Abnormal Cell Lines Research Articles

Confirmatory test versus screening test analyses for fetal mosaic variations; a large scale study

ABSTRACT Background Mosaic genetic anomaly is a problematic and interpretative issue in prenatal diagnosis. Conventional karyotyping, as a confirmatory test traditionally used for detecting mosaic and nonmosiac prenatal disorders. Recently Quantitative Fluorescence PCR (QF-PCR) is used for prenatal testing. We retrospectively assessed the frequency of both mosaic and nonmosaic conditions in a large-scale study and compared the clinical value of confirmatory cytogenetic analysis with QF-PCR and other screening tests. Result Of 6033 cases identified as abnormal conditions by sonography or protein marker screening tests, only 180 nonmosaic and 8 mosaic cases confirmed to be abnormal by confirmatory karyotyping test results. The cytogenetic analysis was correlated with other QF-PCR confirmatory test results for nonmosiac conditions but it was not comparable for mosaic cases. Conclusion The cytogenetic analyses were shown to have the greatest clinical value in revealing the various mosaic conditions. The QF-PCR test is shown to be a reliable confirmatory test for nonmosaic diseases but not for mosaicism, and the screening protein marker test can weakly indicate the presence of abnormal cell lines. Moreover, older mothers (>30 years) are at greater risk for developing mosaic ova.

Recurring germline mosaicism in a family due to reversion of an inherited derivative chromosome 8 from an 8;21 translocation with interstitial telomeric sequences

BackgroundMosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited.MethodsWe performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative...

Rosmarinic acid protects mice from imiquimod induced psoriasis-like skin lesions by inhibiting the IL-23/Th17 axis via regulating Jak2/Stat3 signaling pathway.

IL-23/Th17 (IL-17) axis plays a critical role in psoriasis. Rosmarinic acid (RA) was proved the inhibitory effect of T cell infiltration in the skin. However, whether and how RA has beneficial effects on psoriasis did not really know yet. So lipopolysaccharide (LPS)-induced abnormal proliferation Hacat cell line and Imiquimod (IMQ)-induced psoriasis-like mouse dermatitis were used to assess the pharmacological effects and mechanisms of RA by Psoriasis Area Severity Index (PASI) score, histopathology, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The results showed that RA inhibited LPS-induced aberrant expression of Hacat cell line, and significantly alleviated IMQ-induced skin inflammation. Although RA had no obviously effect on the ratio of epidermal Langerhans cell (LC) and LC migration from the skin to the skin draining lymph nodes, RA inhibited the expression of IL-23 in skin lesions, as well as reduced the differentiation of Th17 cells and producing of IL-17A by down regulating the transcriptor factor RORγt and JAK2/Stat3 signal pathway, comparing to IMQ treated group. The findings suggest that RA inhibits psoriasis-like skin inflammation in vivo and in vitro by reducing the expression of IL-23, inhibiting Th17 dominated inflammation and down regulating the Jak2/Stat3 signal pathway.

A Rare Variant of Turner Syndrome With Isodicentric X Chromosome Resulting in Trisomy: A Case Report

Introduction: Turner syndrome is a genetic disorder caused by the loss of an X-chromosome affecting approximately 1 in every 2,500 females. A constitutional karyotype of 45, X accounts for nearly 50% of patients, while mosaicism and other chromosomal structural abnormalities such as deletions, duplications, ring, isodicentric chromosomes, inversions and translocations, have been reported. Isodicentric X chromosomes are formed presumably by end-to-end fusion of chromatids after a break, with subsequent loss of an acentric fragment. These chromosomes in general have phenotypes characteristic of the resultant X deletions. We present a case of a 14-year-old female diagnosed with Turner syndrome and with 2 abnormal cell lines. Case Presentation: This is a case of a 14-year-old female referred to pediatric endocrinology for concerns of short stature and delayed puberty. She denied any food intolerance, bloating and diarrhea. She is otherwise healthy with unremarkable past medical history. Her weight was normal at 15th percentile. Her height was 137cm or 0.01 percentile with a Z score of –3.6. Work up revealed hypothyroidism with TSH 16.3 mcIU/mL (0.4-4.7 mcIU/mL), positive thyroid peroxidase antibody >900 IU/ml and thyroglobulin antibody 14 IU/mL (< 1.8IUm/mL) and celiac disease (tissue transglutaminase IgA > 100 U/mL) both without associated symptoms. Estradiol level was undetectable, and LH and FSH were 9.89 mIU/ml and 52.69 mIU/ml respectively. The rest of her labs including growth factors were normal. Bone age was normal at 13 years for chronological age of 14 years old. Chromosomal microarray revealed 2 abnormal cell lines: one with monosomy X, the other with a normal X chromosome and an isodicentric X chromosome involving the Xp11.22-q28 region resulting in trisomy of the latter cell line.Levothyroxine was started. Plan is to start growth hormone therapy and initiate puberty after. Patient referred to necessary subspecialties for hearing evaluation as well as cardiac evaluation ConclusionTurner syndrome usually presents as females with short stature, gonadal dysgenesis and 45,X cell line that is either singly or in combination with anothermosaic cell line. Our patient presented with short stature and absence of puberty. Initial investigation revealed hypothyroidism and highly positive celiac antibodies, but unable to attribute her short stature to both diagnoses given the lack of other symptoms. This case emphasizes the importance of checking the karyotype in females presenting with short stature and more importantly delayed puberty as part of the diagnostic algorithm. In addition, checking thyroid and celiac panel are also imperative as treatment of these are treatable etiologies of short stature.

Multifaceted modifications for a cell size-based circulating tumor cell scope technique hold the prospect for large-scale application in general populations.

Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear-cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs onslides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.

Identification and utilization of copy number information for correcting Hi-C contact map of cancer cell lines

BackgroundHi-C and its variant techniques have been developed to capture the spatial organization of chromatin. Normalization of Hi-C contact map is essential for accurate modeling and interpretation of high-throughput chromatin conformation capture (3C) experiments. Hi-C correction tools were originally developed to normalize systematic biases of karyotypically normal cell lines. However, a vast majority of available Hi-C datasets are derived from cancer cell lines that carry multi-level DNA copy number variations (CNVs). CNV regions display over- or under-representation of interaction frequencies compared to CN-neutral regions. Therefore, it is necessary to remove CNV-driven bias from chromatin interaction data of cancer cell lines to generate a euploid-equivalent contact map.ResultsWe developed the HiCNAtra framework to compute high-resolution CNV profiles from Hi-C or 3C-seq data of cancer cell lines and to correct chromatin contact maps from systematic biases including CNV-associated bias. First, we introduce a novel ‘entire-fragment’ counting method for better estimation of the read depth (RD) signal from Hi-C reads that recapitulates the whole-genome sequencing (WGS)-derived coverage signal. Second, HiCNAtra employs a multimodal-based hierarchical CNV calling approach, which outperformed OneD and HiNT tools, to accurately identify CNVs of cancer cell lines. Third, incorporating CNV information with other systematic biases, HiCNAtra simultaneously estimates the contribution of each bias and explicitly corrects the interaction matrix using Poisson regression. HiCNAtra normalization abolishes CNV-induced artifacts from the contact map generating a heatmap with homogeneous signal. When benchmarked against OneD, CAIC, and ICE methods using MCF7 cancer cell line, HiCNAtra-corrected heatmap achieves the least 1D signal variation without deforming the inherent chromatin interaction signal. Additionally, HiCNAtra-corrected contact frequencies have minimum correlations with each of the systematic bias sources compared to OneD’s explicit method. Visual inspection of CNV profiles and contact maps of cancer cell lines reveals that HiCNAtra is the most robust Hi-C correction tool for ameliorating CNV-induced bias.ConclusionsHiCNAtra is a Hi-C-based computational tool that provides an analytical and visualization framework for DNA copy number profiling and chromatin contact map correction of karyotypically abnormal cell lines. HiCNAtra is an open-source software implemented in MATLAB and is available at https://github.com/AISKhalil/HiCNAtra.

Two Abnormal Cell Lines of Trisomy 14 and t(X;14) with Skewed X-Inactivation

Trisomy 14 is incompatible with live, but there are several patients reported with mosaic trisomy 14. We aimed to study the pattern of X inactivation and its effect on a translocated autosome and to find out an explanation of the involvement of chromosome 14 in 2 different structural chromosomal abnormalities. We report on a girl with frontal bossing, hypertelorism, low-set ears, micrognathia, cleft palate, congenital heart disease, and abnormal skin pigmentations. The patient displayed iris, choroidal, and retinal coloboma and agenesis of the corpus callosum and cerebellar vermis hypoplasia. Cytogenetic analysis revealed a karyotype 45,X,der(X)t(X;14)(q24;q11)[85]/46,XX,rob(14;14)(q10;q10),+14[35]. Array-CGH for blood and buccal mucosa showed high mosaic trisomy 14 and an Xq deletion. MLPA detected trisomy 14 in blood and buccal mucosa and also showed normal methylation of the imprinting center. FISH analysis confirmed the cell line with trisomy 14 (30%) and demonstrated the mosaic deletion of the Xq subtelomere in both tissues. There was 100% skewed X inactivation for the t(X;14). SNP analysis of the patient showed no region of loss of heterozygosity on chromosome 14. Also, genotype call analysis of the patient and her parents showed heterozygous alleles of chromosome 14 with no evidence of uniparental disomy. Our patient had a severe form of mosaic trisomy 14. We suggest that this cytogenetic unique finding that involved 2 cell lines with structural abnormalities of chromosome 14 occurred in an early postzygotic division. These 2 events may have happened separately or maybe there is a kind of trisomy or monosomy rescue due to dynamic cytogenetic interaction between different cell lines to compensate for gene dosage.

Characterization of a Rare Mosaicism in Autosomal Translocation of t(5;21) Using Conventional Cytogenetics and FISH Methods

Background:Mosaicism of a normal cell population and an unbalanced autosomal chromosome rearrangement is rarely seen. If the abnormal cell line contributes to a minor part of soma, the phenotype is expected to be normal. Case Report:We report a 29-year-old woman who had balance chromosomal translocation of 46,XX,t(5;21) with a two-year-old affected girl, characterized by mental retardation, dystrophia, hearing impartment, and dysphagia. Methods and Results:Cytogenetic investigation revealed a low mosaic unbalanced translocation of 46,XX,t(5;21)/ 46,XX, which was confirmed by FISH analysis. Studying 200 metaphases and interphases of peripheral blood sample revealed 70% partial monosomy of 21q22 and partial trisomy of 5q(35.3) and 30% of normal pattern. Conclusion:In rare cases such as this study, parents with balanced translocation with no phenotypes may lead to a mosaic unbalanced translocation with abnormal phenotypes in offspring, which underscores the need for prenatal karyotyping and genetics counseling.

Double Autosomal/Gonosomal Mosaic Trisomy 47,XXX/47,XX,+14 in a Newborn with Multiple Congenital Anomalies

Chromosomal trisomies are the most frequent major chromosomal anomalies in humans and can be present in a mosaic or a non-mosaic constitution. We report the first case of a newborn girl presenting with multiple congenital anomalies and a double mosaic trisomy involving chromosome 14 and the X chromosome detected by array CGH. Karyotype analysis revealed a double mosaic with 2 independent abnormal cell lines and the absence of 46,XX and 48,XXX,+14 cell lineages. The patient showed most of the clinical characteristics of mosaic trisomy 14. Analysis of autosomal DNA markers in the proband's blood sample did not support the presence of chimerism. Further analysis of chromosome X DNA markers suggests that the extra X chromosome most probably arose as a consequence of nondisjunction in meiosis II in the maternal lineage.

Improved neural differentiation of normal and abnormal induced pluripotent stem cell lines in the presence of valproic acid.

During the generation of induced pluripotent stem cell (iPSC) lines from cord blood CD34+ cells, a line having complete trisomy of Chromosome 1 and deletion of q23 to qTer of Chromosome 11 was accidentally developed in our lab. The abnormality was consistently detected even at higher passages. These chromosomal anomalies are known to manifest neurological developmental defects. In order to examine if such defects occur during in vitro differentiation of the cell line, we set up a protocol for neural differentiation. Valproic acid (VPA) was earlier reported by us to enhance neural differentiation of placental mesenchymal stem cells. Here, we induced normal and abnormal iPSC lines to neural lineage with/without VPA. Neural differentiation was observed in all four sets, but for both the iPSCs lines, VPA sets performed better. The characteristics tested were morphology, neural filament length, detection of neural markers, and electrophysiology. In summary, the karyotypically abnormal line exhibited efficient neural differentiation. This iPSC line may serve as a useful tool to study abnormalities associated with trisomy 1 and deletion of q23 to qTer of Chromosome 11.

80 mosaic embryo transfers in a single clinic with in-house PGT-A: What we have learned

Introduction IVF clinics are routinely transferring embryos diagnosed through PGT-A as mosaic anueploid with greaterfrequency to patients who lack available euploid embryos. Nevertheless, there is a lack of evidencebasedguidelines that establish which characteristics and genetic abnormalities affect a mosaic embryo'sclinical predictive outcome. Materials & methods 80 mosaic embryos were transferred at a single IVF program from August of 2015 through February of 2018. All embryos were tested in-house by high resolution NGS on trophectoderm biopsies obtained from blastocysts at 5-7 days of development. Karyotype profiles were analyzed for characteristics predictive of improved clinical outcomes. This study followed PGDIS guidelines defining mosaic embryosas containing whole or segmental chromosomal gains or losses between 20% and 80%. Results Of the 80 transferred mosaic embryos, 41 (51.3%) established a chemical pregnancy, 25 (31.3%)developed an embryonic sac (implantation), and 20 (25.0%) developed a fetal heartbeat (FHB). Of these20, 11 resulted in a live birth and at the time of analysis 9 were ongoing pregnancies, meaning that nopost-FHB miscarriages had occurred. Embryos derived from oocytes younger than 35 years old had asignificantly higher implantation rate than embryos from oocytes 35 years or older (50.0% to 21.2%,n=28 and 52 respectively) as well as comparable implantation rates to euploid embryos from across allage groups (younger than 35 years, n=141: 51.1%; greater than 35 years, n=341: 48.4%). There was anoticeable difference in implantation rate between embryos with either a single chromosomal mosaicloss or gain (33.3%, n=45) and mosaic embryos with multiple chromosomal losses or gains (23.1%,n=26). Mosaic embryos displaying only a single segmental chromosomal abnormality had a higherchance of implanting and developing a FHB (n=26, 38.5%, 34.6%) than mosaic embryos with multiplesegmental abnormalities or whole chromosome gains or losses (n=52, 26.9%, 21.2%) and also had a farlower chance of losing a pregnancy after an initial positive beta (23.1% to 46.2%, n=13 and 26respectively). When the embryos were broken down by severity of mosaicism, we found that 20% to50% (n=36) severity mosaic embryos were significantly more likely to implant and develop a FHB thanthe more severe mosaic 50%-80% embryos (n=12). Additionally, mosaic embryos were divided up bynumber of estimated abnormal cell lines present in the trophectoderm biopsy. The implantation rate forembryos estimated with only one aneuploid cell population was significantly higher than those with two(n=12, 33.3% vs n=12, 8.3%). Lastly, embryos diagnosed with mosaic trisomies did not have a significantdifference in implantation or FHB development (n=30, 33.3%, 30.0%) from those with monosomies (n=28, 32.1%, 25.9%). Conclusions Embryos classified as mosaic are capable of producing healthy babies and should be considered for transfer when a euploid embryo is not available. When there are multiple mosaic embryos to select from for any given patient, our data suggests embryos diagnosed with a single segmental abnormality should be preferred, followed by those with less severe mosaicism as measured by number of affected chromosomes and severity of aneuploidy in the mosaic mix.

Identification of molecular processes that differ among Scots pine somatic embryogenic cell lines leading to the development of normal or abnormal cotyledonary embryos

Several coniferous species can today be propagated through somatic embryogenesis, but for species belonging to the Pinus genus, there are still problems related to the small number of genotypes from which embryogenic cultures can be established and the low yield of high-quality cotyledonary embryos. In order to pinpoint molecular processes that might be disturbed during somatic embryogenesis in Scots pine, we have analyzed the expression pattern of selected transcripts during development of somatic embryos in a normal and an abnormal cell line of Scots pine. The selected transcripts have been previously shown to be differentially expressed during early zygotic embryogenesis in Scots pine (Merino et al. 2016). Based on the transcripts that accumulated differentially between the two cell lines, we conclude that the apical–basal polarization in early somatic embryos and the transition from the morphogenic phase to the maturation phase are disturbed in the abnormal cell line. A comparison between transcript accumulation during somatic and zygotic embryogenesis highlighted problems with the continuous embryo degeneration processes in embryogenic cultures of Scots pine. Transcripts that were highly abundant during the cleavage process in zygotic embryos showed different accumulation patterns during somatic embryogenesis in Scots pine compared to those in Norway spruce. Furthermore, altering the expression of these transcripts in embryogenic cultures of Norway spruce resulted in a changed proliferation pattern of the embryos so that they became more similar to proliferating somatic embryos of Scots pine. Taken together, our results provide a deeper understanding of the deviations in abnormal cell lines and indicate that embryogenic cultures of Scots pine are proliferating by a cleavage-like process.

Suspended graphene oxide nanoparticle for accelerated multilayer osteoblast attachment.

Mimicking bone tissues having layered structures is still a significant challenge because of the lack of technologies to assemble osteoblast cell types into bone structures. One of the promising and attractive materials in biomedical and different engineering fields is graphene and graphene-based nanostructures such as graphene oxide (GO) because of their unique properties. In most studies, GO was synthesized using chemical vapor deposition method, and was coated on the substrate. In this study, we proposed a simple technique for assembly of cells that facilitates the construction of osteoblast-like structures using suspended GO synthesized by graphite powder, H2 SO4 , and KMnO4 .Toxicity effects of GO on human mesenchymal stem cells (hMSCs) derived from bone marrow were analyzed. In addition to normal MSCs, toxicity effects of GO on human cancer cell line saos-2 as an abnormal cell line that possess several osteoblastic features, was examined. The attachment and expression of osteoblast cells genes were evaluated after differentiation of MSCs to osteoblast cells in presence of suspended GO by scanning electron microscopy and real time PCR. We found that the toxicity effects of GO are dose dependent and in oseogenic medium containing suspended GO the expression level of osteoblast genes osteopontin and osteocalcin and cell adhesion markers connexin were higher than control group. Interestingly, through this method GO was found to induce multilayer osteoblast cell morphology and enhance the number of cell layer. We expect that the presented method would become a highly useful approach for bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 293-303, 2018.

Plasma and ovarian tissue sphingolipids profiling in patients with advanced ovarian cancer.

The role of lipids in carcinogenesis through induction of abnormal cell lines in the human body is currently undisputable. Based on the literature, bioactive sphingolipids play an essential role in the development and progression of cancer and are involved in the metastatic process. The aim of this study was to determine the concentration of selected sphingolipids in patients with advanced ovarian cancer (AOC, FIGO III/IV, high grade ovarian cancer). Seventy-four patients with ovarian cancer were enrolled. Plasma concentrations of C16-Cer, C18:1-Cer and C18-Cer were assessed by LC/MS/MS. The content of tissue sphingolipids was measured using a UHPLC/MS/MS. Plasma concentration of 3 ceramides: C16-Cer, C18:1-Cer and C18-Cer was significantly elevated in women with advanced ovarian cancer compared to control group (P=0.031; 0.022; 0.020; respectively). There were increases in concentration of 5 ceramides: C16-Cer, C18:1-Cer, C18-Cer, C24:1-Cer, C24-Cer (P=0.025; 0.049; 0.032; 0.005; 0.013, respectively) and S1P (P=0.004) in ovarian tissue of women with advanced ovarian cancer compared to healthy individuals. Importantly, significantly higher risk of ovarian cancer when the plasma concentration of C16-Cer>311.88ng/100μl (AUC: 0.76, P=0.0261); C18:1-Cer>4.75ng/100μl (AUC: 0.77, P=0.0160) and C18-Cer>100.76ng/100μl (AUC:0.77, P=0.0136) was noticed. Bioactive sphingolipids play an essential role in the development and progression of cancer and they also take part in the process of metastasizing. This study suggests that some sphingolipids can be used as potential biomarkers of advanced ovarian cancer and that they can play an important role in the pathogenesis of this disease.

4D nucleome Analysis Toolbox: analysis of Hi-C data with abnormal karyotype and time series capabilities

The availability of powerful analysis tools will further understanding of genome organization and its relationship to phenotype in dynamical settings. The 4D Nucleome Analysis Toolbox (NAT) is a user-friendly and powerful MATLAB toolbox for time series analysis of genome-wide chromosome conformation capture (Hi-C) data and gene expression (RNA-seq). NAT can load and normalize data, define topologically associating domains, analyse translocations, produce visualization, and study time course data. We provide examples that include time series data sets and karyotypically abnormal cell lines demonstrating the flexibility of NAT. https://github.com/laseaman/4D_Nucleome_Analysis_Toolbox. indikar@umich.edu.

Degeneration pattern in somatic embryos of Pinus sylvestris L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.

The Oncolytic Measles Virus Preferentially Infects p53 Abnormal Myeloma Cells

P53 plays a pleiotropic role in cell homeostasis by regulating stress-induced adaptive responses leading to survival or cell death. Although it is well demonstrated that the lack of a functional p53 pathway favors cancers, it remains still unclear whether it is related to its genome guardian or in apoptosis inducer role. Leonova and Gudkov demonstrated that, at least in mice, p53 and CpG methylation prevent expression of the so-called junk DNA, expression of which induces suicidal IFN responses (PNAS 2013;110:E89). Cancer cells mostly harbor hypo methylated DNA, reversible or irreversible silencing of p53 pathway and defects in suicidal IFN responses. Although the molecular bases are still not well understood, oncolytic viruses such as the Measles Virus (MV) preferentially infect and kill tumor cells. The aim of this study was to investigate whether del(17p)+/p53 mutant myeloma cells were preferentially sensitive to the Schwarz vaccine MV strain and whether p53 was involved in. Plasma cells from patients with multiple myeloma (MM) or MGUS over express CD46, the main receptor for the vaccine MV strain, as compared to normal plasma cells and to hematopoietic cells. To assess MV infection and replication, we used the GFP modified strain (MV-GFP) and measured GFP expression at day 4 after infection (MOI=1) using flow cytometry. We showed that p53 abnormal MM cell lines (n=25) over expressed CD46 (p=0.039) and were preferentially infected by MV (p=0.03) as compared with p53 wild type MM cell lines (n=12). Expressions of CD46 and GFP were directly correlated (p=0.014, r=0.505) and GFP expression correlated with cell death (n=37, p<0.0001, r=0.82). CD46 inhibition (using CD46 siRNA or anti-CD46 mAb) fully inhibited infection and death. CProduction of alpha IFN was not frequent (11 out of 37 cell lines), was irrespective of TP53 status and not related to MV resistance (no production of beta IFN was found). To assess whether p53 pathway regulates MV infection/replication, we treated cells 24 hours with the MDM2 inhibitor nutlin3a prior to MV infection. In all wild type and in none p53 abnormal cell lines, nutlin3a-pretreatment induced an increase in DR5 expression (Tessoulin B et al, Blood 2014;124:1626) and a decrease in CD46 expression: the mean of nutlin3a/control DR5 ratio of expression was 1.60 ± 0.27 and 1.04 ± 0.05 in wild type (n=10) and p53 abnormal cell lines (n=4), respectively (p=0.001) while that of nutlin3a/control CD46 ratio expression was 0.55 ± 0.08 and 0.97 ± 0.03, respectively, (p=0.0007). At 24 hours, cells were washed to remove nutlin3a and 9 cell lines sensitive to MV (5 wild type and 4 abnormal) were infected with MV (MOI=1). Decrease in CD46 expression was associated with decrease in MV replication in all p53 wild type (mean nutlin3a/control ratio 0.53 ± 0.05, n=5) but in none p53 abnormal cell lines (1.02 ± 0.18, n=4), p=0.016. We assessed infection of primary cells in unpurified mononuclear cells (MOI=1) from bone marrow or peripheral blood harboring various CD138+ infiltrate. Compared with CD138- cells, CD138+ cells over expressed CD46 (MFI ratio 100 versus 22, p=0.0003) and were preferentially infected by MV (GFP+ 62% versus 20%, p=0.0005, GFP MFI ratio 14 versus 5, p=0.0011, n=17). Assessment of TP53 status in primary cells was determined using FISH (del17p) and the functional assay of DR5 increase in response to nutlin3a. Although del(17p)+ myeloma cells over expressed CD46 and were highly infected by MV when compared to del(17p)- myeloma cells, differences didn’t reach statistical significance. We further showed that nutlin3a induced decrease in CD46 expression and MV infection in 3 primary cells without del(17p) but did not in 2 primary CD138+ cells with del(17p): decrease in CD46 expression correlated with decrease in GFP expression (n=5, p=0.0167, r=1.00) and with increase in DR5 expression (p=0.033, r=-0.89). These data showed that p53 abnormal myeloma cells (cell lines and primary cells) were strongly sensitive to the oncolytic MV, which could be of interest for patients with del(17p) refractory to current therapy. The molecular mechanism of p53-induced decrease in CD46 expression and MV infection is under investigation. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria.

Mosaic trisomy 15 at amniocentesis: Prenatal diagnosis, molecular genetic analysis and literature review

ObjectiveTo present prenatal diagnosis of mosaic trisomy 15 at amniocentesis. Materials and methodsA 37-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Cytogenetic analysis of cultured amniocytes revealed a karyotype of 47,XY,+15[2]/46,XY[17]. She was referred for repeated amniocentesis at 19 weeks of gestation. Array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction assays on uncultured amniocytes, conventional cytogenetic analysis and aCGH on cultured amniocytes, and FISH on uncultured urinary cells after birth were applied. Cordocentesis revealed a karyotype of 46,XY. ResultsAt repeated amniocentesis, cultured amniocytes revealed a karyotypes of 46,XY [22 colonies], FISH on uncultured amniocytes revealed 21.2% (22/104 cells) mosaicism for trisomy 15, aCGH on uncultured amniocytes revealed a genomic gain (log2 ratio = 0.3) in chromosome 15, quantitative fluorescent polymerase chain reaction on uncultured amniocytes excluded uniparental disomy 15 (UPD 15), and aCGH on culture amniocytes revealed no genomic imbalance in chromosome 15. A healthy 3700 g male baby was delivered at 38 weeks of gestation with no phenotypic abnormalities at age 6 months. FISH on uncultured urinary cells at birth and at age 6 months revealed mosaic trisomy 15 levels of 20% (13/65 cells) and 12.2% (6/49 cells), respectively. ConclusionPrenatal diagnosis of mosaic trisomy 15 at amniocentesis should alert doctors about the occurrence of UPD 15 and a clinically significant phenotype. The present case provides evidence for cytogenetic discrepancy between uncultured and cultured amniocytes in mosaic trisomy 15 at amniocentesis. It is possible that the abnormal cell lines of amniocytes with trisomy 15 disappear after long-term cell culture.

Abnormal non-invasive prenatal test results concordant with karyotype of cytotrophoblast but not reflecting abnormal fetal karyotype

We present a unique case in which non-invasive and invasive prenatal diagnoses showed abnormal, but discordant, results. A patient with abnormal non-invasive prenatal test (NIPT) results, indicating a 99% risk for monosomy X, was referred to our center for genetic counseling and confirmatory studies. Cytogenetic analysis of uncultured mesenchymal core of chorionic villi (CV) revealed a mosaic male karyotype consisting of two abnormal cell lines: one with monosomy X and the other with an isodicentric chromosome Y. Array analysis of the trophoblast confirmed the NIPT results. Based on the CV results, the patient opted for termination of pregnancy. After extensive counseling by a clinical geneticist about the possible outcomes and by a gynecologist about the risk of a second-trimester abortion procedure, the patient agreed to undergo early amniocentesis. Amniocentesis confirmed that the fetus had a male karyotype with an isodicentric chromosome Y, and the single nucleotide polymorphism (SNP) array profile suggested absence of the monosomy X cell line. The male infant was expected to be infertile. The patient finally decided to continue the pregnancy. Our case confirms that NIPT results are comparable with those of short-term cultured CV investigating the cytotrophoblast. Our patient was not aware that the NIPT results reveal the placental karyotype, which sometimes may be different from the fetal karyotype. Pretest counseling and providing the risk figures for false-positive and false-negative NIPT results are of great importance in order to discourage women from terminating pregnancies based on NIPT results alone.

Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results

Noninvasive prenatal screening for fetal aneuploidy analyzes cell-free fetal DNA circulating in the maternal plasma. Because cell-free fetal DNA is mainly of placental trophoblast origin, false-positive and false-negative findings may result from placental mosaicism. The aim of this study was to calculate the potential contribution of placental mosaicism in discordant results of noninvasive prenatal screening. We performed a retrospective audit of 52,673 chorionic villus samples in which cytogenetic analysis of the cytotrophoblast (direct) and villus mesenchyme (culture) was performed, which was followed by confirmatory amniocentesis in chorionic villi mosaic cases. Using cases in which cytogenetic discordance between cytotrophoblast and amniotic fluid samples was identified, we calculated the potential contribution of cell line-specific mosaicism to false-positive and false-negative results of noninvasive prenatal screening. The false-positive rate, secondary to the presence of abnormal cell line with common trisomies in cytotrophoblast and normal amniotic fluid, ranged from 1/1,065 to 1/3,931 at 10% and 100% mosaicism, respectively; the false-negative rate was calculated from cases of true fetal mosaicism, in which a mosaic cell line was absent in cytotrophoblast and present in the fetus; this occurred in 1/107 cases. Despite exciting advances, underlying biologic mechanisms will never allow 100% sensitivity or specificity.