No abstract

<p>The cofactor of BRCA1 (COBRA1), which also refers to negative elongation factor polypeptide B (NELF-B), is a negative elongation factor (NELF) subunit that has been implicated in the development and progression of several cancers. While reduced COBRA1 expression has been associated with metastatic breast cancer, COBRA1 negatively regulates the activator protein-1 (AP-1) complex, leading to the down-regulation of trefoil factor-1 (TFF1) expression in gastric cancer cell lines. The involvement of COBRA1 in hepatocellular carcinoma (HCC) tumor formation and progression is not known. Here, we investigated the expression of COBRA1, the AP-1 complex, and TFF1 in several HCC cell lines; ranging from low- to high-grade HCC cell lines generated from patients with different stages of the disease. Our results showed that the COBRA1 protein was highly expressed in the low-grade HCC cell line, while significantly down-regulated in high-grade HCC cell lines. TFF1, previously regarded as a COBRA1 target gene, was only expressed in the low-grade HCC cell line and the control cells. Our results suggest that COBRA1 may play a role in HCC pathogenesis and progression. The TFF1 mRNA expression profile was uncorrelated to that of the AP-1 complex subunit proteins, which suggests the involvement of other regulatory pathways in TFF1 expression; however, this requires further study.</p>

Introduction: Acute myelogenous leukemia (AML) is a fatal disease with dismal outcomes in which most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and can thereby improve clinical decision-making. NPM1 exon 12 frameshift mutations (NPM1mut) are found in ~50% of cytogenetically normal AML and represent a well-established molecular MRD marker typically assessed by RQ-PCR. While assays are readily available for the most common NPM1mut, detection is complicated by hundreds of potential frameshift insertions, reports of NPM1mut type-switching, and a lack of sequence data in patients diagnosed for NPM1mut by capillary electrophoresis. To simplify the deployment and increase the robustness of NPM1mut MRD detection, we developed a digital droplet PCR assay comprised of multiplex primer pools capable of detecting >95% of NPM1mut subtypes without requiring prior knowledge of NPM1 sequence. To assess its potential in patient care, we tested sensitivity and concordance with established type-specific assays and its robustness across different NPM1mut types. We further demonstrate its use in patients lacking NPM1mut sequence information. Results: Detection of 200+ subtypes was evaluated. The assay demonstrated excellent concordance with subtype-specific assays (ρc = 0.97-0.99) as determined by testing in patient samples, cell lines, and synthetic cDNA bearing rare single and multiplex NPM1mut. OCI-AML3/MV-4-11 dilution experiments revealed sensitivity comparable with standard assays (1-2 x 10-5) while retaining specificity for mutant NPM1. Multiplex primer pools produced lower coefficient of variation across digital PCR partitions relative to single primers containing deoxyinosine or 5-nitroindole. To illustrate practical implementation where NPM1mut levels could not otherwise be monitored, we performed longitudinal retrospective MRD analysis of a patient who presented to our center without NPM1mut sequence data having been diagnosed for NPM1mut by capillary electrophoresis at an outside clinic. Monitoring was performed over the course of 356 days. To determine the actual NPM1 subtype, we performed ultra-deep targeted mRNA-seq during a transient spike in NPM1mut levels during remission at day 28. We identified 48/14,271 reads (0.34%) supporting the NPM1 subtype as type D (c.863_864insCCTG) with the incidental finding of IDH1-R132H mutation previously unknown in this patient. Subsequent re-evaluation with the subtype-specific assay (type D) demonstrated agreement with the massively multiplex assay. Steady increases in NPM1mut/104 ABL1 ratios were observed to peak at day 196 when the patient presented 22% blasts in the bone marrow and >28,000 NPM1mut/104ABL1 and re-entered remission with subsequent care. Targeted next generation sequencing at this time further corroborated the NPM1 type D and IDH1-R132H mutation. Conclusions: The new assay demonstrates sensitive and robust quantification of MRD in a variety of NPM1+ AML. Patients lacking in NPM1 sequence information or who harbor rare subtypes with current standard method would greatly benefit from the assay. Moreover, deployment of the NPM1 MRD testing is simplified by covering >95% of NPM1 mutated patients in a single test without requiring plasmid standards or custom assays. Additionally, we show that MRD measurements with the multiplex assay can be used to guide the timing of deep sequencing to capture potential information about imminent relapse and to facilitate intervention at earlier stages in the disease. Disclosures Ritchie: Celgene: Speakers Bureau; Pfizer: Honoraria; Novartis: Honoraria; Arian: Speakers Bureau; Incyte: Speakers Bureau. Guzman:Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy.

Background: Microsatellite markers are highly versatile tools used in a wide array of applications including paternity testing, forensic analysis, and disease markers. However, the allele frequencies of these markers can vary greatly depending on ethnic subpopulations. Thus, the discovery of three distinct Qatari ethnic subpopulations (Bedouin, Persian, and African) has prompted the need to determine the allele frequencies that are more representative of the subpopulations. Objectives: This study aims to analyze 15 autosomal microsatellite loci from individuals belonging to each subpopulation and to generate the corresponding allele frequency tables. Additionally, the microsatellite markers were further investigated to examine the mating behaviors within each subpopulation as well as looking into a possible relationship between the markers and the type 2 diabetes mellitus (T2DM). Methods: DNA samples isolated from 266 individuals of interest (Bedouin, Persian, or African) were amplified using PCR and subjected to electrophoresis. The results were analyzed using various population genetics software such as FSTAT, GenAlEx, and GENEPOP to calculate several population parameters including inbreeding coefficients, allele frequencies, and expected and observed heterozygosity. Results: The results showed that the Bedouin subpopulation was distinct from the other two subpopulations. Furthermore, the Bedouin and African subpopulations were found to have a small positive inbreeding coefficient, while the Persian subpopulation displayed a slight outbreeding pattern. Lastly, two microsatellite loci showed tantalizing differences between diabetic and non-diabetic participants. Conclusions: The distinctness of the Bedouin population could be attributable to the heterogeneity of the Persian and African subpopulations, while the positive inbreeding coefficients could be explained by the relatively high rates of consanguineous marriages. The allele frequency tables generated for each subpopulation through this study can be used to strengthen microsatellite-based genetic testing; however, more studies are needed to further examine the potential relationship between T2DM and microsatellites.

Microsatellites are segments of the DNA comprised of repeated sequences of 4 to 8-base pair units that are found throughout the genome of eukaryotes. Most microsatellites are located at non-coding regions of the genome and consequently mutations in the microsatellite regions are often not causatives of disease. This allows these regions to be highly polymorphic in a population and gives a signature DNA marker for each individual. At the same time, it is often expected to see a wide genetic diversity of alleles present in the populations. In humans, microsatellites, or short tandem repeats (STRs) are standard genetic markers used for human identification in forensic cases and parentage determination.Databases of allele frequencies from various ethnic groups have been established in various parts of the world. In Qatar, as close-kin marriages are customary, homozygosity and possible reduced genetic variability have been a concern. A previous study, however, has concluded that the standard forensic markers are a valid tool for human identification because no substantive reduction of genetic variation has been observed as a result of consanguinity in the Qatari community.In a more recent study, it has been determined that the Qatari population is subdivided into three main ethnic groups, of Bedouin, African or Persian ancestry. This segregation has been genetically significant through studies in single nucleotide polymorphism, or SNP studies. Since SNPs and microsatellite DNA are inherited in a similar fashion, it is expected that there are different allele frequencies for assessed microsatellite loci for each of the populations. Moreover, the allelic heterogeneity in a population is closely linked to interbreeding. Since the Qatari Bedouin population has been closely associated with the practice of consanguinity as evidenced through SNP studies, it is therefore also expected to see higher homozygosity in the Bedouin subpopulation as compared to the other two subpopulations.In recent years diabetes occurrences in the Qatari populations have reached an epidemic level. Like many other diseases where both lifestyle as well as genetics may play a role in the onset of this disease, the microsatellite loci may serve as markers genetically linked to some of the non-communicable disease such as diabetes.The main aim of this study is to understand the genetic variability across the subpopulations of the Qatari nationals. The result can be used to develop new forms of personalized health care that is specific to members of the stratified Qatari subpopulations. The information allows for more efficient treatments and better management to the growing Qatari populations. To accomplish these goals, blood samples are collected from 300 individuals, with 100 from each subpopulation. AmpFISTR® Identifiler® Plus PCR Amplification Kit is used for a multiplex analysis of 15 tetranucleotide loci. The resulting data are analyzed to produce allele frequencies of each of the loci for the corresponding subpopulations. The gene diversity within and among the subpopulations are analyzed and the detection of consanguinity through the application of Hardy-Weinberg is discussed. The sub-profiles for each of the three Qatari subpopulations – Bedouin, African and Persian – are presented. Finally, the concept of personalized health care with respect to diabetes is introduced and the clinical applications relevant to the populations are discussed.