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Genome analysis technologies: towards species identification by genotype.

Traditional identification of species has been based on phenotypic traits, although it is clear that, theoretically, genotype-based classification is more accurate. This is especially the case for microorganisms which possess less identifiable traits and are more easily influenced by environment. Therefore, technology that allows identification of species based on genotype is highly desirable. Whole genome sequencing can provide a sufficient amount of information and can be determinative for this purpose but is very impractical for routine use. Thus, a competent technology is needed that allows a reproducible reduction in the amount of information required about a whole genome, while still providing sufficiently accurate identification. It is almost imperative for such a technology to be of a high cost-performance and of easy handling. Universality and portability are also strongly desired. Based on these criteria, the current state of genome analysis technologies are reviewed. Among various methodologies discussed here, amplified fragment length polymorphism (AFLP), genome profiling (GP) and microarrays are the subject of particular attention. As species identification is a base for most fields of biology including microbiology, ecology, epidemiology and for various biotechnologies, it is of paramount importance to establish a more efficient, easily handled and more objective methodology, in parallel with conventional phenotype-based methodologies. GP is currently considered to have the most optimal nature for identification of species since it can reproducibly reduce a huge amount of genome information to a manageable size by way of random polymerase chain reaction and can extract a sufficient amount of information for species identification from the DNA fragments thus profiled by temperature gradient gel electrophoresis. The potential ability of DNA microarrays for this purpose is also discussed and promises much for the future.

Genome↗

Engineering drought and salinity tolerance in plants: lessons from genome-wide expression profiling in Arabidopsis.

World food security is increasingly dependent on continuous crop improvement and, in particular, the development of crops with increased drought and salinity tolerance. The completed genomic sequence of the model plant Arabidopsis thaliana and the development of whole-genome microarrays, together with increasing repositories of publicly available data and data analysis tools, have opened new avenues to genome-wide systemic analysis of plant stress responses. Here we outline examples of how this full-genome expression profiling can contribute to our understanding of complex stress responses and the identification and evaluation of novel transgenes that could hold the key to the development of commercially viable and sustainable crop plants.

Arabidopsis↗

Tandem Double Inversion Resolves the Structural Paradox Underlying Recurrent KAT6A::NCOA2 Fusion in Acute Myeloid Leukemia.

The KAT6A::NCOA2 (formerly MOZ::TIF2) fusion is an extremely rare recurrent genetic abnormality in acute myeloid leukemia (AML), with only nine cases reported to date. It has consistently been associated with inv(8)(p11q13). However, the genomic mechanism underlying this fusion has remained unresolved. Because both KAT6A and NCOA2 are transcribed in the same reverse orientation on chromosome 8, a simple inversion is structurally insufficient to generate a transcriptionally competent fusion transcript, creating a long-standing cytogenetic paradox. We analyzed an AML case harboring a KAT6A::NCOA2 fusion using targeted genomic profiling and breakpoint-level validation. In addition to the canonical KAT6A::NCOA2 fusion, sequencing identified an unexpected MTFR1::KAT6A rearrangement. Since MTFR1 is located between KAT6A and NCOA2 on chromosome 8, we hypothesized a complex intrachromosomal rearrangement. Genomic breakpoint analysis revealed that the fusion was generated not by a single inv(8), but by two adjacent intrachromosomal inversions forming a tandem double inversion. This rearrangement reoriented genomic segments to place KAT6A and NCOA2 in a transcriptionally compatible configuration, enabling fusion formation. These findings resolve the structural paradox of KAT6A::NCOA2-positive AML by demonstrating that a cytogenetically apparent inv(8)(p11q13) can conceal a tandem double-inversion architecture that reorients KAT6A and NCOA2 into a transcriptionally compatible configuration.

Humans↗

Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli.

The sequence of an unknown PCR product generated by random (and conventional) PCR could be determined without sequencing when it is provided with the template DNA sequence. Theoretically, this was based on formerly established ideas which assert that the amount of random PCR product mainly depends on the stability of the primer-binding structures and that the dynamic solution structure of DNA is essentially governed by the Watson-Crick base pairing. However, it has not been clear whether this holds true for larger genomes of mega- to gigabase size, beside the lambda phage genome (of 50 kb) used previously, nor has it been ascertained to uniquely specify the sequence of a random PCR product. Here, we jointly use two computer programs together with experimental data from Genome Profiling (i.e. TGGE analysis of random PCR products). The first procedure carried out by a newly remodeled computer program (PCRAna-A1) was shown to be competent to calculate a set of random PCR products from Escherichia coli genome DNA (4.7 Mb). The other procedure performed with another program (Poland-H) played a critical role in determining the final candidate sequence by theoretically offering the initial melting temperature and the melting pattern of unspecified candidate sequences. The success attained here not only proved our method to be useful for sequence prediction but also confirmed the above-mentioned ideas as rational. We believe that this is the first case to computer-utilize a genome sequence as a whole.

Base Sequence↗

Integrated molecular profiling of SOD2 expression in multiple myeloma.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2 promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol-induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

2-Methoxyestradiol↗

A Novel BRCA1 Pathogenic Variant in Tunisian Patient With High Grade Ovarian Cancer: Favorable Therapeutic Response to Olaparib.

BACKGROUND: Ovarian cancer is one of the leading causes of death from gynecological cancer worldwide. Genetic mutations in genes involved in key cellular functions such as BRCA1/2 play a central role in tumorigenesis and have major implications for targeted therapeutic strategies, especially the use of poly (ADP-ribose) polymerase (PARP) inhibitors. CASE: Herein, we described a case of a 50-year-old woman diagnosed with severe anemia secondary to heavy menometrorrhagia. Initial gynecological evaluation, including transvaginal ultrasound, was unremarkable, and endometrial biopsy was not indicated. Imaging revealed no ovarian abnormalities; however, exploratory laparotomy identified a peritoneal nodule, leading to further investigation. Targeted NGS was performed on somatic and germline DNA samples and showed a frame shift deletion of 10 bp (c.1256_1265del: p.R419Ter) in the BRCA1 gene. This variant, identified only in tumor tissues, is novel and classified as pathogenic in ClinVar and ACMG databases. Additional somatic alterations were detected in TP53 and MSH6, while germline testing revealed only a variant of uncertain significance in BARD1. After first-line chemotherapy, the patient benefited from olaparib and achieved a progression-free survival of 23 months with good tolerance and no evidence of disease recurrence. CONCLUSION: This finding highlights the importance of integrating tumor-based genomic profiling with germline testing to identify actionable mutations and guide precision oncology. The identification of a novel somatic BRCA1 mutation expands the mutational spectrum of HGSOC and underscores the need to include underrepresented populations, such as those from North Africa, in genomic studies.

Humans↗

Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans.

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).

Animals↗

Two-dimensional gel electrophoretograms of human chromosome specific restriction DNA fragments.

Human genomic DNA can be scanned by a two-dimensional gel electrophoresis of NotI cleaved, end-labelled DNA. This method, called Restriction Landmark Genome Scanning (RLGS) method yields about 2000 landmark spots in such a two-dimensional gel electrophoresis. To assign these spots to chromosomes, DNA from flow-sorted chromosome 3 (7.7 x 10(5) copies) and chromosome 4 (6.7 x 10(5) copies) were subjected to the same analysis. Ninety seven and 122 spots, respectively, were obtained in the electrophoretograms among which 16 spots were common to other chromosomes, and the others were unique to each chromosome. A method has been devised to assign these spots to the whole genomic profile. The ability to assign each spot to a chromosome will render this new mapping technology extremely useful, in which one can detect physical aberrations occurring in chromosomes, or it can be used as an auxiliary method in physical mapping of the human genome.

Autoradiography↗

Genomic characterization of two predominant genotypes of herpes simplex virus type 1.

Genomic profiles of 66 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were investigated with regard to restriction fragment length polymorphism (RFLP) and length variation of fragments containing reiterations. There were two predominant genotypes of F1 and F35, and the genomic characteristics of each were studied. The nucleotide change between F1 and F35 was estimated to be 1.5%. An RFLP marker (VR23) peculiar to genotype F35 was identified as the first case of genomic marker specific to a predominant genotype of HSV-1, and is the diagnostic marker of F35. The a sequences (repeating in an HSV-1 genome and containing reiterations) of F35 were cleaved by SacII on the DR4 (direct repeat 4) stretch, while a sequences of F1 had a rearranged DR4 and were resistant to SacII digestion. Thus, analyses of fragments containing reiterations, such as a sequences, can serve to classify HSV-1 strains as well as for purpose of differentiation. The proportion of strains derived from primary infection to those from recurrent infection was higher in strains of F35 than in those of F1, and this genotypic difference within HSV-1 may possibly influence clinical manifestations.

Animals↗

Detection and characterization of a novel bisegmented double-stranded RNA virus (picobirnavirus) from rabbit faeces.

In two separate studies rabbits were fed orally with human and rabbit "picobirnaviruses". Polyacrylamide gel electrophoresis (PAGE) of nucleic acid extracted from faecal samples collected from inoculated rabbits revealed the presence of discrete equimolar bands, typical of picobirnaviruses, in several specimens. The genome profiles detected in both studies differed significantly from that of the inoculum suggesting that passage of the inoculated picobirnaviruses had not taken place and that the bands were a co-incidental finding. The presence of rabbit picobirnaviruses was confirmed by characterization of the genome bands, as dsRNA by enzyme digestion and by their co-sedimentation in caesium chloride (CsCl) gradients with 32 nm virus particles at a buoyant density of 1.39 g/ml. Picobirnavirus genome segments varied in size in a range between 2.3-2.6 kilo base pairs (kbp) and 1.6-1.9 kbp for the slow and fast migrating bands, respectively. Immune electron microscopy of the picobirnavirus particles revealed round or slightly hexagonal particles with a smooth surface and a mean diameter of 30.7 nm. In one rabbit, an immune response, temporally associated with picobirnaviruses excretion, was demonstrated by immune electron microscopy (IEM) supporting the view that picobirnaviruses may be vertebrate viruses. Two antigenically distinct picobirnavirus strains were defined by IEM.

Animals↗

Machine learning identifies ac4C-related prognostic signature and TUBA1C as therapeutic target in COAD.

To explore the role of N4-acetylcytidine (ac4C)-related genes (acRGs) in colon adenocarcinoma (COAD) and identify reliable prognostic biomarkers and potential therapeutic targets. Multi-source transcriptomic datasets (TCGA-COAD, GSE39582, GSE17536) and single-cell RNA-seq data were analyzed. Ten machine learning algorithms were integrated to construct an acRG-based prognostic signature (acRGBS). Immune microenvironment (TME) and genomic profiling were performed, with in vitro functional experiments validating TUBA1C's role. acRGBS, comprising four hub genes (SARAF, CDC42SE2, TSPYL2, TUBA1C), effectively stratified COAD patients into high- and low-risk groups with distinct survival outcomes and was an independent prognostic factor. High-risk patients exhibited increased genomic instability and immunosuppressive TME, while low-risk patients had favorable immunotherapy response. TUBA1C was overexpressed in COAD cells, and its knockdown inhibited proliferation/migration and induced apoptosis. The acRGBS is a robust prognostic tool for COAD, and TUBA1C serves as a candidate therapeutic target, providing new insights for personalized COAD management.

Humans↗

Distinct chromosomal profiles in metastasizing and non-metastasizing colorectal carcinomas.

BACKGROUND: The prognosis of colorectal cancer patients is to a considerable extent determined by the metastatic potency of the primary tumor. However, despite the fact that liver metastases are the leading cause of death for cancer patients, the molecular basis still remains poorly understood and independent prognostic markers have not been established. MATERIALS AND METHODS: Comparative genomic hybridization (CGH) was used to screen colorectal carcinomas without distant metastases (n=18) and carcinomas synchronously metastatic to the liver (n=18). We aimed to detect distinct chromosomal aberrations indicating a metastatic phenotype. RESULTS AND DISCUSSION: Metastatic tumors exhibited a significantly (P=0.03) higher ANCA value (13.8) if compared with non-metastatic cancers (10.0). Furthermore, we observed that losses of chromosomal regions 1p32-ter and 9q33-ter were present at much higher frequencies in metastatic than in non-metastatic cancers, respectively (P=0.02 and 0.04). CONCLUSION: These data indicate that metastatic tumors may be separated from non-metastatic colorectal cancers based on their genomic profile.

Aged↗

The impact of genomic alterations on the transcriptome: a prostate cancer cell line case study.

Genetic instability may lead to the loss/gain of transcriptional control. Here we investigated the effect of genomic instability, that is loss/gain of chromosomal regions on the global transcriptome of prostate cancer cell line DU145. The genomic loss/gain map obtained through BAC array-based CGH was superimposed on the dynamic transcriptome of DU145 cells treated with serum for 0 h (serum starved), 2 h and 12 h. The genomic analysis suggested that in DU145 cells: (1) chromosomal gains are prominent than losses and (2) copy number changes are associated with chromosome-specific and dynamic gene expression regulatory mechanisms. A significant proportion of the genes in the stable regions of the chromosome were up-regulated whereas a higher proportion of genes were down-regulated at 2 and 12 h in the deleted regions of the chromosomes following serum treatment. No change in expression was observed for the genes in the gained regions over a period of time. This analysis led us to propose that loss of heterozygosity leads to an overall transcriptional down-regulation that may further lead to a decrease in the expression of putative tumor suppressors. The genomic profile of DU145 is similar to pathological specimens of prostate cancer, hence the genomic/transcriptomic signature of DU145 can be used to understand the pathology of prostate cancer. It is expected that this analysis will allow a better understanding of transcriptional regulatory mechanisms in the context of genomic loss and gain and may lead to the discovery of novel oncogenes and tumor suppressors and the underlying regulatory pathways.

Cell Line, Tumor↗

A genomic predictor of oral squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: The objective was to identify a genomic profile that predicts the likelihood of oral squamous cell carcinoma compared with normal oral mucosa in unknown tissue samples. STUDY DESIGN: Using a training set of tissue samples that were histologically classified as oral squamous cell carcinoma or normal mucosa, the authors used principal component analysis to develop a genomic predictor for oral squamous cell carcinoma. On a separate test set of unclassified samples, the authors used the predictor to classify the samples, then evaluated the performance of the predictor using histological diagnosis. METHODS: The authors used a data set consisting of messenger RNA extracted from 29 oral squamous cell carcinoma and 19 normal oral mucosa tissue samples and hybridized to Affymetrix oligonucleotide microarrays containing probe sets for 7070 genes and expressed sequence tags. The samples were divided into a training set of 15 oral squamous cell carcinoma and 10 normal samples and a test set consisting of the remaining samples. Using principal component analysis on the training set, the authors found a composite gene expression vector (principal component vector), which they used to compute likelihood ratios for oral squamous cell carcinoma on the test set. By calculating the contribution of each gene to the principal component vector, the authors identified genes with the greatest predictive value. RESULTS: Using the likelihood ratio, the authors correctly classified all 23 samples in the test set as either oral squamous cell carcinoma or normal. The authors found that many of the most predictive genes are known to be markers of squamous cell carcinoma or normal mucosa. CONCLUSION: Principal component analysis can be used with genomic microarray data to correctly predict the presence of oral squamous cell carcinoma in unknown tissue samples.

Biomarkers, Tumor↗

Age-related genomic characterization and therapeutic targets in Chinese breast cancer: insights from prospective targeted sequencing and clinical data analysis.

BACKGROUND: In China, breast cancer occurs at a much younger age and has a higher recurrence and mortality rate. However, with changes in lifestyle, there has been a trend towards an older age of breast cancer incidence in Chinese women. There is a paucity of large-scale next-generation sequencing cohorts for the analysis of genomic characterization in these populations and the identification of potential therapeutic targets. METHODS: To address this gap, we performed prospective targeted sequencing of tumor and blood samples from Chinese patients and collected detailed clinical information. We then categorized patients into two groups based on age (<&#x2009;40&#xa0;years, n&#x2009;=&#x2009;637;&#x2009;&#x2265;&#x2009;40&#xa0;years, n&#x2009;=&#x2009;3442) and proceeded to provide comprehensive descriptions of somatic and germline mutations in both groups. RESULTS: The somatic mutation analysis revealed that PIK3CA, FOXA1, and TBX3 mutations were more prevalent in elderly patients. By leveraging the aforementioned mutational characteristics, we employed our institution's FUTURE-SUPER clinical trial, an umbrella study targeting metastatic breast cancer, to confirm the potential benefits of PI3K-AKT-mTOR pathway inhibitors among elderly patients with breast cancer. Furthermore, TP53 and ERBB2 were more likely to be co-mutated in young women. Patients with TP53 and ERBB2 co-mutation tend to have a poorer prognosis, but through investigation of the SPARK cohort, patients carrying the TP53 and ERBB2 co-mutation are more likely to benefit from immune checkpoint inhibitor combination with tyrosine kinase inhibitor therapy. In our study, we observed a higher frequency of mutations in the DNA homology-dependent recombination pathway in young patients with breast cancer, which was associated with an elevated Ki67 index. Additionally, we confirmed a significant prevalence of germline breast cancer susceptibility gene 1 (gBRCA1) mutations in young patients, whereas germline checkpoint kinase 2 (gCHEK2) mutations are more common in elderly patients. CONCLUSIONS: Our study, which makes use of the largest Chinese breast cancer sequencing cohort, sought to characterize the age-related genomic profile of breast cancer patients and identify novel therapeutic opportunities for individuals with breast cancer.

Adult↗

The Great Island subgroup of tick-borne orbiviruses represents a single gene pool.

The geographical distribution of members of the Great Island (GI) subgroup in the Kemerovo serogroup of orbiviruses extends from the Arctic to the Sub-antarctic. To examine the gene pool size of this group, five topotypes whose origins ranged from Iceland in the northern hemisphere to Macquarie Island in the Southern Ocean were tested for their ability to reassort in vitro. All the isolates were distinguishable by plaque reduction neutralization tests, and their genome profile in polyacrylamide gels. They showed high frequency reassortment following dual infection of cell cultures with temperature-sensitive (ts) mutants and/or wild-type virus. Analysis of the dsRNA profile of the reassortants by PAGE confirmed the observation from reassortment assays that the Great Island subgroup constitutes a single gene pool. A seventh reassortment group was identified, distinct from the six groups previously described. The ts lesions for reassortment groups I, V and VII were considered to be in genome segments 9, 3 and 2, respectively. Segment 6 of GI virus (in contrast to segment 5 of Broadhaven and Wexford viruses) was shown to be the major genetic determinant of serotype specificity.

Animals↗

Evaluation of DNA "fingerprinting" for predicting the potential of E. coli O157:H7 isolates to cause hemolytic uremic syndrome (HUS).

Escherichia coli O157:H7 has been recognized since 1982 as a serious human pathogen spread by contaminated food and water. Pulsed-field gel electrophoresis has proven useful for identification of specific isolates/strains of this organism. Hemolytic uremic syndrome (HUS), generally occurring in children or the aged, is the most severe sequela associated with E. coli O157:H7 infection. The presently described work was designed to compare the genomic profile of isolates known to have caused HUS with those having had no such involvement. We asked the question: "Can we develop the means to recognize an 'HUS-prone' E. coli isolate and thereby alert medical personnel to the increased risk?" Twenty-two HUS-related and 10 HUS-unrelated E. coli O157:H7 samples were chosen for genomic analysis. Isolates were cultured overnight prior to being embedded in agarose gel plugs. Plugs were digested, using Xbal restriction endonuclease, and subjected to pulsed-field gel electrophoresis (PFGE) for 20 hours. Gels were stained with ethidium bromide, photographed under ultraviolet light, and Southern blotted. Radiolabeled toxin gene probes were used for hybridization assays. The two classes of isolates were compared by optical imaging software. A computer-generated dendrogram, based on restriction profiles, offered strong initial evidence that the HUS sequela may be produced by a particularly virulent and identifiable clone. The predictive value of this finding appears to be substantial.

DNA Fingerprinting↗

Pathognomonic genetic expression profile within peripheral blood mononuclear cells of rheumatic heart disease patients.

The present study was addressed to understand as to how the expression of genes, that play crucial role in both inflammation and autoimmune process, within blood mononuclear cells are effected by the molecular mimicry between streptococcal antigen and heart tissue recognized as main contributor towards the genesis of rheumatic heart disease (RHD). Such a study for the first time revealed that as compared to genomic profile within normal blood mononuclear cells, the cells derived from rheumatic heart disease patients exhibited significantly higher expression of genes coding for IL-8, IFN-gamma and CX3CR1 coupled with significant downregulation of CD36 mRNA expression. Based upon these results, we propose that maintenance of such a pathognomonic transcriptome within blood mononuclear cells may be responsible for the initiation and progression of rheumatic heart disease.

Adult↗