A refined method to study gene dosage changes in-vitro using CRISPR/Cas9

Aims: Gene dosage can have a major impact on cell biology although, hitherto, is has been difficult to study using in-vitro models. We sought to refine and accelerate the development of “gene dosage” models through using CRISPR/Cas9 (a gene-editing technology) for sequential knockout of gene alleles. Methods: Our method involved (i) using Cas9 nuclease mRNA rather than expression plasmids, (ii) using a fluorescently labelled FAM-6 tracr complexed with guide RNA and (iii) using High Resolution Melting (HRM) analysis to screen for mutations. HCT116 cells, wild-type for TP53, were transfected with different molarities of FAM-6 tracr labelled and guide RNA targeting different exons of TP53 and selected by Fluorescence Associated Cell Sorting (FACS). Single-cell colonies were then isolated, expanded and tested for mutation in the targeted region by PCR/HRM. Results: Out of 32 clones tested, 12 have shown aberrant melting by HRM, giving a targetting efficiency of 37.5%. One clone was sequenced and a heterozygous mutation found - in this case comprising a single base deletion in exon 3. mRNA sequencing confirmed the mutation was expressed and western blotting for p53 showed the presence of both wild type and truncated protein bands. Changes in expression of MDM-2 isoforms suggested a functional effect of the induced TP53 mutation. Conclusions: We have developed an in-vitro model to study TP53 gene dosage effects. The protocol is efficient and applicable to any gene. Importantly, we have used Cas9 mRNA and labelled tracr/guideRNA to isolate likely-mutated cells and HRM for rapid mutation detection

Clinicopathological and molecular characteristics of Ku 70/80 expression in Nigerian breast cancer and its potential therapeutic implications

Ku 70/80 is a regulator of the Non-Homologous End Joining (NHEJ) roles in clinicopathological features, and has prognostic significance in breast cancer (BC) in Caucasian populations. However, its significance in the Nigerian BC population, which is characterized by a higher rate of the triple-negative and basal phenotype, p53 mutation rate and BRCA1 deficiency, still needs to be investigated. We hypothesize that Ku70/80 expression shows adverse expression in Nigerian BC and, furthermore, that it is likely to have a therapeutic implication for Black BC management.This study investigated the biological, clinicopathological and prognostic significance of Ku 70/80 expression in a BC cohort from a Nigerian population. Ku 70/80 expression was determined in 188 well-characterized formalin-fixed, paraffin-embedded (FFPE) BC samples using tissue microarray and immunohistochemistry. Ku 70/80 expression was correlated with clinicopathological, molecular and prognostic characteristics of patients.Ku 70/80 was expressed in 113 (60.1%) tumors, and was positively associated with metastatic disease, triple-negative and basal phenotype, BRCA1 down regulators (MTA-1 and ID4), p-cadherin, PI3KCA and p53 expression. It inversely correlated with BRCA1, BRCA2, BARD1 and p27. Ku 70/80 was predictive of breast cancer-specific survival in multivariate analysis, but not of disease-free interval.This study demonstrated that Ku 70/80 expression is associated with triple negativity and down-regulation of the homologous recombination pathway of DNA repair. Therefore, the development of novel drugs to target KU70/80 may improve the patients’ outcome in the treatment of Black BC

N_LyST: a simple and rapid screening test for Lynch Syndrome

Aims: We sought to use PCR followed by high-resolution melting (HRM) analysis to develop a single closed-tube screening panel to screen for Lynch Syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation.Methods:For MSI-testing, 5 mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n = 99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS). Results:All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation whilst 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases. Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, 5 were identified as MSS, 4 as MSI/non-LS and 3 as MSI/possible LS. These results were concordant with the previous data.Conclusion: We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick simple cheap method for screening for Lynch Syndrome

Semantic annotation for computational pathology : multidisciplinary experience and best practice recommendations

Recent advances in whole‐slide imaging (WSI) technology have led to the development of a myriad of computer vision and artificial intelligence‐based diagnostic, prognostic, and predictive algorithms. Computational Pathology (CPath) offers an integrated solution to utilise information embedded in pathology WSIs beyond what can be obtained through visual assessment. For automated analysis of WSIs and validation of machine learning (ML) models, annotations at the slide, tissue, and cellular levels are required. The annotation of important visual constructs in pathology images is an important component of CPath projects. Improper annotations can result in algorithms that are hard to interpret and can potentially produce inaccurate and inconsistent results. Despite the crucial role of annotations in CPath projects, there are no well‐defined guidelines or best practices on how annotations should be carried out. In this paper, we address this shortcoming by presenting the experience and best practices acquired during the execution of a large‐scale annotation exercise involving a multidisciplinary team of pathologists, ML experts, and researchers as part of the Pathology image data Lake for Analytics, Knowledge and Education (PathLAKE) consortium. We present a real‐world case study along with examples of different types of annotations, diagnostic algorithm, annotation data dictionary, and annotation constructs. The analyses reported in this work highlight best practice recommendations that can be used as annotation guidelines over the lifecycle of a CPath project

Prevalence, Predictive Factors, and Characteristics of Osteoporosis in Hyperthyroid Patients

Objective. The osteoporosis in thyroid disorder has the lowest report especially in sub-Saharan Africa. This study aims to determine the prevalence, predictive factors, and characteristics of osteoporosis in hyperthyroid patients. Method. Forty (40) hyperthyroid patients and healthy controls ages 21–50 years were recruited in this study. Questionnaires were administered to capture bio- and clinical data. Biochemical tests included blood, thyroid functions, intact parathyroid hormone, corrected calcium, and 25-hydroxyvitamin D tests. Bone mineral density (BMD) was also evaluated. Data were analyzed using the SPSS 21. A p value < 0.05 was regarded as significant. Results. Osteoporosis was observed in 18 (45%) of study subjects, 13 (72.2%) females and 5 (27.8%) males, respectively. The BMD of the hyperthyroid patients had a negative correlation with free triiodothyronine, FT3 (r=−0.49, p=0.005), FT4 (r=−0.33, p=0.009), corrected calcium (r=−0.31, p=0.039), alkaline phosphatase (r=−0.53, p<0.001), and osteocalcin (r=−0.61, p<0.001). Conversely, a positive association with thyroid-stimulating hormone (TSH) (r=0.54, p<0.001) was observed. Multiple regression showed osteocalcin (p<0.001) and TSH (p=0.015) as independent predictors of osteoporosis. Conclusion. Thyrotoxicosis is a risk factor for osteoporosis occurrence, and we recommend routine screening for this bone disease in persons over 20 years old with this disorder

Targeted next generation sequencing reveals a common genetic pathway for colorectal cancers with chromosomal instability and those with microsatellite and chromosome stability

Introduction Microsatellite stable sporadic colorectal cancers (CRCs) can be classified as either tumours with chromosomal instability (CIN+) or tumours that are ‘Microsatellite and Chromosomal Stable’ (MACS). The CIN + tumours are aneuploid whilst MACS are near-diploid; little else is known about their differences. We compared the mutation profiles of CIN + and MACS CRCs. Method Targeted Next Generation Sequencing for mutation in 26 driver genes (TruSight-26 kit) was undertaken in 46 CIN + and 35 MACSCRCs. Tumours were compared for mutation frequency, allelic imbalance and clonal heterogeneity. Results Mutations were detected in 58% genes and, overall, mutation in driver genes was at expected frequencies. Comparison of classes revealed similar mutation frequencies in most genes and allelic imbalance atAPC and TP53. Differences were seen in mutation frequency in KRAS (41% CIN+ vs 68% MACS, p = 0.015) and GNAS (0% CIN+ vs 12% MACS, p = 0.032). Twenty percent CIN + CRCs harboured mutations only in TP53 - a profile not seen in the MACS tumours (p = 0.009). None of the differences were significant after multiple testing corrections. Conclusions The mutation profiles of CIN and MACS CRCs are similar. The events allowing aneuploidy (or forcing retention of diploidy) remain unknown