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Background: Multiple pterygium syndrome (MPS) is a genetically heterogeneous rare form of arthrogryposis multiplex congenita characterized by joint contractures and webbing or pterygia, as well as distinctive facial features related to diminished fetal movement. It is divided into prenatally lethal (LMPS, MIM253290) and nonlethal (Escobar variant MPS, MIM 265000) types. Developmental spine deformities are common, may present early and progress rapidly, requiring regular fo llow-up and orthopedic management. Methods: Retrospective chart review and prospective data collection were conducted at three hospital centers. Molecular diagnosis was confirmed with whole exome or whole genome sequencing. Results: This case series describes the clinical features and scoliosis treatment on 12 patients from 11 unrelated families. A molecular diagnosis was confirmed in seven, two with MYH3 variants and five with CHRNG. Scoliosis was present in all but our youngest patient. The remaining 11 patients spanned the spectrum between mild (curve ≤ 25°) and malignant scoliosis (≥50° curve before 4 years of age), the two patients with MYH3 mutations presented with malignant scoliosis. Bracing and serial spine casting appear to be beneficial for a few years, non-fusion spinal instrumentation may be needed to modulate more severe curves during growth and spontaneous spine fusions may occur in those cases. Conclusions: Molecular diagnosis and careful monitoring of the spine is needed in children with MPS.

Arthrogryposis multiplex congenita (AMC) describes a group of conditions characterized by the presence of non-progressive congenital contractures in multiple body areas. Scoliosis, defined as a coronal plane spine curvature of ≥10 degrees as measured radiographically, has been reported to occur in approximately 20% of children with AMC. To identify genes that are associated with both scoliosis as a clinical outcome and AMC, we first queried the DECIPHER database for copy number variations (CNVs). Upon query, we identified only two patients with both AMC and scoliosis (AMC-SC). The first patient contained CNVs in three genes (FBN2, MGF10, and PITX1), while the second case had a CNV in ZC4H2. Looking into small variants, using a combination of Human Phenotype Ontogeny and literature searching, 908 genes linked with scoliosis and 444 genes linked with AMC were identified. From these lists, 227 genes were associated with AMC-SC. Ingenuity Pathway Analysis (IPA) was performed on the final gene list to gain insight into the functional interactions of genes and various categories. To summarize, this group of genes encompasses a diverse group of cellular functions including transcription regulation, transmembrane receptor, growth factor, and ion channels. These results provide a focal point for further research using genomics and animal models to facilitate the identification of prognostic factors and therapeutic targets for AMC.

SummaryLeukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

A major challenge in the comprehension of the endometrial transformations leading to the completion of each menstrual cycle in humans is in the identification of specific molecular pathways underlying these monthly turnovers. Towards this goal we compared, by the differential display technique, the relative expression of mRNA in endometrial biopsies harvested in individuals (n = 48) either at the proliferative or the secretory phase of the menstrual cycle. We isolated a cDNA fragment homologous to NDRG1 (N-myc Downstream-Regulated Gene-1) that is present in markedly higher amounts in the secretory phase. Northern blot analysis and quantitative real time PCR experiments confirmed this result in distinct cohorts of individuals (44 and 560 respectively). A closer examination of data showed that the highest mRNA levels were found during the range of 25-28 days of the uterine cycle. Consistent with the mRNA data, the temporal profile of the NDRG1 protein showed a 15-fold increase during the secretory phase, as demonstrated by using semi-quantitative dot blot analyses (n = 92). Immunohistochemical localization revealed that NDRG1 was expressed both in epithelial and stromal cells. This large scale validation of the NDRG1 mRNA and protein increase in endometrium during the secretory phase is consistent with its differentiation-related function described in other tissues and its potential involvement in the window of implantation of the human endometrium, as suggested by previous chip-based evidence.

Objective To determine whether the proportion of several leukocyte subsets is modulated in the endometrium of patients with endometriosis and, if yes, whether it can be used for diagnostic purposes. Design: Case-control study. Setting: Eight clinical institutions of the Montreal area. Patient(s): Women who underwent laparoscopy or laparotomy between 1997 and 2001, who had regular menstrual cycles and were not under hormone treatment for the previous 3 months were selected. This study included 368 women, 173 with surgically confirmed endometriosis and 195 controls with no surgical evidence of endometriosis. Intervention(s): Main outcome measure(s): Cytometry analysis was used to measure the proportion of several leukocyte subsets among CD45 + endometrial cells. Result(s): The proportion of CD3 + , CD16 + , CD3 − HLADR − , CD3 - CD45RA − , CD3 + CD16 − , CD3 + CD56 − , CD56 − CD16 + , and CD16b + leukocytes was significantly altered in the endometrium of cases compared with controls. A multiple logistic regression model was adjusted with these endometrial leukocytes, serum CA-125 levels, risk factors, and confounders. The diagnostic performance of this predictive model was defined by a specificity of 95% and a sensitivity of 61%. Furthermore, the positive and negative predictive values were 91% and 75%, respectively. Conclusion(s) This predictive model represents a novel diagnostic tool to identify women with a high likelihood of suffering from endometriosis.

We have identified a human gene encoding a 48-kDa protein that specifically interacts with the peptidyl prolyl isomerase FK506-binding protein 59 (FKBP59) and also with the well known FKBP12. FKBP59 and FKBP12 belong to the large family of immunophilins that bind the macrolide immunosuppressant drugs FK506 and rapamycin. The yeast two-hybrid system was used to isolate target proteins that interact with the immunosuppressant drug binding domain of the rabbit FKBP59. The cDNA for an as yet unidentified protein was isolated and cloned from a Jurkat cell library. The cDNA sequence of 1804 base pairs reveals an open reading frame of 417 amino acids. In vitro experiments suggest a direct interaction between FKBP59 and this new target protein. This specific association seems to be restricted to the FKBP family, since it also occurs both in vivo and in vitro with FKBP12 but not with cyclophilin 40. This novel protein was named FKBP-associated protein (FAP48). The formation of the complexes between FKBP59 or FKBP12 and FAP48 is prevented by FK506 and rapamycin in a dose-dependent manner. These results suggest that FAP48 shares or overlaps the macrolide binding site on FKBP59 as well as on FKBP12 and therefore may represent a natural common ligand of these immunosuppressant drug receptors.

Cancer is a genetic disease, caused by a variety of alterations to a cell's genome. One specific type of alteration associated with cancer is point mutations, and Next-generation sequencing (NGS) is a key tool to identify those mutations. Unlike inherited mutations, cancer mutations can be found at very low frequencies, mainly because of tumor heterogeneity and their dilution in the germline background. Cell-free DNA (cfDNA) derived from blood plasma, is a promising material for cancer research and diagnosis, and can be readily obtained via non-invasive procedures. However, the amount of tumor DNA and, hence, the amount of rare allele in the cfDNA are typically very low. Sensitive methods are therefore needed for the detection of somatic, cancer-associated mutations in cfDNA isolated from liquid biopsies to support both discovery and clinical decision making in oncology. To overcome these challenges, we developed at Rubicon Genomics an NGS library preparation kit, namely ThruPLEX® Plasma-seq, optimized specifically for cfDNA at input levels from less than 1 ng to 30 ng. We designed experiments to determine the lower limit of SNV detection under these assay conditions. Six individual ThruPLEX® Plasma-seq libraries generated from 10 ng cfDNA were enriched with the Agilent SureSelect XT2 reagents and ClearSeq Comprehensive Cancer panel (151 genes). After sequencing to >500X coverage, SNP calling was performed using Agilent SureCall software to identify the SNPs specific to each sample. In a second step, the cfDNA isolated from two of these plasma samples with known SNP differences were mixed to generate various spike-in frequencies (10%, 5%, 2% and 0.5%) prior to preparing libraries and enrichment. This is to simulate low allele frequency, and to determine the sensitivity of ThruPLEX® Plasma-seq under the outlined assay conditions. Our results showed that it was possible to detect specific alleles down to an actual 0.6% with the theoretical 0.5% spike-in ratio. The percent of on­target bases was over 45%. The sensitivity (SNPs detected/total SNPs analyzed) of our test was above 97% for 10%, 5%, and 2% frequencies and 70% for 0.5%. This is a demonstration of ThruPLEX® Plasma-seq to produce libraries with high enough complexity to detect low frequency alleles from limited amounts of cfDNA from plasma. Citation Format: Kamran Shazand, Jing Ning, Anthony Popkie, Egon Ranghini, John Paul Jerome. High efficiency detection of low frequency alleles in cell-free DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3621.

We reported previously the cloning and sequence of the Bacillus subtilis infB gene which encodes the essential IF2 factor required for initiation of translation (K. Shazand, J. Tucker, R. Chiang, K. Stansmore, H. U. Sperling-Petersen, M. Grunberg-Manago, J. C. Rabinowitz, and T. Leighton, J. Bacteriol. 172:2675-2687, 1990). The location of the 5' border of the infB operon was investigated by using integrative plasmids carrying various DNA fragments from the region upstream of the infB gene. The lethal effect of disruption of the infB transcriptional unit could be suppressed when the integrated plasmid introduced the spac promoter upstream of the infB operon and transformants were selected in conditions of induction of spac expression. Such an integrated plasmid was used as a starting point to clone the promoter of the infB operon. Primer extension mapping suggests that a single sigma A-type promoter controls transcription of the infB operon. The sequence of a 5,760-bp region encompassing the infB gene was determined. The infB operon is located immediately downstream of the polC gene and comprises seven open reading frames, four of which appear to be the homologs of genes present in the same order in the Escherichia coli infB operon, including nusA. The striking similarity between the E. coli and B. subtilis infB operons suggests that the function of each gene pair is conserved and that the B. subtilis NusA homolog, which is 124 residues shorter than its E. coli counterpart, could play a role similar to its role in E. coli.

Endometriosis is a polygenic gynaecological condition affecting 5-15% of women of childbearing age. Major symptoms of the disease are pelvic pain and infertility. No clear link has been established between symptoms and the stage of the disease. Although some aspects have begun to be clarified, clinical understanding of endometriosis remains partial at the molecular level. In this perspective, we targeted isolation of differentially expressed genes in the eutopic endometrial tissue. Our assumption was that the endometrial cells of patients presented an unusual gene expression profile, allowing their implantation and survival in an ectopic site, leading to endometriotic lesions. Here, we report that mRNA steady-state levels of two key transcription factors are modulated in endometriosis. FOXO1 (also known as FKHR) levels were 1.6-fold lower in endometriosis compared to the control group at the onset of the secretory phase (day 15-21), while c-jun mRNA was present at higher amounts in endometriosis (1.5-fold) at the proliferative phase of the menstrual cycle. These results were derived from a large sample composed of 157 control subjects and 209 patients with endometriosis. Gene profiling was conducted by real-time quantitative PCR, and data were quality controlled before statistical analysis. Whether protein levels are affected as well remains to be investigated.

Abstract 3776 Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.

Objective To determine whether blood leukocyte populations could be modulated in endometriosis. Design Case-control study. Setting Eight clinical institutions of the Montreal area. Patient(s) Women with regular menstrual cycles who underwent laparoscopy or laparotomy between 1997 and 2001 and who were not under hormonal treatment for at least 3 months were selected. This study includes 175 cases and 131 controls. Main outcome measure(s) The proportion of blood leukocytes expressing markers for T, B lymphocytes, monocytes, or natural killer (NK) cells were compared by flow cytometric analysis between cases and controls. Result(s) Age and parity were identified as important confounders. Given that smoking, history of acute infection, and previous use of oral contraceptives strongly correlate with the level of some blood leukocyte populations, these parameters were taken into account in addition with age and parity when the level of blood leukocyte subsets were evaluated in cases and controls. Blood monocytes expressing CD14 and CD44 molecules were increased in patients with endometriosis. Alternatively, B lymphocytes were shown to be significantly decreased in cases compared with controls. Conclusion(s) Although these results suggest that endometriosis is associated with some systemic manifestations, the exact role of these modulations remains unclear.

Development in Bacillus subtilis involves the formation of two cell types with activation of the transcription factors sigma F in the forespore and sigma E in the mother cell. Activation of sigma E is due to the processing of the inactive precursor pro-sigma E, which requires the putative protease SpoIIGA and the presence of active sigma F. We have introduced missense mutations altering the promoter recognition properties of sigma F. These mutations abolish pro-sigma E processing, suggesting that sigma F is involved through its transcriptional activity and that the processing machinery responds to a signal generated by the product(s) of some unidentified gene(s) transcribed in the forespore. The role of the septum in transducing this signal was investigated. Induction of sigma F during exponential growth in cells producing SpoIIGA and pro-sigma E led to a high level of processing and sigma E activity. Moreover, pro-sigma E was efficiently processed in a mutant strain blocked prior to septation and synthesizing sigma F in active form at the onset of sporulation. Therefore, the sporulation septum is not required for induction of pro-sigma E processing and pro-sigma E can be processed in the same cell in which sigma F is active. These results suggest that some unknown mechanism must exist to prevent sigma E from becoming active in the forespore.

Abstract 2735 Leukemia stem cells (LSC) play a crucial role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI), such as dasatinib, can eradicate the majority of CML cells, they frequently fail to eliminate the dormant, niche-resident LSC that are hypothesized to drive CML relapse. Cumulative evidence from CML cell lines and CD34+ primary patient cells suggests that increased expression of pro-survival BCL2 family members contributes to TKI resistance and CML progression. However there is a relative dearth of data on BCL2 family expression in primary CML LSC and on the role of these proteins in TKI resistance in selective niches. Full transcriptome RNA sequencing revealed that LSC switch from pro-apoptotic to pro-survival BCL2 family member splice isoform expression during progression from chronic phase to blast crisis CML. Using splice isoform-specific qRT-PCR, we identified overrepresentation of long (pro-survival) compared with short (pro-apoptotic) MCL1, BCLX, and BCL2 isoforms in blast crisis LSC compared with chronic phase and normal progenitors. Following intrahepatic transplantation of blast crisis LSC into neonatal RAG2−/−gc−/− mice, LSC engrafted in the marrow niche were quiescent, were dasatinib resistant and upregulated BCL2 expression. These data led us to speculate that inhibition of BCL2 in dasatinib-resistant LSC may sensitize LSC to TKI therapy. Treatment with a high-potency, novel pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptosis along with a decrease in the frequency of leukemic progenitors compared to vehicle treated controls. Normal human cord blood progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately five times higher than that for blast crisis CML cells (210 nM versus 43 nM). Moreover, sabutoclax treatment did not inhibit cord blood colony formation or colony replating in vitro. Treatment of CML LSC-transplanted mice with sabutoclax led to a significant reduction in LSC burden in all hematopoietic organs analyzed. Sabutoclax treatment in vivo also sensitized surviving bone marrow blast crisis LSC to dasatinib treatment ex vivo. Importantly, there was no reduction in normal progenitor engraftment in bone marrow following sabutoclax treatment. These results demonstrate that marrow niche blast crisis CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan, BCL2 antagonist, sabutoclax, at doses that spare normal hematopoietic progenitors. While BCL2 splice isform switching promotes LSC survival and TKI resistance, pan-BCL2 family member inhibition with sabutoclax eliminates LSC and may form the cornerstone of a clinical strategy to avert cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Background ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo “library preparation” to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. Results In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a “gold standard” reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. Conclusions We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3135-y) contains supplementary material, which is available to authorized users.

International audience; The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumors from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of over 25,000 cancer genomes at the genomic, epigenomic, and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically-relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.