodes Contract, ESMO Translational Research Fellowship Programme. Disclosure A. Cervantes: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Beigene; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Servier; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy, Research grant / Funding (institution): Astellas; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Fibrogen; Research grant / Funding (institution): Amcure; Research grant / Funding (institution): Sierra Oncology; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): BMS; Research grant / Funding (institution): MSD; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Foundation Medicine. All other authors have declared no conflicts of interest.

Genetic testing in fetuses with isolated agenesis of the corpus callosum

Purpose The objectives of this study were to explore genetics pathogenesis of isolated agenesis of corpus callosum (ACC) and assess the utility of chromosomal microarray analysis (CMA) for genetic diagnosis of isolated ACC. Methods We analyzed the genomes of 16 fetuses with isolated ACC using Afymetrix CytoScan HD arrays and conducted further bioinformatic analysis for one proband fetus with an abnormal copy number variation (CNV). Results Of the 16 fetal samples examined, two (12.5%) had pathogenic CNVs and three (18.75%) had variants of unknown significance. Two cases, case 2 and case 9, were found to have pathogenic CNVs. Bioinformatic analyses indicated that the CNV of one fetus (case 9) contained 115 annotated coding genes, five of which (SLC6A5, BDNF, ELP4, PAX6, and SLC1A2) have been associated with neurodevelopment. Three of these genes (SLC1A2, BDNF, and PAX6) may play a key role in ACC development. GO cluster analysis of the implicated genes revealed strong representations of protein binding and metal ion binding functions. KEGG pathway analysis pointed to four pathways: longevity regulating pathway, amyotrophic lateral sclerosis, cocaine addiction, and autophagy-animal. Conclusions BDNF, SLC1A2, and PAX6 may be involved in the development of isolated ACC. CMA is a feasible technology for prenatal diagnosis of isolated ACC.

Screening of urine identifies PLA2G16 as a field defect methylation biomarker for prostate cancer detection.

BackgroundProstate cancer (PC) is a multifocal disease. DNA methylation alterations are not restricted to the immediate peritumor environment, but spatially widespread in the adjacent and distant histologically normal prostate tissues. In the current study, we utilized high-throughput methylation arrays to identify epigenetic changes in the urine from men with and without cancer.Design, setting, and participantsDNA urine samples were enriched for methylated fragments using MBD methyl-binding antibodies and applied to high density CytoScanHD arrays. Significant loci were validated using quantitative pyrosequencing and binary logistic regression modeling applied to urine sample analyses in a training (n = 83) and validation approach (n = 84). Methylation alterations in prostate tissues using pyrosequencing at the PLA2G16 locus were examined in 38 histologically normal specimens from men with (TA, n = 26) and without (NTA, n = 12) cancer and correlated to gene expression.ResultsMethylation microarrays identified 3,986 loci showing significantly altered methylation in the urine samples from patients with PC compared to those without (TA vs NTA; p<0.01). These loci were then compared against subjects with their prostates removed to exclude non-prostate cell markers yielding 196 significant regions. Multiple CpGs adjacent to PLA2G16 CpG island showed increased methylation in TA compared to NTA (p<0.01) in a large validation study of urine samples. The predictive accuracy of PLA2G16 methylation at CG2 showed the highest predictive value at 0.8 (odds ratio, 1.37; 95% confidence interval, 1.16–1.62; p<0.001). Using a probability cutoff of 0.065, the sensitivity and specificity of the multivariate model was 92% and 35%. When histologically normal prostate tissues/biopsies from patients with PC (TA) were compared to subjects without cancer, significant hypermethylation of PLA2G16 was noted (odds ratio, 1.35; 95% confidence interval, 1.07–1.71; p = 0.01).ConclusionPLA2G16 methylation defines an extensive field defect in histologically normal prostate tissue associated with PC. PLA2G16 methylation in urine and prostate tissues can detect the presence of PC.

PS997 A NEW BIOMARKER OF RESPONSE TO 5‐AZACITIDINE THERAPY IN MDS AND AML PATIENTS: SIRPB1

Background:Myelodisplastic syndromes (MDS) and Acute Myeloid Leukemia (AML) are a group of diseases of the elderly that initiates in a hematopoietic stem cell and are characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background. In both diseases, 5‐Azacitidine (5‐Aza) has been successful, inducing prolonged survival and delayed AML evolution.Aims:To identify the genes mostly predictive of treatment response, we use high‐throughput genomic analysis (SNP arrays and/or NGS‐RNA‐seq and/or NGS‐WES and/or GEP) in azacitidine‐sensitive and resistant MDS/AML patients.Methods:NGS‐WES or RNA seq HiSeq 2000 (Illumina) was positively done in 35/214 AML samples (16%), GEP (GeneChip Human Transcriptome Array 2.0, Affymetrix Inc.) was performed in 65/214 AML samples (30%). SNPs arrays (CytoScan HD Array, Affymetrix Inc.) in 125/214 AML samples (58%) and 18/32 MDS samples (56%) at diagnosis, then analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.), Nexus Copy Number™ v7.5 (BioDiscovery) and GeneGo MetaCore™ software.Results:We treated 246 adult pts with MDS or AML: 214 were AML and 32 were MDS with a median age of 59 and 70 years, respectively. 45 were treated with 5‐Aza (32 MDS/13 AML), while 201 AML with conventional chemotherapy. 45 MDS/AML were treated with at least one complete cycle of 5‐Aza (75 mg/sqm/daily). SNP arrays was done in 22/45 (49%), 13 pts were defined “insensitive/resistant”, ie. never achieving clinical complete remission (CCR) and 9 were defined “sensitive”, ie. all of them obtaining CCR. Copy Number Alterations (CNAs) ranged from loss or gain of complete chr arms to focal deletions and gains targeting one or few genes involving macroscopic (&gt;1.5 Mbps), submicroscopic genomic intervals (50 Kbps‐1.5 Mbps) and LOH (&gt;5 Mbps) events. Macroscopic CNAs affecting a complete chromosome or its arms were detected in 5 of 22 pts (23%), while classical cytogenetic was able to detect only two cases of trisomy 8 (9%), suggesting superiority of SNPs array for CNAs identifications. Microscopic CNAs abnormalities were detected in all of the patients affecting all the chromosomes. Chromosomic aberrations disease‐related are more statistically frequent on pts “insensitive” versus pts. “sensitive” (64% vs. 35%) (p≤0.01). Moreover we found that from the median of chromosomic alterations lenghts (in kbp) the group of “insensitive” MDS/AML patients to 5‐Aza present more gains and losses than “sensitive” ones. By Nexus Copy Number software, we identify 137 genes highly differentially gain (SIRPB1 and KIT with p ≤ 0.05) or loss (SIRPB1, LCE1C, BCAS1, EXD3 with p ≤ 0.05) or LOH between “insensitive” versus “sensitive” to 5‐Aza (p ≤ 0.05). Among these, we focused on SIRPB1 (cytoband 20p13, 56Kbps), since it was loss on 14/22 (64%) “insensitive” pts (p = 0,023) and gain on 7/22 (70%) “sensitive” ones with a significantly (p = 0,0324), respectively. SIRPB1 common deletion region goes from 1571 to 1598 (27 kbps) and common amplified region goes from 1561 to 1591 (30 kbps). By NGS‐WES we analyzed 35/214 (16%) AML samples at diagnosis, finding mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR and again in SIRPB1 an heterozigosity frameshift deletion (c. 388delC; p. H130fs) in exone 2 in a AML pts with normal karyotype. By GEP we further analyzed 48/214 (22%) AML samples for SIRPB1 in order to correlate and confirm the expression or loss of expression of these genes, in correlation with 5‐Aza response.Summary/Conclusion:We conclude that SIRPB1 is a promising marker of response to 5‐Aza treatment in MDS and AML.

PS1137 COMPLEX STRUCTURAL VARIANTS LEAD TO GENE INACTIVATION RATHER THAN EXPRESSION OF DE NOVO FUSION GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Complex structural variants (cSVs) represented by chromothripsis have recently been identified in various tumor types, including chronic lymphocytic leukemia (CLL). They lead to extensive chromosomal rearrangements due to chromosome shattering and consequent random re‐joining of scattered fragments. Substantial parts of DNA are typically lost, and other chromosomal parts potentially containing oncogenes are amplified. Importantly, albeit not common in CLL, de novo fusion gene variants with novel functions can be formed. Overall, chromothripsis is associated with unfavorable prognosis, which can be in part attributed to the frequent co‐occurrence with TP53 gene defects.Aims:To perform a detailed study of cSVs in CLL and to identify putative de novo fusion genes and their possible functions.Methods:cSVs were screened among TP53‐aberrant CLL cases using CytoScanHD arrays (ThermoFisher Scientific). In parallel, conventional cytogenetics, mFISH, iFISH and chromosome painting methods were used to supplement array findings. Transcriptome analysis was performed using TruSeq Stranded Total RNA Library Prep Kit on HiSeq 2000–1T (Illumina). Obtained data were analyzed with in‐house bioinformatic pipelines developed for specific detection of fusion gene variants in transcriptomic data.Results:cSVs suggestive of chromothripsis were screened among 93 CLL patients (111 samples in total) with various types of wild‐type TP53 gene inactivation using CytoScan assay. Out of them, cSVs were identified in 20 patients (21.5%; 26 samples) and were associated with defect of both TP53 alleles (p = 0.0006). In 13/20 patients we predominantly observed clustered deletions; in the remaining 7/20 patients, deletions and also amplifications were present in localized regions. The number of breakpoints ranged from 30 to 154 per case (median 54); 10 to 65 coding genes (median 24) were present in the breakpoints. cSVs were clustered in one chromosome in 9/20 cases, whereas in the rest they were detected in ≥ 2 chromosomes. Comparing the array results to cytogenetic findings, typically a higher number of chromosomes (up to 8) was involved in cSVs within the genome. It can be explained by balanced abnormalities undetectable by the array. Several chromosomes were affected recurrently with the most frequent being chr6 and chr13 in 8 cases, and chr8 and chr9 in 6 cases, respectively. Ten cases with cSVs were subjected to the transcriptome analysis. Although a high number of genomic breakpoints was detected in these cases (555 breakpoints in total affecting 236 genes), they mostly did not lead to formation of expressed fusion genes. Altogether 15 fusion gene transcripts were noted in 8/10 cases (range 1 to 4 per case); 10 fusions were validated by PCR. They mostly consisted of two fusion gene partners, however, in 2 instances, fusions of 3 and 5 genes were detected, respectively. Using online tools, we tested whether the 10 validated fusion transcripts could be translated in proteins and found out that 4 of them encoded abnormal proteins containing domains with predicted functions.Summary/Conclusion:Using our comprehensive approach we showed that chromothripsis affects over a fifth of CLL cases with aberrant TP53 and mostly leads to inactivation of genes present in genomic breakpoints. De novo fusion transcripts supposedly encoding abnormal proteins are expressed only to a limited extent.Supported by CEITEC2020 LQ1601, MZCR‐RVO 65269705, MUNI/A/1105/2018, MZCR‐AZV 16–34272A, GACR 19–11299S, H2020 116026 and 825872.

PS1138 TUMOR MUTATIONAL BURDEN ASSESSED BY TARGETED HIGH THROUGHPUT SEQUENCING PREDICTS TREATMENT FREE SURVIVAL IN CLL: RESULTS FROM A BICENTER STUDY

Background: Complex structural variants (cSVs) represented by chromothripsis have recently been identified in various tumor types, including chronic lymphocytic leukemia (CLL). They lead to extensive chromosomal rearrangements due to chromosome shattering and consequent random re-joining of scattered fragments. Substantial parts of DNA are typically lost, and other chromosomal parts potentially containing oncogenes are amplified. Importantly, albeit not common in CLL, de novo fusion gene variants with novel functions can be formed. Overall, chromothripsis is associated with unfavorable prognosis, which can be in part attributed to the frequent co-occurrence with TP53 gene defects. Aims: To perform a detailed study of cSVs in CLL and to identify putative de novo fusion genes and their possible functions. Methods: cSVs were screened among TP53-aberrant CLL cases using CytoScanHD arrays (ThermoFisher Scientific). In parallel, conventional cytogenetics, mFISH, iFISH and chromosome painting methods were used to supplement array findings. Transcriptome analysis was performed using TruSeq Stranded Total RNA Library Prep Kit on HiSeq 2000–1T (Illumina). Obtained data were analyzed with in-house bioinformatic pipelines developed for specific detection of fusion gene variants in transcriptomic data. Results: cSVs suggestive of chromothripsis were screened among 93 CLL patients (111 samples in total) with various types of wild-type TP53 gene inactivation using CytoScan assay. Out of them, cSVs were identified in 20 patients (21.5%; 26 samples) and were associated with defect of both TP53 alleles (p = 0.0006). In 13/20 patients we predominantly observed clustered deletions; in the remaining 7/20 patients, deletions and also amplifications were present in localized regions. The number of breakpoints ranged from 30 to 154 per case (median 54); 10 to 65 coding genes (median 24) were present in the breakpoints. cSVs were clustered in one chromosome in 9/20 cases, whereas in the rest they were detected in ≥ 2 chromosomes. Comparing the array results to cytogenetic findings, typically a higher number of chromosomes (up to 8) was involved in cSVs within the genome. It can be explained by balanced abnormalities undetectable by the array. Several chromosomes were affected recurrently with the most frequent being chr6 and chr13 in 8 cases, and chr8 and chr9 in 6 cases, respectively. Ten cases with cSVs were subjected to the transcriptome analysis. Although a high number of genomic breakpoints was detected in these cases (555 breakpoints in total affecting 236 genes), they mostly did not lead to formation of expressed fusion genes. Altogether 15 fusion gene transcripts were noted in 8/10 cases (range 1 to 4 per case); 10 fusions were validated by PCR. They mostly consisted of two fusion gene partners, however, in 2 instances, fusions of 3 and 5 genes were detected, respectively. Using online tools, we tested whether the 10 validated fusion transcripts could be translated in proteins and found out that 4 of them encoded abnormal proteins containing domains with predicted functions. Summary/Conclusion: Using our comprehensive approach we showed that chromothripsis affects over a fifth of CLL cases with aberrant TP53 and mostly leads to inactivation of genes present in genomic breakpoints. De novo fusion transcripts supposedly encoding abnormal proteins are expressed only to a limited extent. Supported by CEITEC2020 LQ1601, MZCR-RVO 65269705, MUNI/A/1105/2018, MZCR-AZV 16–34272A, GACR 19–11299S, H2020 116026 and 825872.

Genetic anomalies in fetuses with tetralogy of Fallot by using high-definition chromosomal microarray analysis

BackgroundThe etiology of TOF is complex and the genesis of TOF has been associated with environmental factors and genetic disorders, including chromosomal anomalies, aneuploidies, 22q11.2 deletion and single-gene disease. Previous literatures have shown that a chromosome alteration in about 30% patients with TOF and recently published articles reported that 22q11.2 deletion syndrome accounts for 16% cases with TOF diagnosed postnatally. CMA now is considered as gold standard for detecting genetic anomalies in fetuses with congenital malformations. CMA could detect a 6.6–25% incremental yield of CNVs in CHDs. The aim of this study was to assess the genetic anomalies in fetal tetralogy of Fallot (TOF) by using high-definition CMA.MethodsThis retrospective study reviewed all the fetuses diagnosed with TOF between 2013 and 2018. Prenatal ultrasongraphic findings, including cardiac angle, and the findings of CMA using Affymetrix CytoScan HD array were collected.ResultsNinety-six fetuses with TOF and known genetic results were enrolled. Right aortic arch was the most common associated anomalies (22.9%). One fetus with trisomy 18, one with 46, XX, t (7;10)(q36;q22), one with 47, XYY and five with trisomy 21 were identified. Clinically significant CNVs occurred in 6.8% and uncertain significant CNVs in 3.4% fetal TOF with normal karyotype. A total of four cases with 22q11.2 microdeletion and two fetuses with Yq11.223q11.23 microduplication have been identified. Genetic anomalies, including chromosomal aberrations and pathogenic CNVs, were significantly higher in the TOF with extracardiac anomaly group than in the TOF without extracardiac anomaly group (P = 0.005). Abnormal cardiac angle was noticed in 24.0% fetal TOF. Genetic anomalies were more common in the TOF with abnormal cardiac angle than with normal cardiac angle (P = 0.001). On the other hand, abnormal cardiac angle was noticed in 64.3% fetal TOF with genetic anomalies while abnormal cardiac angle occurred in 17.1% fetal TOF with normal genetic results (P = 0.001).ConclusionsGenetic testing should be offered, specially using microarray analysis, for the fetal TOF with abnormal cardiac angle or extracardiac defects.

SA25 - AN INHERITED DISTAL 16P11.2 DELETION DEMONSTRATES VARIABLE EXPRESSIVITY AND INCOMPLETE PENETRANCE FOR PSYCHIATRIC ILLNESS AS WELL AS ASSOCIATION WITH RHIZOMELIC SHORTENING: A CASE REPORT

Background The most commonly reported 16p Copy Number Variant (CNV) - 16p11.2 deletion syndrome (16pDS) (~600 kb; 29.5 -30.1 Mb) – has been robustly associated with schizophrenia (SZ), as well as Intellectual Disabilities (ID)/Developmental Delays (DD), congenital anomalies, dysmorphic features and obesity. Non-overlapping “distal” 16p11.2 deletions (~200 kb; 28.7 -28.9 Mb) are less commonly reported, but have a variable phenotype similar to that of 16pDS, including emerging data suggesting a psychiatric phenotype. We report here on a parent/child dyad who share a distal 16p11.2 deletion. Methods Patient 1 (index) was assessed (for indications including seizures, DD, and short limbs) at ages 2, 17, and 36, when she had clinical Chromosomal Microarray (CMA) (Affymetrix Cytoscan HD array, clinical thresholds: 200 kb (deletions), 400 kb (duplications), Chromosome Analysis suite (v2.0.0 195)). Fluorescence in Situ Hybridization (FISH) was used to confirm the deletion and determine origin. Results Patient 1 was born at term to non-consanguineous parents. A 46XX karyotype was confirmed at age 2 and 17 (testing indications: myoclonic seizures, DD, rhizomelic shortening of upper limbs). At age 17, she was also noted to have small hands ( Discussion While distal 16p11.2 deletions have been shown to increase risk for schizophrenia, this report contributes to the emerging evidence that this CNV is also associated with broader psychiatric phenotypes, such as bipolar disorder. The father's milder cognitive and metabolic phenotype supports variable expressivity of distal 16p11.2 deletions, even when inherited; his negative psychiatric history reinforces the incomplete penetrance of this CNV for psychiatric disorders. This report also contributes to the generation of a more thorough clinical description of the phenotypic range of distal 16p11.2 deletion, which we suggest, includes rhizomelic limb shortening.

Copy number variation in the susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a strong genetic component and etiology characterized by chronic inflammation and autoantibody production. The purpose of this study was to ascertain copy number variation (CNV) in SLE using a case-control design in an admixed Brazilian population. The whole-genome detection of CNV was performed using Cytoscan HD array in SLE patients and healthy controls. The best CNV candidates were then evaluated by quantitative real-time PCR in a larger cohort or validated using droplet digital PCR. Logistic regression models adjusted for sex and ancestry covariates was applied to evaluate the association between CNV with SLE susceptibility. The data showed a synergistic effect between the FCGR3B and ADAM3A loci with the presence of deletions in both loci significantly increasing the risk to SLE (5.9-fold) compared to the deletion in the single FCGR3B locus (3.6-fold). In addition, duplications in these genes were indeed more frequent in healthy subjects, suggesting that high FCGR3B/ADAM3A gene copy numbers are protective factors against to disease development. Overall, 21 rare CNVs were identified in SLE patients using a four-step pipeline created for identification of rare variants. Furthermore, heterozygous deletions overlapping the CFHR4, CFHR5 and HLA-DPB2 genes were described for the first time in SLE patients. Here we present the first genome-wide CNV study of SLE patients in a tri-hybrid population. The results show that novel susceptibility loci to SLE can be found once the distribution of structural variants is analyzed throughout the whole genome.

Partial Deletion of the Long Arm of Chromosome 7: A Case Report.

Study advances with a childhood case of partial deletion of the long arm of chromosome 7. The patient is a 36-month-old girl with growth retardation, mild mental retardation and delayed bone age. She showed no signs of hypotelorism, upslanting palpebral fissures, epicanthal folds, low-set ears, or flat and broad nasal bridge. Microarray testing using the Affymetrix CytoScan HD array revealed an approximately 58 kb deletion at 7q31.1 in the girl and her father, suggesting paternal origin. As the patient had no characteristic facial features, 7q deletions had not been considered. This case broadens the range of case presentations for microdeletions of chromosome 7.

Prognostic implications of additional genomic lesions in adult Philadelphia chromosome-positive acute lymphoblastic leukemia.

To shed light onto the molecular basis of Philadelphia chromosome-positive acute lymphoblastic leukemia and to investigate the prognostic role of additional genomic lesions, we analyzed copy number aberrations using the Cytoscan HD Array in 116 newly diagnosed adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia enrolled in four different GIMEMA protocols, all based on a chemotherapy-free induction strategy. This analysis showed that patients with Philadelphia chromosome-positive acute lymphoblastic leukemia carry an average of 7.8 lesions/case, with deletions outnumbering gains (88% versus 12%). The most common deletions were those targeting IKZF1, PAX5 and CDKN2A/B, which were detected in 84%, 36% and 32% of cases, respectively. Patients carrying simultaneous deletions of IKZF1 plus CDKN2A/B and/or PAX5 had a significantly lower disease-free survival rate (24.9% versus 43.3%; P=0.026). The only IKZF1 isoform affecting prognosis was the dominant negative one (P=0.003). Analysis of copy number aberrations showed that 18% of patients harbored MEF2C deletions, which were of two types, differing in size: the longer deletions were associated with the achievement of a complete molecular remission (P=0.05) and had a favorable impact on disease-free survival (64.3% versus 32.1% at 36 months; P=0.031). These findings retained statistical significance also in multivariate analysis (P=0.057). KRAS deletions, detected in 6% of cases, were associated with the achievement of a complete molecular remission (P=0.009). These results indicate that in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia a detailed evaluation of additional deletions - including CDKN2A/B, PAX5, IKZF1, MEF2C and KRAS - has prognostic implications and should be incorporated in the design of more personalized treatment strategies.

Large-scale deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia

Copy-number variations (CNVs) have been studied in the context of familial hypercholesterolemia but have not yet been evaluated in patients with extreme levels of HDL cholesterol. We evaluated targeted, next-generation sequencing data from patients with very low levels of HDL cholesterol (i.e., hypoalphalipoproteinemia) with the VarSeq-CNV® caller algorithm to screen for CNVs that disrupted the ABCA1, LCAT, or APOA1 genes. In four individuals, we found three unique deletions in ABCA1: a heterozygous deletion of exon 4, a heterozygous deletion that spanned exons 8 to 31, and a heterozygous deletion of the entire ABCA1 gene. Breakpoints were identified with Sanger sequencing, and the full-gene deletion was confirmed by using exome sequencing and the Affymetrix CytoScan HD array. Previously, large-scale deletions in candidate HDL genes had not been associated with hypoalphalipoproteinemia; our findings indicate that CNVs in ABCA1 may be a previously unappreciated genetic determinant of low levels of HDL cholesterol. By coupling bioinformatic analyses with next-generation sequencing data, we can successfully assess the spectrum of genetic determinants of many dyslipidemias, including hypoalphalipoproteinemia.

A novel dNTP-limited PCR and HRM assay to detect Williams-Beuren syndrome

BackgroundWilliams-Beuren syndrome (WBS) is caused by a microdeletion of chromosome arm 7q11.23. A rapid and inexpensive genotyping method to detect microdeletion on 7q11.23 needs to be developed for the diagnosis of WBS. This study describes the development of a new type of molecular diagnosis method to detect microdeletion on 7q11.23 based upon high-resolution melting (HRM). MethodsFour genes on 7q11.23 were selected as the target genes for the deletion genotyping. dNTP-limited duplex PCR was used to amplify the reference gene, CFTR, and one of the four genes respectively on 7q11.23. An HRM assay was performed on the PCR products, and the height ratio of the negative derivative peaks between the target gene and reference gene was employed to analyze the copy number variation of the target region. ResultsA new genotyping method for detecting 7q11.23 deletion was developed based upon dNTP-limited PCR and HRM, which cost only 96 min. Samples from 15 WBS patients and 12 healthy individuals were genotyped by this method in a blinded fashion, and the sensitivity and specificity was 100% (95% CI, 0.80–1, and 95% CI, 0.75–1, respectively) which was proved by CytoScan HD array. SignificanceThe HRM assay we developed is an rapid, inexpensive, and highly accurate method for genotyping 7q11.23 deletion. It is potentially useful in the clinical diagnosis of WBS.

GENIPAC: A Genomic Information Portal for Head and Neck Cancer Cell Systems

Head and neck cancer (HNC)–derived cell lines represent fundamental models for studying the biological mechanisms underlying cancer development and precision therapies. However, mining the genomic information of HNC cells from available databases requires knowledge on bioinformatics and computational skill sets. Here, we developed a user-friendly web resource for exploring, visualizing, and analyzing genomics information of commonly used HNC cell lines. We populated the current version of GENIPAC with 44 HNC cell lines from 3 studies: ORL Series, OPC-22, and H Series. Specifically, the mRNA expressions for all the 3 studies were derived with RNA-seq. The copy number alterations analysis of ORL Series was performed on the Genome Wide Human Cytoscan HD array, while copy number alterations for OPC-22 were derived from whole exome sequencing. Mutations from ORL Series and H Series were derived from RNA-seq information, while OPC-22 was based on whole exome sequencing. All genomic information was preprocessed with customized scripts and underwent data validation and correction through data set validator tools provided by cBioPortal. The clinical and genomic information of 44 HNC cell lines are easily assessable in GENIPAC. The functional utility of GENIPAC was demonstrated with some of the genomic alterations that are commonly reported in HNC, such as TP53, EGFR, CCND1, and PIK3CA. We showed that these genomic alterations as reported in The Cancer Genome Atlas database were recapitulated in the HNC cell lines in GENIPAC. Importantly, genomic alterations within pathways could be simultaneously visualized. We developed GENIPAC to create access to genomic information on HNC cell lines. This cancer omics initiative will help the research community to accelerate better understanding of HNC and the development of new precision therapeutic options for HNC treatment. GENIPAC is freely available at http://genipac.cancerresearch.my/.

Clinical and genetic characteristics of 15 families with hereditary hypophosphatemia: Novel Mutations in PHEX and SLC34A3

BackgroundHereditary hypophosphatemia is a group of rare renal phosphate wasting disorders. The diagnosis is based on clinical, radiological, and biochemical features, and may require genetic testing to be confirmed.MethodologyClinical features and mutation spectrum were investigated in patients with hereditary hypophosphatemia. Genomic DNA of 23 patients from 15 unrelated families were screened sequentially by PCR-sequencing analysis for mutations in the following genes: PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. CytoScan HD Array was used to identify large deletions.ResultsGenetic evaluation resulted in the identification of an additional asymptomatic but intermittent hypophosphatemic subject. Mutations were detected in 21 patients and an asymptomatic sibling from 13 families (86.6%, 13/15). PHEX mutations were identified in 20 patients from 12 families. Six of them were novel mutations present in 9 patients: c.983_987dupCTACC, c.1586+2T>G, c.1206delA, c.436+1G>T, c.1217G>T, and g.22,215,887–22,395,767del (179880 bp deletion including exon 16–22 and ZNF645). Six previously reported mutations were found in 11 patients. Among 12 different PHEX mutations, 6 were de novo mutations. Patients with de novo PHEX mutations often had delayed diagnosis and significantly shorter in height than those who had inherited PHEX mutations. Novel compound heterozygous mutations in SLC34A3 were found in one patient and his asymptomatic sister: c.1335+2T>A and c.1639_1652del14. No mutation was detected in two families.ConclusionsThis is the largest familial study on Turkish patients with hereditary hypophosphatemia. PHEX mutations, including various novel and de novo variants, are the most common genetic defect. More attention should be paid to hypophosphatemia by clinicians since some cases remain undiagnosed both during childhood and adulthood.

Chromothripsis in acute myeloid leukemia: biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

A rare 19p13.11-p13.12 deletion in a patient with microcephaly and developmental delay

Submicroscopic deletions in chromosome 19 have been rarely reported. The aim of this study was to describe the genotype-phenotype correlation of a 19p13.11-p13.12 deletion. A male patient was referred for genetic evaluation by presenting neurodevelopmental delay and facial dysmorphisms. He is the first child of non-consanguineous parents. Pregnancy and delivery were uneventful. Anthropometrical data at seven-year-old were weight 25,4 kg (p75), length 120 cm (p50) and head circumference 47,3 cm (&lt; -2SD). It was also observed malar hypoplasia, low-set and dysmorphic ears, bilateral preauricular tags, synophrys, short nose with anteverted nares and flat nasal bridge, down-turned corners of the mouth, long philtrum, micrognathia, high palate and congenital clubfoot. Abdominal ultrasonography showed pyelocaliceal ectasia, transfontanellar ultrasound revealed mild enlargement of magna cisterna and a small cyst ate nucleous caudatus. GTG-banding was normal and chromosomal microarray analysis (CMA), using the CytoScan HD Array (Affymetrix®, Santa Clara, CA, USA) showed an interstitial