The Efficacy of Whole Genome Sequencing and RNA-Seq in the Diagnosis of Whole Exome Sequencing Negative Patients with Complex Neurological Phenotypes

Whole-genome sequencing (WGS) is being increasingly utilized for the diagnosis of neurological disease by sequencing both the exome and the remaining 98 to 99% of the genetic code. In addition to more complete coverage, WGS can detect structural variants (SVs) and intronic variants (SNVs) that cannot be identified by whole exome sequencing (WES) or chromosome microarray (CMA). Other multi-omics tools, such as RNA sequencing (RNA-Seq), can be used in conjunction with WGS to functionally validate certain variants by detecting changes in gene expression and splicing. The objective of this retrospective study was to measure the diagnostic yield of duo/trio-based WGS and RNA-Seq in a cohort of 22 patients (20 families) with pediatric onset neurological phenotypes and negative or inconclusive WES results in lieu of reanalysis. WGS with RNA-Seq resulted in a definite diagnosis of an additional 25% of cases. Sixty percent of these solved cases arose from the identification of variants that were missed by WES. Variants that could not be unequivocally proven to be causative of the patients' condition were identified in an additional 5% of cases.

Molecular Characterization Using Oncoscan Chromosome Microarray in an International Cohort of 51 Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy with multi-organ and frequent skin involvement, and poor clinical outcomes. Based on the limited available data, the estimated incidence is 0.44% of all hematologic malignancies, representing less than 1% of acute leukemias, and 0.7% of cutaneous lymphomas. Due to the rarity of this entity, there have been relatively few studies characterizing the molecular profile of BPDCN. We examined a cohort of 51 patients with BPDCN using OncoScan chromosome microarray, which provides genome-wide copy number abnormality (CNA) analysis. Methods An international cohort of BPDCN cases were collected from centers in Brazil (Laboratorio de Patologia, Botucatu), Swtizerland (University of Zurich), France (Hospital St. Louis, Paris), Peru (Instituto Nacional de Enfermedades Neoplasicas, Lima), Canada (Department of Pathology, University of Montreal), Italy (Derpartment of Pathology, University of Bologna), and US (Department of Pathology - The Ohio State University, Department of Hematopathology - MD Anderson Cancer Center; and Department of Pathology - University of Virginia). A total of 58 tissue blocks from 51 patient samples were retrieved. The diagnosis of BPDCN was done and confirmed by at least three independent hematopathologists or dermatopathologists in accordance with the WHO classification (Lyon 2017). For the purpose of the molecular analysis substratification, cases were classified as 'BPDCN' if they were positive for TCF4, and 'BPDCN-like' if they were negative for TCF4. Immunohistochemistry for CD123, CD4, and CD56 was performed in all cases. Exclusion criteria included expression of MPO, lysozyme, CD3, CD19, CD20, CD22, and/or EBV. DNA was extracted from FFPE samples via standard techniques and processed on OncoScan CNV Plus microarray (ThermoFisher Scientific) according to manufacturer's recommended protocol. Copy number abnormalities and select single nucleotide variants and insertions/deletions (74 mutations in 9 genes) were analyzed on Chromosome Analysis Suite software (ChAS v4.1; ThermoFisher Scientific). Additional analysis was performed using Nexus Copy Number (BioDiscovery, version 10.0). Results To date, we have successfully analyzed 45 cases of BPDCN with Oncoscan, revealing widespread CNA in the vast majority of cases (44/45; 98%). Alterations of chromosome 9 were common in this cohort, particularly CNAs involving CDKN2A/B at 9p21.3. Twenty-five cases (56%) demonstrated CNA including CDKN2A/B, with ten of these cases demonstrating a homozygous loss of CDKN2A/B (22%). Alterations of chromosome 13 were also frequently detected with loss of RB1 (located at 13q14.2) detected in 24 cases (53%). The RUNX1 gene (21q22.12) was a common target of CNAs in this cohort, seen in nine cases (20%). Eight of these cases showed a copy number gain of RUNX1, which is a recurrent finding in a variety of hematological malignancies, particularly myeloid neoplasms. The remaining case with RUNX1 CNA showed a focal, homozygous loss of the gene, demonstrating that dysregulation of RUNX1 through CNA is a common event in BPDCN. We observed frequent deletions of ETV6 (53%), IKZF1 (33%), and TP53(16%) in our cohort. The ARHGAP26 gene (5q31.3), which is associated primarily with juvenile myelomonocytic leukemia, was included in CNA in 13 cases (29%), with both gains and losses observed in this cohort. Oncoscan can detect a limited number of single nucleotide variants in nine genes that are frequently mutated in cancers (BRAF, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, PTEN, and TP53). Mutations were detected in ten cases (22%), with NRAS and TP53 variants detected in three cases each and KRAS and IDH2 variants detected in two cases each. Conclusions Our preliminary data demonstrates complex genomic alterations in BPDCN, with the RB1 locus on chromosome 13, the CDKN2A/B locus on chromosome 9, and the ETV6 locus on chromosome 12 most commonly detected. However, widespread genomic alterations were detected involving a variety of cancer-associated genes further characterizing CNA in BPDCN. Analysis of additional BPDCN cases is progress. Disclosures Khoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Gru: StemLine: Honoraria, Research Funding, Speakers Bureau; CRISPT Therapeutics: Research Funding; Innate Pharma: Research Funding.

Prenatal Diagnosis of True Fetal Mosaicism with Small Supernumerary Marker Chromosome Derived from Chromosome 16 by Funipuncture and Molecular Cytogenetics Including Chromosome Microarray

This study examined the molecular characterization of a prenatal case with true fetal mosaicism of small supernumerary marker chromosome 16 (sSMC(16)). A 41-year-old female underwent amniocentesis at 19 weeks of gestation due to advanced maternal age. Chromosomal analysis for cultured amniocytes revealed a karyotype of 47,XY,+mar[4]/46,XY[16]. Spectral karyotyping and metaphase fluorescence in situ hybridization (FISH) demonstrated that the sSMC was derived from chromosome 16 (47,XY,+mar.ish der(16)(D16Z1+)[13/20]). Confined placental mosaicism was initially suspected because the prenatal ultrasound revealed a normal structure and the pregnancy was uneventful. However, interphase FISH of cord blood performed at 28 weeks of gestation showed 20% mosaicism of trisomy chromosome 16 (nuc ish(D16Z2×3)[40/200]). Chromosome microarray analysis further demonstrated 55% mosaicism of an 8.02 Mb segmental duplication at the subcentromeric region of 16p12.1p11.1 (arr[GRCh37] 16p12.1p11.1(27021975_35045499)×3[0.55]). The results demonstrated a true fetal mosaicism of sSMC(16) involving chromosome16p12.1p11.1 that is associated with chromosome 16p11.2 duplication syndrome (OMIM #614671). After non-directive genetic counseling, the couple opted for late termination of pregnancy. This case illustrated the use of multiple molecular cytogenetic tools to elucidate the origin and structure of sSMC, which is crucial for prenatal counseling, decision making, and clinical management.

Prenatal Diagnosis of Microduplication of Fetal Chromosome 17 Following Noninvasive Prenatal Testing

Background Chromosome 17q12 duplication syndrome is a disease caused by the complete or partial duplication of q12 in the long arm of chromosome 17, there were no cases reported about the prenatal diagnosis of the syndrome. Most of the fetal phenotype of the syndrome may not be evident during the pregnancy, which means the syndrome was only be discovered accidentally or missed during the prenatal examination. Objective Noninvasive prenatal testing (NIPT) is widely used in the screening of common fetal chromosome aneuploidy. However, reports on chromosomal microduplication and microdeletion are rare. The aim of the study was to investigate the application value of NIPT for the detection of chromosomal microduplication. Case presentations: We found two cases of microduplication in the long arm of chromosome 17(17q12), they were first detected by NIPT and then were further diagnosed by copy number variation (CNV) analysis based on chromosome microarray analysis (CMA). The CMA results of prenatal diagnosis showed that the microduplications in 17q12 (one was 1.5Mb, the other was 1.9Mb) were consistent with the NIPT results. The amniotic fluid karyotype analysis showed no abnormalities. Finally, because it was pathogenic copy number variant, both of the parents chose to terminate the pregnancy. Conclusion In the study, two cases of microduplication fragment in the long arm of chromosome 17 were detected by NIPT and were confirmed by CMA. To our knowledge, this is the first report of prenatal diagnosis of chromosome 17q12 duplication syndrome following NIPT. This suggests that NIPT is an effective method to screen chromosome microduplications in prenatal diagnosis, especially for the chromosome 17q12 duplication syndrome.

Chromosomal Microarray Analysis on Intergenerational Effects of Oligomeric Proanthocyanidins Against Bisphenol-A Induced Brain Deformities

Oligomeric Proanthocyanidins (OPC) is a type of polyphenolic compound which have been demonstrated to have anti-cancer, anti-oxidant, anti-inflammatory and anti-mutagenic properties that may have the potential to reduce intergenerational effect of BPA towards DNA. This study was conducted to determine the effect of OPC on the DNA damage of BPA-induced rats using microarray CGH Chromosome Karyotyping. Adult Male Sprague Dawley rats were divided into six groups which are Normal, BPA, OPC10, OPC20, BPA+OPC10 and BPA+OPC20. The administration of BPA and/or OPC were conducted for 21 days using oral gavage before being mated with female rats of the same age at 1:1 mating ratio. Once the female rats were confirmed pregnant, the male rats were decapitated and their blood were collected for chromosome microarray analysis. The male offspring (F1 generation) were allowed to grow until 10 weeks old and their blood were also collected for chromosome microarray analysis. BPA group had a deletion of Vomeronasal receptor genes in which the deletion magnitude increased from P to F1 generation may affect the ability of the rats to detect chemosensory cues during neurobehavioral test. The amplification of Major Histocompatibility complex (MHC) class I gene in BPA+OPC20 group may aid in a better performance during hippocampal-dependent memory tests. These results suggested that OPC could be a potential agent in reducing the intergenerational effect of BPA. Current finding may enrich our understanding particularly in the possible mechanism of OPC on BPA-induced memory impairment Keywords: Bisphenol-A, Brain, microarray CGH Chromosome Karyotyping, Oligomeric Proanthocyanidins, Intergenerational

SINO Syndrome Causative KIDINS220/ARMS Gene Regulates Adipocyte Differentiation

Nonsense variants in KIDINS220/ARMS were identified as the main cause of spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO) syndrome, a rare disease with birth defects in brachycephaly, neurological disorder, and obesity. The cause of neural cell dysfunction by KIDINS220/ARMS were extensively studied while the cause of obesity in SINO syndrome remains elusive. Here, we identified KIDINS220/ARMS as an adipocyte differentiation-regulating gene. A Chinese family, mother and her two sons, all showed severe symptoms of SINO syndrome. G-banding karyotyping, chromosome microarray analysis, and whole exome sequencing revealed a novel amber mutation, c.3934G>T (p. E1312X), which was close to the C-terminal region of KIDINS220/ARMS and resulted in the premature of the protein. Both the mRNA and protein levels of KIDINS220/ARMS gradually decreased during adipocyte differentiation. Knockdown of KINDINS220/ARMS could prompt adipocyte differentiation and lipid accumulation while overexpression of KIDINS220/ARMS decrease the rate of matured adipocytes. Furthermore, we demonstrated that KIDINS220/ARMS inhibits adipocyte maturation through sustained extracellular signal-regulated kinase signaling. In conclusion, this is the first report about a vertical heredity of severe dominant pathogenic mutation of KIDINS220/ARMS, suggested that KIDINS220/ARMS played a negative role in adipocyte maturation, explained the cause of obesity in SINO syndrome and could highlight the importance of adipocyte differentiation in neuron functions.