SOLiD 5500xl Research Articles

Abstract 142: The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells

Abstract Introduction. We previously demonstrated that the activation of the epidermal growth factor receptor (EGFR) in mesenchymal stem cells (MSCs) modulates the expression of a number of genes coding for secreted proteins that promote breast cancer cell progression. As different microRNAs (miRNAs) have been shown to be involved in the cross-talk between MSCs and tumor cells, we hypothesized that the EGFR might regulate in MSCs the expression of miRNAs that might affect breast cancer progression through paracrine circuits. To this aim, we analysed the whole small RNA transcriptome of MSCs stimulated or not with transforming growth factor α (TGFα), one of the main ligands of the EGFR. Materials and methods. Small RNA sequencing was performed using the SOLiD 5500xl System. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was used for pathway analysis. Exosomes containing miRNAs were isolated from conditioned media with the ExoQuick reagent. MiRNA expression levels in conditioned medium were analyzed by Real Time PCR. Anchorage-dependent growth assays and Boyden-chamber based colorimetric migration assays were used to assess the proliferation and the migration of breast cancer cells, respectively. Results. Following small RNA sequencing, we identified 36 miRNAs differentially expressed between untreated and TGFα-treated MSCs with a fold change (FC) <0.56 or FC ≥1.90 (Confidence Interval, CI, 95%) and a threshold of sequence counts≥150 in at least one sample. In particular, 18 miRNAs resulted up-regulated with FC≥1.90 and 18 miRNAs down-regulated with FC<0.56. Pathways analysis of the target genes of the differentially expressed miRNAs revealed a significant enrichment in different KEGG pathways involved in cancer development and progression, including TGFβ signaling, focal adhesion, Rap1 signaling, Hippo signaling, mTOR and RAS signaling. To study miRNAs involved in the TGFα-mediated interaction between MSCs and breast cancer cells, we collected conditioned media from MSCs and analysed the expression levels of selected miRNAs. Real Time PCR analysis revealed the presence of several miRNAs in the conditioned medium from MSCs, including miR-23c, miR379-3p and miR-432-5p. The biological activity of the identified miRNAs was explored in a panel of breast cancer cell lines. MiR-23c was found to regulate the proliferation and migration of triple negative MDA-MB-468 and MDA-MB-231 breast cancer cells. Conclusion. Collectively, our data suggest that the EGFR signaling regulate in MSCs a wide number of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cross-talk. Citation Format: Marianna Gallo, Marianeve Carotenuto, Cristin Roma, Francesca Bergantino, Pasqualino de Antonellis, Nicola Normanno, Antonella De Luca. The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 142.

Methodology for Y Chromosome Capture: A complete genome sequence of\xa0 Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads

This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin–biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

Whole-Exome Sequencing Identifies One De Novo Variant in the FGD6 Gene in a Thai Family with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) has a strong genetic basis, although the genetics of autism is complex and it is unclear. Genetic testing such as microarray or sequencing was widely used to identify autism markers, but they are unsuccessful in several cases. The objective of this study is to identify causative variants of autism in two Thai families by using whole-exome sequencing technique. Whole-exome sequencing was performed with autism-affected children from two unrelated families. Each sample was sequenced on SOLiD 5500xl Genetic Analyzer system followed by combined bioinformatics pipeline including annotation and filtering process to identify candidate variants. Candidate variants were validated, and the segregation study with other family members was performed using Sanger sequencing. This study identified a possible causative variant for ASD, c.2951G>A, in the FGD6 gene. We demonstrated the potential for ASD genetic variants associated with ASD using whole-exome sequencing and a bioinformatics filtering procedure. These techniques could be useful in identifying possible causative ASD variants, especially in cases in which variants cannot be identified by other techniques.

Sequencing on the SOLiD 5500xl System – in-depth characterization of the GC bias

ABSTRACT Different types of sequencing biases have been described and subsequently improved for a variety of sequencing systems, mostly focusing on the widely used Illumina systems. Similar studies are missing for the SOLiD 5500xl system, a sequencer which produced many data sets available to researchers today. Describing and understanding the bias is important to accurately interpret and integrate these published data in various ongoing research projects. We report a particularly strong GC bias for this sequencing system when analyzing a defined gDNA mix of 5 microbes with a wide range of different GC contents (20–72%) when comparing to the expected distribution and Illumina MiSeq data from the same DNA pool. Since we observed this bias already under PCR-free conditions, changing the PCR conditions during library preparation – a common strategy to handle bias in the Illumina system - was not relevant. Source of the bias appeared to be an uneven heat distribution during the SOLiD emulsion PCR (ePCR) - for enrichment of libraries prior loading – since ePCR in either small pouches or in 96-well plates improved the GC bias. Sequencing of chromatin immunoprecipitated DNA (ChIP-seq) is a common approach in epigenetics. ChIP-seq of the mixed source histone mark H3K9ac (acetyl Histone H3 lysine 9), typically found on promoter regions and on gene bodies, including CpG islands, performed on a SOLiD 5500xl machine, resulted in major loss of reads at GC rich loci (GC content ≥ 62%), not explained by low sequencing depth. This was improved with adaptations of the ePCR.

Stressors alter intercellular communication and exosome profile of nasopharyngeal carcinoma cells.

Head and neck cancers comprise the sixth most common cancer type worldwide. One of the most remarkable malignancies of the head and neck is the cancer of the nasopharynx, with a strong metastatic tendency already in the early stage. Besides the conventional pathways of metastasis formation, the information content of exosomes produced by the cancer cells may play a key role in metastatic transformation. The aim of this study was to investigate how stressors alter the characteristic of tumor derived exosomes. In our experimental model, we compared the quantity and content of exosomes produced by a nasopharyngeal carcinoma cell line (5-8F) under conventional (chemotherapy) and alternative (Ag-TiO2 -catalyzed reactive oxygen species generation) cytostatic treatment. After isolation, exosomes were identified by atomic force microscopy and quantified with Nanosight NS500 device. MicroRNA content of them was analyzed using SOLiD 5500xl technology. The sequences were annotated in CLC Genomics Workbench version 5.5.1. Beyond the classic chemotherapeutic agent (doxorubicin), Ag-TiO2 in a photo-catalytic process also showed cytostatic activity. Tumor cell damage induced by the cytostatic treatments significantly altered the number of released exosomes and led to the predominance of tumor suppressors in the exosomal miRNA profile. Our results suggest that the intercellular communication between tumor cells and surrounding stroma cells can be altered by microenvironment which increased quantity of exosomes and diversity of miRNAs in this study. Imbalance of oncogenic and tumor suppressor miRNAs caused by cytostatic treatments may influence the antiproliferative and metastasis inhibitory effect of cytostatic agents.

Validation of two-channel sequencing-by-synthesis for noninvasive prenatal testing of fetal whole and partial chromosome aberrations.

To validate Illumina's two-channel NextSeq 500 sequencing system for noninvasive prenatal testing (NIPT) of fetal whole chromosome and partial aberrations. A total of 162 plasma samples, previously sequenced for NIPT on a SOLiD 5500xl platform, were sequenced on the NextSeq 500 using 75-bp single-end sequencing, followed by analysis using the WISECONDOR algorithm. For whole chromosome aneuploidy detection, all samples were classified correctly (in total 3× T13, 3× T18, 8× T21 and 145× euploid). Three partial aberrations (36-Mb terminal loss of 5p, 14-Mb gain on 18p and 33-Mb terminal loss of 13q) were also correctly identified. Fetal fractions in 34 male samples sequenced on both the SOLiD 5500xl and NextSeq 500 platform showed no significant difference. To test robustness, two sample sets, containing both euploid and aneuploid samples, were sequenced on different NextSeq 500 machines, revealing identical results. With unchanged laboratory flow, the NIPT turnaround time could be reduced from 15-16 calendar days to 7-8 calendar days, after switching from the SOLiD 5500xl to the NextSeq 500 platform. The NextSeq 500 platform can be used for NIPT to detect both whole and partial chromosome aberrations. It has fast turnaround times and is suitable for mid-sized laboratories.

76 Comprehensive Assessment of Rare Genetic Variation in Dilated Cardiomyopathy Genes in Patients and Controls: Abstract 76 Table 1

<h3>Introduction</h3> Dilated cardiomyopathy (DCM) is a genetically heterogenous condition with mutations reported in at least 50 associated genes.¹ Our consortium has demonstrated the presence of truncating mutations in the giant sarcomeric gene titin (TTN) in up to 27% of DCM² making this the commonest genetic cause of DCM. Here we conducted a comprehensive analysis of the frequency of rare coding variants in cardiomyopathy genes in DCM patients and ethnically-matched healthy controls. <h3>Methods</h3> We sequenced 64 cardiomyopathy genes in 651 individuals, comprising 234 patients referred for prospective evaluation of DCM with cardiac MRI (54 ± 14 yrs at scan, 70.5% males), 98 end-stage DCM (39 ± 16 yrs on date of transplant/left-ventricular assist device implantation, 83.7% males), and 319 prospectively recruited adult healthy volunteers (HVOL, 42 ± 14 yrs at scan, 46.1% males), who underwent detailed phenotyping with cardiac MRI. Next-generation targeted exon sequencing was performed on the SOLiD 5500xl platform. Variant calling was performed using Lifescope v2.5.1. Data were mapped to the Hg19 (GRCh37) human genome reference. Caucasian ethnicity was confirmed using Principal Component Analysis. Burden testing for potentially disease-causing variation was performed on each gene. <h3>Results</h3> DCM patients were enriched for novel protein-altering variants in 28 genes (Table 1: top 7 genes), but only 5 achieved nominal significance. Truncating variants in TTN (TTNtv) remained significant after multiple testing correction (12% DCM, 1.9% controls, p = 8.1 × 10–6). Rare variation in SCN5A and MYH6, previously reported to play a key role in DCM, were not enriched in DCM (SCN5A – 2.1% DCM, 2.8% controls, p = 0.80; MYH6 –0.9% DCM, 2.2% controls, p = 0.95) and nor were non-synonymous SNPs in TTN (TTNns, 25.6% DCM, 25.7% controls, p = 0.55). Importantly, an additive effect of variation in multiple DCM genes on DCM risk was identified using logistic regression models (p = 5.7 × 10 x -4), demonstrating a multi-genic basis for DCM in some cases. <h3>Conclusions</h3> To the best of our knowledge, this is the first comprehensive study of the genetic architecture of DCM compared to an ethnically-matched healthy control cohort. The majority of genes reported to contain rare variants in DCM have similar burdens of variation in controls. Some variants may contribute to disease, but excluding TTNtv the majority of such variants are uninterpretable in the absence of functional or segregation data. <h3>References</h3> Mestroni L, Taylor MR. Genetics and genetic testing of dilated cardiomyopathy: a new perspective. Discov Med. 2013;15(80):43-9 Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, <i>et al</i>. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366(7):619-28

Exome sequencing reveals novel and recurrent mutations with clinical significance in inherited retinal dystrophies.

This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in RHO, c.937-2_944del, (ii) one rare homozygous mutation in C2orf71, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in ABCA4, c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of RPGR. The molecular findings for RHO and C2orf71 confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two ABCA4 mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the RPGR mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.

Abstract 305: Ultra-low coverage exome sequencing of FFPE tumor specimens identifies exposure to carcinogenic aristolochic acid

Abstract Dietary intake of Aristolochia plants containing aristolochic acid (AA, IARC Group 1 carcinogen), leads to renal disease and to recurrent urinary tract transitional cell carcinomas (TCC). Mutation screens identified frequent A&amp;gt;T transversions, alterations underrepresented in other cancer types, as the signature of AA exposure. AA-associated TCC is a preventable cancer yet millions are estimated at risk due to unregulated medicinal use of Aristolochia. Here we present an ultra-low coverage modification of whole-exome sequencing (ULC-WES) as a novel molecular epidemiology method that reliably detects the AA mutation signature in small amounts of DNA from FFPE tumor tissues and can identify new exposed populations. DNA was isolated from paraffin sections of 18 TCC tumors from endemic nephropathy (EN) patients from regions in Croatia and Bosnia linked to exposure to AA. Four non-EN upper tract TCCs were used as controls. 250 ng of DNA was used for sequencing libraries and exome enrichment (Nimblegen, Roche). Libraries were sequenced on Illumina HiSeq2500 at 10-12x coverage. After data processing, GATK was used to call single nucleotide variants (SNV). To assess platform independence, 7 samples were run on SOLID 5500XL (Life), using proprietary chemistry and pipeline for data processing and variant calling. Normal genetic variants were filtered out after SNV annotation. ULC-WES at 10x identified the A&amp;gt;T predominance in 17 of 18 EN tumors (94%), and at &amp;lt;10x coverage (3-9 reads per SNV) still detected the major A&amp;gt;T pattern in 14 (78%) samples. In contrast, the A&amp;gt;T presence was &amp;lt;8% in the control TCCs. We observed stochastic distribution of the A&amp;gt;T mutations in the genome (R^2=0.83 for mutation count correlated with the chromosome length) but also identified recurrent non-synonymous A&amp;gt;T mutations in tumor suppressors and oncogenes including TP53, ARID1A, BRCA1/2, AXIN1, GATA3 and NRAS, DNMT1, FLT3, CSF1R and PIK3CA. We next detected recurrent A&amp;gt;T mutations in a number of histone and DNA modifiers suggesting epigenetic deregulation of genome activity in the AA-associated TCCs. In 2 patients, pairs of distinct TCCs from the same surgery had markedly overlapping mutation patterns (624 SNVs shared between same-side pelvic and ureteric tumors in one case, and 191 shared between renal pelvic and bladder tumors in the other). Thus, ULC-WES provided evidence supporting the concept of tumor cell seeding along the urinary tract as a mechanism of TCC spread and recurrence. We conclude that ULC-WES is a sensitive and specific method to identify AA exposure in tumor tissue and is directly applicable to identification of new populations at risk. The method can ultimately facilitate evidence-based preventive measures in the affected regions. Acknowledgments: Genetic Services Platform, IARC; Genome Technology Center, NYU Langone Medical Center; NIH P01 ES04068, NIH Fogarty International Center 1R03TW007042, Croatian Ministry of Science. Citation Format: Xavier Castells, Sandra Karanovic, Magali Olivier, Maude Ardin, Florence Le Calvez-Kelm, Catherine Voegele, James McKay, Damir Dittrich, Karla Medak Tomic, Kathleen Dickman, Arthur P. Grollman, Bojan Jelakovic, Jiri Zavadil. Ultra-low coverage exome sequencing of FFPE tumor specimens identifies exposure to carcinogenic aristolochic acid. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2014-305

Transcriptome resources for the white‐footed mouse (Peromyscus leucopus): new genomic tools for investigating ecologically divergent urban and rural populations

Genomic resources are important and attainable for examining evolutionary change in divergent natural populations of nonmodel species. We utilized two next-generation sequencing (NGS) platforms, 454 and SOLiD 5500XL, to assemble low-coverage transcriptomes of the white-footed mouse (Peromyscus leucopus), a widespread and abundant native rodent in eastern North America. We sequenced liver mRNA transcripts from multiple individuals collected from urban populations in New York City and rural populations in undisturbed protected areas nearby and assembled a reference transcriptome using 1 080 065 954 SOLiD 5500XL (75 bp) reads and 3 052 640 454 GS FLX + reads. The reference contained 40 908 contigs with a N50 = 1044 bp and a total content of 30.06 Megabases (Mb). Contigs were annotated from Mus musculus (39.96% annotated) Uniprot databases. We identified 104 655 high-quality single nucleotide polymorphisms (SNPs) and 65 single sequence repeats (SSRs) with flanking primers. We also used normalized read counts to identify putative gene expression differences in 10 genes between populations. There were 19 contigs significantly differentially expressed in urban populations compared to rural populations, with gene function annotations generally related to the translation and modification of proteins and those involved in immune responses. The individual transcriptomes generated in this study will be used to investigate evolutionary responses to urbanization. The reference transcriptome provides a valuable resource for the scientific community using North American Peromyscus species as emerging model systems for ecological genetics and adaptation.

Platform comparison of detecting copy number variants with microarrays and whole-exome sequencing.

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders, Mendelian diseases, and common/complex traits. Genomic microarrays are often employed for CNV detection. More recently, whole-exome sequencing (WES) has enabled detection of clinically relevant point mutations and small insertion—deletion exome wide. We evaluated (de Ligt et al. 2013) [1] the utility of short-read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compared these results to data from three independent high-resolution microarray platforms. Calls made by the different platforms and detection software are available at dbVar under nstd84.

Deciphering intrafamilial phenotypic variability by exome sequencing in a Bardet–Biedl family

Bardet–Biedl syndrome (BBS) is a model ciliopathy characterized by a wide range of clinical variability. The heterogeneity of this condition is reflected in the number of underlying gene defects and the epistatic interactions between the proteins encoded. BBS is generally inherited in an autosomal recessive trait. However, in some families, mutations across different loci interact to modulate the expressivity of the phenotype. In order to investigate the magnitude of epistasis in one BBS family with remarkable intrafamilial phenotypic variability, we designed an exome sequencing–based approach using SOLID 5500xl platform. This strategy allowed the reliable detection of the primary causal mutations in our family consisting of two novel compound heterozygous mutations in McKusick–Kaufman syndrome (MKKS) gene (p.D90G and p.V396F). Additionally, exome sequencing enabled the detection of one novel heterozygous NPHP4 variant which is predicted to activate a cryptic acceptor splice site and is only present in the most severely affected patient. Here, we provide an exome sequencing analysis of a BBS family and show the potential utility of this tool, in combination with network analysis, to detect disease-causing mutations and second-site modifiers. Our data demonstrate how next-generation sequencing (NGS) can facilitate the dissection of epistatic phenomena, and shed light on the genetic basis of phenotypic variability.

Abstract 9444: Study of Natriuretic Peptides Levels in the Left Ventricular Tissue of Patients With Dilated Cardiomyopathy Using RNA-Seq

Background: Natriuretic peptides (NPs) levels have been extensively studied in plasma of heart failure (HF) patients. Currently, BNP and proBNP are used for diagnosis and prognosis. Our objective is to evaluate the tissue levels of these peptides and several molecules involved in their processing and biological activity at mRNA level, in the left ventricle of patients with dilated cardiomyopathy (DCM) undergoing heart transplantation. Methods: We analyze the mRNA levels of 14 left ventricular samples from patients with nonischemic DCM undergoing heart transplantation and 7 control subjects (CNT), using massive sequencing, RNAseq, (SOLID 5500XL) and RT-qPCR (ViiATM 7 Real-Time PCR System). Results: ANP and BNP tissue levels are elevated in DCM compared with CNT group (fold change (FC)=37, p&lt;0.01 and FC=26, p&lt;0.05, respectively). CNP was not detected by RNAseq in the left ventricular samples of patients with DCM or in CNT group. NPs receptors (NPR1, NPR2 and NPR3) show no significant differences in mRNA levels between the two study groups. Finally, regarding the enzymes involved in the cleavage of these peptides we observed that corin is significantly decreased in the DCM group compared to CNT (FC=-2.5, p&lt;0.01), while no significant changes are observed in furin. Conclusions: RNA-Seq analysis confirm the presence of ANP in adult ventricular tissue, suggesting that the elevations observed in plasma could be originated in part in the left ventricle. CNP was not detected in cardiac tissue. Changes are specific since there were no modifications in the levels of any of the receptors and only in one of the enzymes involved.

Stop-Codon Mutation In The Extracellular Part Of G-CSFR Leading To G-CSF Unresponsiveness Due To Defects In Erk1/2 Phopsphorylation In a Patient With Severe Congenital Neutropenia

We identified a 10 year old female severe congenital neutropnia patient who did not respond to the treatment with G-CSF (Filgrastim) up to 100 µg/kg/day, s.c. By search of possible reasons of G-CSF unresponsiveness, we detected by deep sequencing of the CSF3R (SOLID 5500xl, 3125 reads) a novel mutation within the extracellular part of CSF3R. The mutation was also confirmed by convential Sanger sequencing. It is a heterozygous stop-codon mutation C1641T (NM_000760), p.W547* (NP_000751) which is localized proximal to the cell membrane in the fifth fibronectin type III domain of the CSF3R. This is a first report describing a stop-codon mutation in the extracellular part of CSF3R in severe congenital neutropenia. FACS analysis revealed severely diminished surface expression levels of G-CSFR on CD33+ bone marrow myeloid progenitor cells of this patient as compared to G-CSFR levels on CD33+ cells of healthy individuals. In line with reduced expression of G-CSFR, we found disturbed G-CSFR-triggered intracellular signaling pathways activated by G-CSFR. MAPK/Erk1/2 activation by phosphorylation is known to be crucial for G-CSF- induced myeloid differentiation and STAT5 signaling is important for proliferation and survival of myeloid cells upon stimulation with G-CSF. We demonstrated abrogated phosphorylation of ERK1/2 and of STAT5 in bone marrow CD33+ myeloid cells of CN patient treated with G-CSF in vitro for 2, 5, 10 and 15 minutes, comparing with healthy donor. Since this patient has no known inherited CN-associated mutations (e.g. ELANE, HAX1, G6PC3), it may represent a new subgroup of CN patients characterized by non-responsiveness to G-CSF due to lack of binding of G-CSF to its receptor. Since the majority of investigations on CSF3R mutations are targeting the cytoplasmic part of the receptor only, we suggest that CN patients who do not respond to G-CSF treatment should be tested also for mutations within the extracellular part of the CSF3R. Disclosures:No relevant conflicts of interest to declare.

High Insta High Instability Of The CSF3R Gene In Severe Congenital Neutropenia Patients As Judged By Multiple CSF3R Mutations In The Majority Of Patients

▪It has been reported by us and others that approx. 30 % of all patients with congenital neutropenia (CN) acquire CSF3R mutations in their life time. More than 80 % of the CN patients who develop myeloid leukemia (CN/AML) harbour CSF3R mutations. This suggests that they are the first hit in leukemogenesis. However, detecting sequence changes e.g. by Sanger sequencing reveals only mutations presented in more than 20 % of the cells due to its technical detection limit. Therefore, we asked whether there is a systematic underestimation of cell clones harbouring CSF3R mutations, which might have been traditionally overlooked. We applied the deep-sequencing technology (SOLID 5500xl) to identify CSF3R mutations in myeloid cells from 158 patients with different types of neutropenia (86 severe congenital neutropenia (CN) patients with known inherited mutations (ELANE, HAX1, G6PC3), 21 cyclic neutropenia (CyN) patients, 28 patients with severe chronic neutropenia with so far unknown inherited mutations, 11 patients with SBDS-associated neutropenia) as well as a group of 12 healthy individuals. All neutropenia patients were treated with G-CSF and notably 21 CN patients developed leukemia or MDS. Deep sequencing data were processed according to our custom NGS pipeline (annotation of sequences and prediction of damaging effects on the coding sequence by Polyphen2, removal of known dbSNP variants, and accepting significant Phred-scores at the variant calling stage). Overall the read numbers ranged between 18 and 128069 (median 716), while only variants with at least two percent of the reads were accepted for further consideration (the statistically significant limit is between one and two percent of all reads). All together, we detected 92 CSF3R mutations in 42 CN patients leading to 49 distinct amino acid exchanges (38 missense and 11 stop-codon mutations). The frequency of the mutant alleles ranged from 2 to 96 %. In contrast, in CyN only five out of 21 patients harbour CSF3R mutations; interestingly, two of them in isoform IV of CSF3R (p.P752T). Most notably, whereas 18 patients displayed only one CSF3R mutation, 24 individuals had more than one CSF3R mutation (2-10 mutations, in total 74 mutations). During follow up of some patients, we could demonstrate that the number of mutations increased over time. The majority of mutations were located in the cytoplasmatic region (aa 651-831) of CSF3R, while 15 patients presented mutations within the extracellular region of CSF3R. Intriguingly, in 16 patients we detected 23 non-sense mutations, where 20 of these are stop-codon mutations affecting glutamine (Q) 768, 770, 776, and 781. This suggests that this part of CSF3R is highly instable. In two patients who did not respond to Filgrastim treatment, we detected a stop codon at aa 546 and 547, respectively, affecting the Fibronectin type-III like part of the CSF3R. Twelve patients who developed leukemia (CN/AML) had more than one CSF3R mutations (two to ten) , whereas eight with CN/AML harbored only one mutation.None of the healthy controls, only three neutropenia patients with unknown inheritance, and only one SBDS patient revealed mutations in CSF3R. Taken together, this data suggests that CSF3R is highly prone to genetic instability in severe congenital neutropenia, because more than one mutation in half of the patients was observed and various CSF3R mutations during the course of life accumulated. Once a cell clone harboring CSF3R mutation obtains a second hit (e.g. RUNX1 mutation), they are prone to undergo leukemic transformation. Disclosures:No relevant conflicts of interest to declare.

Detection of Clinically Relevant Copy Number Variants with Whole-Exome Sequencing

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders. This has resulted in the widespread application of genomic microarrays as a first-tier diagnostic tool for CNV detection. More recently, whole-exome sequencing (WES) has been proven successful for the detection of clinically relevant point mutations and small insertion-deletions exome wide. We evaluate the utility of short-read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compare these results to data from two independent high-resolution microarrays. Eleven of the 12 clinically relevant CNVs were detected via read-depth analysis of WES data; a heterozygous single-exon deletion remained undetected by all algorithms evaluated. Although the detection power of WES for small CNVs currently does not match that of high-resolution microarray platforms, we show that the majority (88%) of rare coding CNVs containing three or more exons are successfully identified by WES. These results show that the CNV detection resolution of WES is comparable to that of medium-resolution genomic microarrays commonly used as clinical assays. The combined detection of point mutations, indels, and CNVs makes WES a very attractive first-tier diagnostic test for genetically heterogeneous disorders.

Abstract 1509: Transcriptome analysis reveals significant effects of EGFR signaling on the secretome of mesenchymal stem cells.

Abstract Mesenchymal stem cell (MSCs) are recruited by developing tumors, where MSCs release factors that enhance tumor cell proliferation and migration. Since different carcinomas synthesize epidermal growth factor (EGF)-like peptides and MSCs express the EGF receptor (EGFR), we hypothesize that EGFR signaling might mediate the interaction between MSCs and tumor cells. In order to comprehensively assess the mechanisms and factors that are regulated by EGFR signaling in MSCs and are involved in the interaction between MSCs and tumor cells, we analysed the transcriptome of MSCs cultured in absence or in presence of transforming growth factor α (TGFα), one of the main ligands of the EGFR, by using massively parallel sequencing. Libraries were prepared from poly-A RNA from not-stimulated (MSC-CTRL) or TGFα-stimulated (MSC-TGFα) MSCs. Sequencing was performed on the SOLiD 5500xl platform using the paired-end protocol (75bp + 35bp). The experiments were repeated in quadruplicate, and the average number of reads obtained in the 4 replicates resulted of about 37 million for MSC-CTRL, and 47 million for MSC-TGFα. The quality analysis of raw data showed that 75% of reads at the 75th position had quality values above 14. The mapping of the sequence reads to human hg19 reference genome evidenced a coverage ranging between 79.5% and 81.7% for MSC-CTRL, and between 76.3% and 79.0% for MSC-TGFα. The number of observed counts was normalized for the length of the element and the number of mapped reads, namely RPKM (Reads Per Kilobase per Million of mapped reads). The RPKM values obtained in the 4 replicates were compared by ANOVA test and the very high p-value (of about 1) demonstrated that the replicates were not statistically different. The differential gene expression in MSC-CTRL and MSC-TGFα was represented as fold induction (FI). When selecting genes for which the average count value of the 4 replicates was equal or higher than 1, 8946 genes resulted to have FI&amp;gt;1 and 10305 genes FI&amp;lt;1 according to RPKM values. Next, we focused the attention on 274 genes coding for secreted proteins, including interleukins, chemokines, angiogenesis factors, growth factors of the EGF, IGF and FGF families and their receptors. Using as cutoff a count value equal or higher than 1, 47 genes were considered not expressed, 126 genes resulted to have FI&amp;gt;1.0, 91 genes FI&amp;lt;1.0 and 10 genes FI=1.0. Among the secreted proteins investigated, a number of angiogenic factors were significantly modulated in MSCs by TGFα treatment. Analysis of secreted proteins, performed with XMAP based assays, confirmed that activation of EGFR signaling induces in MSCs the secretion of several pro-angiogenic factors, including VEGF, IL6, IL8, HGF and G-CSF. These findings strongly suggest that the EGFR-mediated interaction between MSCs and tumor cells induces in MSCs the secretion of factors that play an important role in tumor progression. Citation Format: Antonella De Luca, Cristin Roma, Francesca Fenizia, Francesca Bergantino, Marianna Gallo, Daniela Frezzetti, Susan Costantini, Nicola Normanno. Transcriptome analysis reveals significant effects of EGFR signaling on the secretome of mesenchymal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2013-1509

Regulation of Gene Expression by Short Poly‐N‐Acetyl Glucosamine (sNAG) Nanofibers in Endothelial Cells and Keratinocytes

O‐GlcNAcylation is a catalyzed transfer of N‐acetylglucosamine to protein substrates. This modification aids cells in regulation of many crucial cellular processes. Short poly poly‐N‐acetyl glucosamine nanofibers (sNAG) enhance wound healing, by cell migration, and hemostasis, by promoting fibrin matrix and clot formation.This study investigated the effects of sNAG on global gene expression using the SOLiD 5500xl Next generation sequencing system in both HUVEC (Human Umbilical Vein Endothelial Cells) and HaCaT (Human keratinocytes) focussing on those having roles in angiogenesis and wound healing.In HUVEC cells, 1,705 genes had at least 2‐fold up‐or down‐regulation upon sNAG treatment. Genes which remained up‐regulated at both 1 and 6 hours of treatment were NFKBIZ, defensins and wound heaIing responses. Many of the collagen family of proteins, E‐Selectin, ICAM1, and cytokines, CXCL1 and CCL2, were up‐regulated transiently.In HaCaT cells, 2,329 genes showed 2‐fold up regulation in response to sNAG (1 or 6 hours) and 2,487 genes showed 2‐fold down regulation. Genes that were up‐regulated over the 6 hour treatment include desmogleins and keratins, suggesting regulation of keratinocyte differentiation and re‐epithelialization.Responses to sNAG observed in this study add support to its role in angiogenesis and wound healing pathways at the level of regulation of gene expression.

Palindromic sequence impedes sequencing-by-ligation mechanism

BackgroundCurrent next-generation sequencing (NGS) platforms adopt two types of sequencing mechanisms: by synthesis or by ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed.MethodsTo test the effect of the palindromic region on sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from MmeI digestion of the amplified clone to generate a sequencing library for SOLiD 5500xl sequencer.ResultsResults showed that, traditional ABI sequencers, which adopt sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region.ConclusionsTaken together, we demonstrate that SOLiD machines, which employ sequencing-by-ligation mechanism, are unable to read through the palindromic region. On the other hand, sequencing-by-synthesis sequencers had no difficulty in doing so.

Beyond mRNAs and Mirnas: Unraveling the Full-Spectrum of the Normal Human Platelet Transcriptome Through Next-Generation Sequencing

AbstractAbstract 3298The anucleate platelets play a critical role in the formation of thrombi and prevention of bleeding. While the repertoire of platelet transcripts is a reflection of the megakaryocyte at the time of platelet differentiation, post-transcriptional events are known to occur. Furthermore, a strong correlation between the expressed mRNAs and proteome has been identified. Having a complete understanding of the platelet transcriptome is important for generating insights into the genetic basis of platelet disease traits.To capture the complexity of the platelet transcriptome, we performed RNA sequencing (RNA-seq) in leukocyte-depleted platelets from 10 males, with median age of 24.5 yrs and unremarkable medical history. Their short and long RNA platelet transcriptomes were analyzed on the SOLiD 5500xl sequencing platform. We generated ∼3.5 billion sequence reads ∼40% of which could be mapped uniquely to the human genome.Our analysis revealed that ∼9,000 distinct protein-coding mRNAs and ∼800 microRNAs (miRNAs) were present in the transcriptome of each of the 10 sequenced individuals. Comparison of the levels of mRNA expression across the 10 individuals showed an exceptional level of consistency with pair-wise Pearson correlation values ≥0.98. The miRNA expression profiles across the 10 individuals showed a similar consistency with pair-wise Pearson correlation values ≥0.98.Surprisingly, we found that these mRNAs and miRNAs accounted for a little over 1/2 of all of the uniquely mapped sequence reads suggesting the abundant presence of additional non-protein coding RNA (ncRNA) transcripts. Using the annotated entries of the latest release of the ENSEMBL database, we investigated the genetic make-up of these other transcripts.We found that ∼25% of each individual’s uniquely mapped reads corresponded to non-protein coding transcripts from mRNA-coding loci. These reads accounted for more than 10,000 distinct such transcripts. In addition, each of the individuals in our cohort expressed an average of ∼1,500 pseudogenes and ∼200 long intergenic non-coding RNAs (lincRNAs).The short RNA profiles of the ten individuals revealed an abundance of diverse categories of ncRNAs including the signal recognition particle RNA (srpRNA), small nuclear RNA (snRNA) and small cytoplasmic RNAs (scRNA). These ncRNAs are involved in the processing of pre-mRNAs and their presence and prevalence in the anucleate platetet suggests the existence of a complex network of mRNA processing that persists after the megakaryocyte fragmentation.We also investigated the RNA-omes of the ten individuals for evidence of transcription of the pyknon category of ncRNAs. Pyknons are of particular interest because each has numerous intergenic and intronic copies whereas nearly all known human protein-coding genes contain one or more pyknons in their mRNA. Recent experimental work has shown that intergenic instances of the pyknons are transcribed in a tissue- and cell-state specific manner. An average of ∼100,000 pyknons are transcribed in each of the 10 sequenced individuals suggesting the possibility of a far-reaching network of interactions that link exonic space to distant non-exonic regions and are active in platelets.Lastly, we found that a large variety of distinct repeat element categories are expressed in the RNA-omes (both short and long) of these individuals. Among the most abundantly represented categories of repeat elements were DNA transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons such as long interspersed elements (LINEs) and short interspersed elements (SINEs).In summary, our RNA-seq analyses have revealed a spectrum of platelet transcripts that transcends protein-coding genes and miRNAs. Indeed, the transcripts that have their source in genomic features not previously discussed or analyzed in the platelet context represent a very significant portion of all platelet transcripts. This in turn suggests an unanticipated richness, and presumably commensurate complexity, for the platelet transcriptome. While the role of these novel non-protein coding RNAs is currently unknown it is expected that at least some of them may be of functional significance which will in turn permit a better understanding of the molecular mechanisms that regulate platelet physiology and may contribute to processes beyond thrombosis and hemostasis. Disclosures:No relevant conflicts of interest to declare.