Genetic Mutation Analysis Research Articles

An amino acid metabolism-based seventeen-gene signature correlates with the clinical outcome and immune features in pancreatic cancer.

Background: Pancreatic cancer is an aggressive tumor with a low 5-year survival rate and primary resistance to most therapy. Amino acid (AA) metabolism is highly correlated with tumor growth, crucial to the aggressive biological behavior of pancreatic cancer; nevertheless, the comprehensive predictive significance of genes that regulate AA metabolism in pancreatic cancer remains unknown. Methods: The mRNA expression data downloaded from The Cancer Genome Atlas (TCGA) were derived as the training cohort, and the GSE57495 cohort from Gene Expression Omnibus (GEO) database was applied as the validation cohort. Random survival forest (RSF) and the least absolute shrinkage and selection operator (LASSO) regression analysis were employed to screen genes and construct an AA metabolism-related risk signature (AMRS). Kaplan-Meier analysis and receiver operating characteristic (ROC) curve were performed to assess the prognostic value of AMRS. We performed genomic alteration analysis and explored the difference in tumor microenvironment (TME) landscape associated with KRAS and TP53 mutation in both high- and low-AMRS groups. Subsequently, the relationships between AMRS and immunotherapy and chemotherapy sensitivity were evaluated. Results: A 17-gene AA metabolism-related risk model in the TCGA cohort was constructed according to RSF and LASSO. After stratifying patients into high- and low-AMRS groups based on the optimal cut-off value, we found that high-AMRS patients had worse overall survival (OS) in the training cohort (a median OS: 13.1months vs. 50.1 months, p < 0.0001) and validation cohort (a median OS: 16.2 vs. 30.5 months, p = 1e-04). Genetic mutation analysis revealed that KRAS and TP53 were significantly more mutated in high-AMRS group, and patients with KRAS and TP53 alterations had significantly higher risk scores than those without. Based on the analysis of TME, low-AMRS group displayed significantly higher immune score and more enrichment of T Cell CD8+ cells. In addition, high-AMRS-group exhibited higher TMB and significantly lower tumor immune dysfunction and exclusion (TIDE) score and T Cells dysfunction score, which suggested a higher sensitive to immunotherapy. Moreover, high-AMRS group was also more sensitive to paclitaxel, cisplatin, and docetaxel. Conclusion: Overall, we constructed an AA-metabolism prognostic model, which provided a powerful prognostic predictor for the clinical treatment of pancreatic cancer.

Analysis of Recurrent DNA Base Pair Mismatches in Eye Cancer Patient Genomes

This research investigates potential genetic drivers for uveal melanoma (UM), the most frequent primary cancer of the eye, through a frequency and ontology analysis of recurrent genetic mutations in UM patients. Genome data of 32 patients, 23 primary and 9 metastatic, was acquired from the U.S. National Institute of Health (NIH) and obtained from samples that were surgically collected from eye enucleations or resected from liver metastases. It was hypothesized that chromosomes 15 and 16 would predominantly exhibit genetic alterations linked to UM due to their role in the expression of eye color. During analysis, DNA from cancerous tumor cells was compared to a reference DNA sequence (extracted from normal tissue) to identify nucleotide base pair mismatches. The locus of each mutation noted to determine what genes mutated. Pareto analysis of cross-patient data performed to identify chromosomes with the most genetic mutations along with any recurrent genetic mutations across patients. A gene ontology (GO) analysis conducted to study the functions of mutated genes and investigate possible links to cancer, such as anomalies in genes with a known role in tumor suppression. A total of 130 genetic mutations were identified (7 recurrent and 123 non-recurrent), with most mutations occurring in chromosomes 3 and X. Recurrent mutations varied from 8.7% to 17.39% occurrence in the UM patient sample. The recurring mutations observed as missense mutations in seven genes. These findings suggest that UM is a recessive heterogeneous disease with selected homozygous mutations. Notably, this study has potential wider significance because the seven genes targeted by recurrent mutations affected in other cancers. It concluded that immunotherapy is a highly promising treatment for Uveal Melanoma due to the disease’s heterogeneous nature

Expert opinion on the diagnostic odyssey and management of late-onset Pompe disease: a neurologist's perspective.

This consensus statement by a panel of neurology experts aimed to provide a practical and implementable guidance document to assist clinicians with the best clinical practice in terms of diagnosis, treatment, and monitoring of late-onset Pompe disease (LOPD). The participating experts consider the clinical suspicion of LOPD by the physician to be of utmost importance in the prevention of diagnostic and therapeutic delay in LOPD patients. A diagnostic algorithm is proposed to facilitate the diagnosis of LOPD in patients presenting with unexplained proximal/axial weakness (with or without respiratory symptoms) or restrictive respiratory insufficiency with hyperCKemia and/or exercise intolerance as the red flag symptoms/signs that raise the index of suspicion for LOPD diagnosis. The diagnosis is based on the subsequent use of dried blood spot (DBS) assay, and the DBS assay can be confirmed by acid alpha-glucosidase (GAA) tissue analysis in leukocytes, fibroblasts, or muscle fibers and/or genetic mutation analysis. Accordingly, experts consider increased awareness among physicians about potential presenting characteristics with a high index of suspicion for LOPD to be crucial to suspect and consider LOPD in the differential diagnosis, while strongly suggesting the use of a diagnostic algorithm combined with DBS assay and confirmatory tests in the timely diagnosis of LOPD and implementation of best practice patterns.

Iterative-Acting Thioesterase from Polyketide Biosynthesis Accepts Diverse Nucleophilic Alcohols to Yield Oxazole-Containing Esters.

The biosynthesis of antitumor oxazole-containing conglobatin is directed by a multienzyme assembly line of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), in which an uncanonical iterative-acting C-terminal thioesterase domain, Cong-TE, ligated two fully elongated chains/conglobatin monomers on the terminal acylcarrier protein and subsequently cyclized the resulting dimer to a C2-symmetric macrodiolide. Screening of the conglobatin producer for secondary metabolites led to the discovery of two new compounds conglactones A (1) and B (2), possessing inhibitory activities to phytopathogenic microorganisms and cancer cells, respectively. The compounds 1 and 2 feature the ester bond-linked hybrid structures consisting of an aromatic polyketide benwamycin I (3) and one (for 1)/two (for 2) molecules of the conglobatin monomer (5). Genetic mutational analysis revealed that the production of 1 and 2 was correlated with the biosynthetic pathways of 3 and 5. Biochemical analysis indicated that 1 and 2 were produced by Cong-TE from 3 and an N-acetylcysteamine thioester form of 5 (7). Furthermore, the substrate compatibility of Cong-TE was demonstrated by enzymatically generating a bunch of ester products from 7 and 43 exotic alcohols. This property of Cong-TE was further validated by producing 36 hybrid esters in the fermentation of conglobatin producer fed with nonindigenous alcohols. This work shows a prospect of developing Cong-TE for green synthesis of valuable oxazole-containing esters, thus complementing the environmentally unfriendly chemosynthesis strategies.

Genetic mutation analysis of hereditary spherocytosis in Guangxi Zhuang Autonomous Region.

Hereditary spherocytosis (HS) is a common, hereditary hemolytic anemia (HHA) that is attributed to the disturbance of five erythrocyte membrane proteins. HS is also common in Guangxi, China. Target region capture high-throughput sequencing technology was used to analyze genetic mutations found in HS patients. Pedigree analysis was also performed, in some cases, to provide an optimized approach for the etiological diagnosis of complex, hereditary hemolytic anemia. Blood samples from the probands and their families were assessed by laboratory tests, target region capture high-throughput sequencing technology, and Sanger sequencing. We detected 79 HS patients from 37 unrelated families. The mutations observed in these patients were found mainly in four HS-related genes. These included SLC4A1, which was mutated in 31.65% of patients (25/79), SPTA1 (30.78% (24/79)), EPB42 (6.33% (5/79)), and SPTB (5.06% (4/79)). Composite genotype was observed in 26.58% (21/79) of patients and included mutations in two or more HS-related genes or mutations in HS-related genes combined with thalassemia or G6PD deficiency. No significant differences in clinical symptoms were found among patients of various genotypes except total bilirubin. Mean reticulocyte volume (MRV) and mean sphered cell volume (MSCV) of the composite genotype were significantly different from other groups. A total of 28 mutation types were found in HS-related genes. Using high-throughput sequencing technology, we also found some cases that had been misdiagnosed. MRV and MSCV are more significant in compound mutations as sensitive determinants of HS. High-throughput sequencing technology can be used to provide a more effective etiological diagnostic method for HS, with high efficiency and specificity.

Impact of Molecular Status on Metastasectomy of Colorectal Cancer Liver Metastases.

Although surgical resection could provide better survival for patients with colorectal cancer liver metastases (CRLM), the recurrence rate after resection of CRLM remains high. The progress of genome sequencing technologies has greatly improved the molecular understanding of colorectal cancer. In the era of genomics and targeted therapy, genetic mutation analysis is of great significance to guide systemic treatment and identify patients who can benefit from resection of CRLM. RAS and BRAF mutations and microsatellite instability/deficient deoxyribonucleic acid (DNA) mismatch repair status have been incorporated into current clinical practice. Other promising molecular biomarkers such as coexisting gene mutations and circulating tumor DNA are under active investigation. This study aimed to review the prognostic significance of molecular biomarkers in patients with CRLM undergoing metastasectomy based on the current evidence.

Clinicopathologic Features and Genetic Alterations in Mixed-Type Ampullary Carcinoma

Mixed-type ampullary carcinoma is a subtype that combines intestinal-type (I-type) and pancreatobiliary-type (PB-type) lesions, but few studies have examined its clinicopathologic features and genetic alterations. The differences in genetic alterations between mixed type and other subtypes, as well as the genetic differences between I-type and PB-type lesions in the mixed type, remain unclear. In this study, we compared the clinicopathologic features and prognosis of 110 ampullary carcinomas classified by hematoxylin and eosin and immunohistochemical staining as follows: 63 PB-type, 35 I-type, and 12 mixed-type carcinomas. A comparative analysis of genetic mutations by targeted sequencing of 24 genes was also performed in 3 I-type cases, 9 PB-type cases, and I and PB-type lesions of 6 mixed-type cases. The mixed subtype had a poorer prognosis than the other subtypes, and there was also a similar tendency in the adjuvant group (n = 22). A total of 49 genetic mutations were detected in all 18 lesions for which genetic alteration was analyzed. No genetic mutations specific to the mixed type were found, and it was not possible to determine genetically whether the mixed type had originally been I or PB type. However, 5 of 6 cases had mutations common to both I and PB-type lesions, and additional mutations were found only in either I or PB-type lesions. In support of this, the mixed type more frequently exhibited genetic heterogeneity intratumorally than the other subtypes. Mixed-type tumors are histologically, immunohistochemically, and genetically heterogeneous, and this heterogeneity is associated with poor prognosis and may affect treatment resistance.

Genetic Analysis of HIBM Myopathy-Specific GNE V727M Hotspot Mutation Identifies a Novel COL6A3 Allied Gene Signature That Is Also Deregulated in Multiple Neuromuscular Diseases and Myopathies

The GNE-associated V727M mutation is one of the most prevalent ethnic founder mutations in the Asian HIBM cohort; however, its role in inducing disease phenotype remains largely elusive. In this study, the function of this hotspot mutation was profoundly investigated. For this, V727M mutation-specific altered expression profile and potential networks were explored. The relevant muscular disorder-specific in vivo studies and patient data were further analyzed, and the key altered molecular pathways were identified. Our study found that the GNEV727M mutation resulted in a deregulated lincRNA profile, the majority of which (91%) were associated with a down-regulation trend. Further, in silico analysis of associated targets showed their active role in regulating Wnt, TGF-β, and apoptotic signaling. Interestingly, COL6a3 was found as a key target of these lincRNAs. Further, GSEA analysis showed HIBM patients with variable COL6A3 transcript levels have significant alteration in many critical pathways, including epithelial-mesenchymal-transition, myogenesis, and apoptotic signaling. Interestingly, 12 of the COL6A3 coexpressed genes also showed a similar altered expression profile in HIBM. A similar altered trend in COL6A3 and coexpressed genes were found in in vivo HIBM disease models as well as in multiple other skeletal disorders. Thus, the COL6A3-specific 13 gene signature seems to be altered in multiple muscular disorders. Such deregulation could play a pivotal role in regulating many critical processes such as extracellular matrix organization, cell adhesion, and skeletal muscle development. Thus, investigating this novel COL6A3-specific 13 gene signature provides valuable information for understanding the molecular cause of HIBM and may also pave the way for better diagnosis and effective therapeutic strategies for many muscular disorders.

Genomic profiles of Indonesian colorectal cancer patients.

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide and genetic mutation plays a vital role in CRC development. A previous study has suggested that genetic alterations among Indonesian patients with CRC might differ from those known in developed countries. This study aimed to describe the genomic profiles of Indonesian patients with CRC. Methods: A total of 13 patients were recruited for this study from May to July 2019. Tissue samples were collected, and genomic DNA was extracted from the samples. AmpliSeq for Illumina Cancer HotSpot Panel v2 Next-generation sequencing was used for DNA sequencing and a genome analysis toolkit was used for local realignment around the discovered variants. Results: A total of 45 genes comprising 391 single nucleotide variants (SNVs) with a depth >10 were observed. The genes with the most variants were STK11, SMAD4, EGFR, and ERBB4 and the genes with the most non-synonymous variants were SMAD4, TP53, FGFR3, CDKN2A, and STK11. Genes and SNVs in at least 90% of all samples consisted of 43 genes comprising 286 variants. Genes with the most non-synonymous SNVs were EGFR, SMO, FGFR3, TP53, STK11, CDKN2A. Genes related to the chromosomal instability pathway, such as TP53, SMAD4, KRAS, and APC, are also found in the analysis. Conclusions: Our findings showed that all patients with CRC in this study had genetic mutations in the chromosomal instability pathway. Analysis of genetic mutation of Indonesian patients with CRC might be crucial for advanced targeted therapy and for better clinical outcomes.

Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene

To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis. The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases. We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin. The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

Aminoacyl-tRNA Synthetase-based Prognosis Model and Exploration of Potential Mechanisms in Hepatocellular Carcinoma.

Aminoacyl-tRNA synthetases (ARSs) participate in tumor initiation and progression but their involvement in hepatocellular carcinoma (HCC) is not clear. This study aimed to investigate the prognostic value and underlying mechanisms of ARS in HCC. Data were obtained from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium, Gene Expression Omnibus and Human Protein Atlas databases. The prognostic model was constructed with the use of Cox regression and least absolute shrinkage and selection operator regression. Kaplan-Meier survival analysis, enrichment analysis, single sample gene set enrichment analysis and tumor mutation burden calculation were performed with R to evaluate the model and explore the underlying mechanism. Wilcoxon tests were used for comparisons between groups. Aspartyl-tRNA synthetase 2 (DARS2), tyrosyl-tRNA synthetase 1 (YARS1) and cysteinyl-tRNA synthetase 2 (CARS2) were identified as prognostic biomarkers and enrolled in model construction. The area under receiver operating characteristic curve of the model was 0.775. The model was used to assign patients from TCGA into low- and high-risk groups. Those in the high-risk group had a worse prognosis (p<0.001). The clinical significance of the model was tested in different clinical subgroups. Genetic mutation analysis had a higher TP53 mutation frequency in high-risk group. Enrichment analysis and study of immune-related cells and molecules found that the high-risk group was characterized by immune-cell infiltration and immunosuppression states. A novel ARS family-based model of HCC prognosis was constructed. TP53 mutation frequency and immune-suppressive status accounted for a worse prognosis in patients included in the high-risk group.

Clinical and cytogenetic profile of Fanconi anemia diagnosed after implementation of mitomycin C cytogenetic test in the state of Pernambuco, Brazil

IntroductionFanconi anemia (FA) is a rare autosomal recessive disease characterized by chromosomal instability and increased predisposition to malignancy. The diagnosis of FA requires clinical evaluation, confirmation of chromosomal fragility and/or analysis of genetic mutations. Therefore, this study aims to identify the clinical profile of patients with FA in the state of Pernambuco, Brazil. MethodWe analyzed 100 individuals referred from the major hematology and bone marrow (BM) transplant centers in the state of Pernambuco, Brazil, between the years 2018 and 2022. The diagnosis of FA was performed using the mitomycin C chromosomal fragility test, clinical data and classical and molecular cytogenetic analyses. ResultsWe enrolled a total of 16 patients with FA to comprise this study. Most of these individuals (87.5%) came from the Agreste and Sertão regions of Pernambuco. We observed a slight female prevalence of FA (1.3:1). The primary clinical and laboratory findings were café au lait spots (62.5%) and bone abnormalities (53%, mainly thumb deformities [40%]). We performed BM cytogenetic analysis for eight patients – seven showed no chromosomal abnormalities and one presented the karyotype 47,XY,+21 [15]. ConclusionsOur results are important to promote public health measures for the early diagnosis of FA, as well as to foster the engagement of a multidisciplinary group in the treatment of this disease.

Microstructural evaluation of the brain with advanced magnetic resonance imaging techniques in cases of electrical status epilepticus during sleep (ESES).

The cause and treatment of electrical status epilepticus during sleep (ESES), one of the epileptic encephalopathies of childhood, is unclear. The aim of this study was to evaluate possible microstructural abnormalities in the brain using advanced magnetic resonance imaging (MRI) techniques in ESES patients with and without genetic mutations. This research comprised 12 ESES patients without structural thalamic lesions (6 with genetic abnormalities and 6 without) and 12 healthy children. Whole-exome sequencing was used for the genetic mutation analysis. Brain MRI data were evaluated using tractus-based spatial statistics, voxel-based morphometry, a local gyrification index, subcortical shape analysis, FreeSurfer volume, and cortical thickness. The data of the groups were compared. The mean age in the control group was 9.05 ± 1.85 years, whereas that in the ESES group was 9.45 ± 2.72 years. Compared to the control group, the ESES patients showed higher mean thalamus diffusivity (p < 0.05). ESES patients with genetic mutations had lower axial diffusivity in the superior longitudinal fasciculus and gray matter volume in the entorhinal region, accumbens area, caudate, putamen, cerebral white matter, and outer cerebellar areas. The superior and middle temporal cortical thickness increased in the ESES patients. This study is important in terms of presenting the microstructural evaluation of the brain in ESES patients with advanced MRI analysis methods as well as comparing patients with and without genetic mutations. These findings may be associated with corticostriatal transmission, ictogenesis, epileptogenesis, neuropsychiatric symptoms, cognitive impairment, and cerebellar involvement in ESES. Expanded case-group studies may help to understand the physiology of the corticothalamic circuitry in its etiopathogenesis and develop secondary therapeutic targets for ESES.

Single-Cell Transcriptome Reveals Comprehensive Immune Profiles of T Follicular Helper Cell Lymphoma

Background: Angioimmunoblastic T-cell lymphoma and other nodal lymphomas of T follicular helper (TFH) cell origin (TFH lymphoma) have a poor prognosis and are associated with immune dysregulation. However, comprehensive immune profiles of TFH lymphoma have not been fully investigated. The RHOA G17V mutation (G17V) is a disease-specific mutation found in 60-70% of TFH lymphoma and considered to gain a novel function through binding to VAV1 protein. Nevertheless, the prevalence of homozygous (Homo) and heterozygous (Hetero) G17V mutations and the difference in their oncogenicity due to its zygosity have not been elucidated. Here, we performed single-cell transcriptomic analysis to characterize tumor and immune cells of TFH lymphoma. Methods: We performed single-cell RNA sequencing (scRNA-seq) and T-cell/B-cell receptor sequencing (scTCR/BCR-seq) for 9 lymph nodes (LNs) and 16 peripheral blood (PB) from 14 TFH lymphoma patients (9 at initial diagnosis [ID] and 5 at relapsed/refractory diagnosis [RR]), as well as 7 homeostatic LNs (HLNs) from patients with solid cancers (Fig. 1). Whole-exome sequencing (WES) was also performed for genetic mutation analysis using bulk DNA extracted from tumor tissues and PB of the same cohort as scRNA-seq. scRNA-seq and scTCR/BCR-seq libraries were constructed using the Chromium system (10x Genomics). Publicly available scRNA-seq data from 5 healthy donors (HDs) were also integrated as control. scRNA-seq data analysis was performed by Seurat, Scater, and Batchelor for quality control, integration, and clustering; SingleR for annotation; Immunarch for TCR/BCR repertoire analysis; gene set variation analysis (GSVA) for pathway analysis, and CellPhoneDB for cell-cell interaction analysis. Genomon2 pipeline was used for calling somatic mutations in WES data. Subsequently, VarTrix was applied to estimate mutations detected by WES in scRNA-seq data. Results: In total, 68,738 cells from LNs and 98,891 cells from PB were analyzed. Unsupervised clustering and annotation by canonical markers identified 4 major cell types: CD4/CD8+ T, natural killer (NK), B, and myeloid cells. Tumor cells were identified by the expanded TCR clonality and the TFH-like gene expression signatures. Next, using scRNA-seq data, we reanalyzed a total of 393 somatic mutations discovered by WES and identified 15 mutations in more than 5 cells of scRNA-seq data, of which only RHOA G17V was identified in multiple samples. G17V-mutated cells were detected in 4 LN and 6 PB samples, mostly restricted to tumor cells. G17V-Homo and Hetero cells were identified based on frequency of mutant reads. Differentially expression gene (DEG) analysis and GSVA in G17V-mutated tumor cells of LNs revealed that the regulatory signature was significantly activated in Homo, whereas the proliferation signature was up-regulated in Hetero. Additionally, DEG analysis identified three tumor-specific gene candidates and the expression of cell surface protein PLS3 was validated by flow cytometry and immunohistochemistry. Subsequently, we performed sub-clustering and GSVA of immune cells and found that dysfunctional CD8 T cells were significantly increased in LNs and PB of patients compared to controls and exhibited oligoclonal expansion by TCR repertoire analysis. Notably, TREG, complement component 1q positive macrophages (C1Q+ Mφ), and LAMP3+ dendritic cells (DCs) were significantly increased in LNs from patients with RR diseases than HLNs. DEG analysis and GSVA revealed high expression of PD-L1 and regulatory signatures in LAMP3+ DCs. Moreover, genes related to immune suppression were significantly upregulated in TREG of patients compared to those of controls. Finally, cell-cell interaction analysis uncovered that tumor cells and TREG of RR patients strongly interacted with C1Q+ Mφ and LAMP3+ DCs via CXCR3-CXCL9 and CCR4-CCL17 interactions, respectively, indicating that these myeloid cells not only induced TREG into tumor tissues but also actively interacted with tumor cells. Conclusion: Tumor cells of TFH lymphoma acquired the immunosuppressive signature in the clonal evolution process from the G17V Hetero to Homo. Furthermore, comprehensive immune profiling of TFH lymphoma revealed expansion of cells involved in the immune escape. These results unveiled the enrichment of immune evasive phenotypes in TFH lymphoma and suggested their contribution to treatment resistance in RR patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunogenic cell death-related gene landscape predicts the overall survival and immune infiltration status of ovarian cancer.

Background: Ovarian cancer (OC) is the most troubling malignant tumor of the female reproductive system. It has a low early diagnosis rate and a high tumor recurrence rate after treatment. Immunogenic cell death (ICD) is a unique form of regulated cell death that can activate the adaptive immune system through the release of DAMPs and cytokines in immunocompromised hosts and establish long-term immunologic memory. Therefore, this study aims to explore the prognostic value and underlying mechanisms of ICD-related genes in OC on the basis of characteristics. Methods: The gene expression profiles and related clinical information of OC were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. ICD-related genes were collected from the Genecards database. ICD-related prognostic genes were obtained by intersecting ICD-related genes with the OC prognostic-related genes that were analyzed in the TCGA database. Functional enrichment, genetic mutation, and immune infiltration correlation analyses were further performed to identify underlying mechanisms. Subsequently, we developed a TCGA cohort-based prognostic risk model that included a nine-gene signature through univariate and multivariate Cox regression and LASSO regression analyses. Meanwhile, external validation was performed on two sets of GEO cohorts and the TCGA training cohort for three other common tumors in women. In addition, a nomogram was established by integrating clinicopathological features and ICD-related gene signature to predict survival probability. Finally, functional enrichment and immune infiltration analyses were performed on the two risk subgroups. Results: By utilizing nine genes (ERBB2, RB1, CCR7, CD38, IFNB1, ANXA2, CXCL9, SLC9A1, and SLAMF7), we constructed an ICD-related prognostic signature. Subsequently, patients were subdivided into high- and low-risk subgroups in accordance with the median value of the risk score. In multivariate Cox regression analyses, risk score was an independent prognostic factor (hazard ratio = 2.783; p < 0.01). In the TCGA training cohort and the two GEO validation cohorts, patients with high-risk scores had worse prognosis than those with low-risk scores (p < 0.05). The time-dependent receiver operating characteristic curve further validated the prognostic power of the gene signature. Finally, gene set enrichment analysis indicated that multiple oncological pathways were significantly enriched in the high-risk subgroup. By contrast, the low-risk subgroup was strongly related to the immune-related signaling pathways. Immune infiltration analysis further illustrated that most immune cells showed higher levels of infiltration in the low-risk subgroup than in the high-risk subgroup. Conclusion: We constructed a novel ICD-related gene model for forecasting the prognosis and immune infiltration status of patients with OC. In the future, new ICD-related genes may provide novel potential targets for the therapeutic intervention of OC.

Genetic Analysis of TP53 Gene Mutations in Exon 4 and Exon 8 among Esophageal Cancer Patients in Sudan

Genetic Analysis of TP53 Gene Mutations in Exon 4 and Exon 8 among Esophageal Cancer Patients in Sudan

The Potential Role of Cytotoxic Immune Effectors in Induction, Progression and Pathogenesis of Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is an auto-immune neurodegenerative disorder affecting the motor-neuron system. The causes of ALS are heterogeneous, and are only partially understood. We studied different aspects of immune pathogenesis in ALS and found several basic mechanisms which are potentially involved in the disease. Our findings demonstrated that ALS patients' peripheral blood contains higher proportions of NK and B cells in comparison to healthy individuals. Significantly increased IFN-γ secretion by anti-CD3/28 mAbs-treated peripheral blood mononuclear cells (PBMCs) were observed in ALS patients, suggesting that hyper-responsiveness of T cell compartment could be a potential mechanism for ALS progression. In addition, elevated granzyme B and perforin secretion at a single cell level, and increased cytotoxicity and secretion of IFN-γ by patients' NK cells under specific treatment conditions were also observed. Increased IFN-γ secretion by ALS patients' CD8+ T cells in the absence of IFN-γ receptor expression, and increased CD8+ T cell effector/memory phenotype as well as increased granzyme B at the single cell level points to the CD8+ T cells as potential cells in targeting motor neurons. Along with the hyper-responsiveness of cytotoxic immune cells, significantly higher levels of inflammatory cytokines including IFN-γ was observed in peripheral blood-derived serum of ALS patients. Supernatants obtained from ALS patients' CD8+ T cells induced augmented cell death and differentiation of the epithelial cells. Weekly N-acetyl cysteine (NAC) infusion in patients decreased the levels of many inflammatory cytokines in peripheral blood of ALS patient except IFN-γ, TNF-α, IL-17a and GMCSF which remained elevated. Findings of this study indicated that CD8+ T cells and NK cells are likely culprits in targeting motor neurons and therefore, strategies should be designed to decrease their function, and eliminate the aggressive nature of these cells. Analysis of genetic mutations in ALS patient in comparison to identical twin revealed a number of differences and similarities which may be important in the pathogenesis of the disease.

Isolated unconjugated hyperbilirubinemia in adults: the Gilbert’s versus Criggler Najar Syndrome Type 2 conundrum.

Gilbert’s syndrome is a genetic disorder characterised by non-hemolytic unconjugated hyperbilirubinemia. It is caused by mutations in the UGT1A1 gene which codes for the enzyme uridine diphosphate glucoronosyl transferase-1, which conjugates bilirubin for excretion. Affected individuals are usually asymptomatic apart from a mild jaundice and investigations reveal a mild isolated indirect hyperbilirubinemia. This may be exacerbated in the face of environmental and physical stressors. It is very similar in presentation to Criggler-Najjar syndrome (CNS) type 2. There is a small risk of kernicterus in patients with CNS type 2 needing daily phenobarbitone therapy. This risk is miniscule in Gilbert’s syndrome. Genetic testing for polymorphisms of the UGT1A1 gene is the diagnostic clincher for Gilbert’s syndrome, but it can also be picked up by evaluating the response to phenobarbitone and fasting, particularly in resource poor settings. Due to limited availability, case reports documenting the genetic mutational analysis are sparse. We reported one such rare case with an unusually high indirect hyperbilirubinemia in Gilbert’s syndrome confirmed by both phenobarbitone response and genetic analysis.

High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9.

The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.

Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.

Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n = 21, lymphatic malformations n = 27, lymphatic venous malformations n = 1, and Klippel-Trenaunay syndrome n = 10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). In total, 37 of 59 patients (62.7%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.