Next Generation Sequencing in Oncological Diagnostics: Hype or Hope?

The understanding of how genetic and epigenetic factors influence tumorigenesis, progression and invasion, is vastly growing since new technologies allow the analysis of the functional genome namely the exome, the transcriptome and the epigenome, besides enabling genome-wide assessment of genetic variations. With the advent of new drugs that are indicated tissue agnostic, depending on certain mutations, there is a growing demand for fast and cost-effective genetic diagnosis. The method in focus that already became an indispensable tool in viral diagnosis is next-generation sequencing (NGS). This approach allows sequencing of literally every DNA molecule in the sample and can either be used to assess numerous genetic markers of one patient at a time, or to assess fewer markers of many patients in parallel, which reduces costs. We submitted 23 samples of different tumor entities to four diagnostic companies with different analysis profiles. The results as disclosed and discussed in this report indicate that so far, the main application of NGS is rather in cancer research than in diagnosis, as none of the reports had a real impact on the therapeutic scheme. We are perfectly aware that such a small cohort cannot be generalized, but considering the costs vs. benefits, NGS should be engaged upon a very stringent evaluation only. However, in cases where obtaining a tissue biopsy is impossible or unfavorable, analysis of liquid biopsy by NGS provides a vital alternative.

A novel PIK3CD C896T mutation detected in bilateral sudden sensorineural hearing loss using next generation sequencing:An indication of primary immunodeficiency

Objective:To investigate immune-related genetic background in bilateral sudden sensorineural hearing loss (SSNHL). Case report and methods: The case is a 45-year-old man presenting with a 7-year history of bilateral profound SSNHL. Blood biochemical testing demonstrated increased levels of total cholesterol (5.88 mmol/L). Tests for hepatitis B showed a positive antibody against the hepatitis B core antigen. Complement C3 was below the normal value, and complement C4 and IgG were in the lower range of normal values. CT images showed a normal inner ear and vestibular aqueduct but round window membranous ossification on both sides. A total number of 232 immune-associated genes were sequenced using the next generation sequencing technique. Results: Mutations were detected in 5 genes, including the phosphoinositide 3-kinase catalytic subunit delta (PIK3CD), caspase recruitment domain-containing protein 9 (CARD9), complement factor H-related (CFHR2), immunoglobulin lambda-like polypeptide 1 Protein (IGLL1), and transmembrane channel-like gene family 8 (TMC8). In the PIK3CD gene, a C896T substitute in exon 7 was detected. This mutation causes primary immunodeficiency and is an autosomal dominant disease. Conclusion: The PIK3CD C896T mutation responsible for primary immunodeficiency may contribute to the onset of bilateral SSNHL with subsequent rapid progression.

Current scenario of the genetic testing for rare neurological disorders exploiting next generation sequencing

Next generation sequencing is currently a cornerstone of genetic testing in routine diagnostics,allowing for the detection of sequence variants with so far unprecedented large scale,mainly in genetically heterogenous diseases,such as neurological disorders.It is a fast-moving field,where new wet enrichment protocols and bioinformatics tools are constantly being developed to overcome initial limitations.Despite the as yet undiscussed advantages,however,there are still some challenges in data analysis and the interpretation of variants.In this review,we address the current state of next generation sequencing diagnostic testing for inherited human disorders,particularly giving an overview of the available high-throughput sequencing approaches;including targeted,whole-exome and whole-genome sequencing;and discussing the main critical aspects of the bioinformatic process,from raw data analysis to molecular diagnosis.

Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.Given its insidious onset,the condition often already progresses to advanced stage when symptoms occur.Thus,early diagnosis is of great significance for timely clinical intervention,efficacy enhancement,and prognostic improvement.Featuring high throughput,fastness,and rich information,next generation sequencing(NGS)can greatly shorten the detection time,which is a widely used detection technique at present.AIM To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC,and to establish a technological platform for CRC screening,diagnosis,and efficacy monitoring through fecal DNA detection.METHODS NGS was used to sequence the stool DNA of patients with CRC,which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease,as well as the tumor tissues of CRC patients.Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened,and their significances in diagnosing CRC and predicting patients'prognosis were comprehensively evaluated.RESULTS High mutation frequencies of TP53,APC,and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery.Contrastively,no pathogenic mutations of the above three genes were noted in the postoperative stools,the normal controls,or the benign intestinal disease group.This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients.The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent.Compared to the postoperative stools and the stools in the two control groups,the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools(χ^(2)=7.328,P<0.05;χ^(2)=4.219,P<0.05),suggesting that fecal TP53 and KRAS genes can be used for CRC screening,diagnosis,and prognostic prediction.No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups(χ^(2)=0.878,P>0.05),so further analysis with larger sample size is required.Among CRC patients,the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools,with a true positive rate of 59.26%,while the pathogenic mutation sites of KRAS occurred in 10 stools,with a true positive rate of 37.04%.The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67%(18/27)and 68.97%,respectively,both of which were higher than those of TP53 or KRAS mutation detection alone,suggesting that the combined genetic testing can improve the CRC detection rate.The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T(mutation start and stop positions were both 7579436 for the former,while 55249164 for the latter)were found in the preoperative stools and tumor tissues.These"undetected"mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression,which needs to be confirmed through further research.Some mutations of"unknown clinical significance"were found in such genes as TP53,PTEN,KRAS,BRAF,AKT1,and PIK3CA,whose clinical values is worthy of further exploration.CONCLUSION NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis.Fecal TP53 and KRAS can be used as specific genes for the screening,diagnosis,prognostic prediction,and recurrence monitoring of CRC.Moreover,the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing:A cohort analysis

BACKGROUND Genetic tests are increasingly performed for the management of unresectable pancreatic cancer.For genotyping aimed samples current guidelines recommend using core specimens,although based on moderate quality evidence.However,in clinical practice among the endoscopic ultrasound(EUS) guided tissue acquisition methods,fine needle aspiration(FNA) is the most widely performed.AIM To assess the adequacy for next generation sequencing(NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma(PDAC) samples.METHODS Between November 2018 and December 2021,105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center.Either 22 gauge(G) or 19G FNA needles were used.One pass was dedicated to DNA extraction.DNA concentration and purity(A260/280,A260/230) were assessed by spectrophotometry.We assessed the differences in DNA parameters according to needle size and tumor characteristics(size,location) and the adequacy of the extracted DNA for NGS(defined as A260/280 ≥ 1.7,and DNA yield:≥ 10 ng for amplicon based NGS,≥ 50 ng for whole exome sequencing [WES],≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and ttest respectively.Moreover,we compared DNA purity parameters across the different DNA yield categories.RESULTS Our cohort included 49% male patients,aged 67.02 ± 8.38 years.The 22G needle was used in 71%of the cases.The DNA parameters across our samples varied as follows:DNA yield:1289 ng(inter quartile range:534.75-3101),A260/280 = 1.85(1.79-1.86),A260/230 = 2.2(1.72-2.36).DNA yield was > 10 ng in all samples and > 100 ng in 93% of them(one sample < 50 ng).There were no significant differences in the concentration and A260/280 between samples by needle size.Needle size was the only independent predictor of A260/230 which was higher in the 22G samples(P =0.038).NGS adequacy rate was 90% for 19G samples regardless of NGS type,and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS.Samples with DNA yield > 100 ng had significantly higher A260/280(1.89 ± 0.32 vs 1.34 ± 0.42,P = 0.013).Tumor characteristics were not corelated with the DNA parameters.CONCLUSION EUS-FNA PDAC samples yield DNA adequate for subsequent NGS.DNA amount was similar between 22G and 19G FNA needles.DNA purity parameters may vary indirectly with needle size.

Next Generation Transcriptome Sequencing and Quantitative Real-Time PCR Technologies for Characterisation of the Bemisia tabaci Asia 1 mtCOI Phylogenetic Clade

A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.

The role of gene variants in the pathogenesis of neurodegenerative disorders as revealed by next generation sequencing studies:a review

The clinical diagnosis of neurodegenerative disorders based on phenotype is difficult in heterogeneous conditions with overlapping symptoms.It does not take into account the disease etiology or the highly variable clinical course even amongst patients diagnosed with the same disorder.The advent of next generation sequencing(NGS)has allowed for a system-wide,unbiased approach to identify all gene variants in the genome simultaneously.With the plethora of new genes being identified,genetic rather than phenotype-based classification of Mendelian diseases such as spinocerebellar ataxia(SCA),hereditary spastic paraplegia(HSP)and Charcot-Marie-Tooth disease(CMT)has become widely accepted.It has also become clear that gene variants play a role in common and predominantly sporadic neurodegenerative diseases such as Parkinson’s disease(PD)and amyotrophic lateral sclerosis(ALS).The observation of pleiotropy has emerged,with mutations in the same gene giving rise to diverse phenotypes,which further increases the complexity of phenotype-genotype correlation.Possible mechanisms of pleiotropy include different downstream effects of different mutations in the same gene,presence of modifier genes,and oligogenic inheritance.Future directions include development of bioinformatics tools and establishment of more extensive public genotype/phenotype databases to better distinguish deleterious gene variants from benign polymorphisms,translation of genetic findings into pathogenic mechanisms through in-vitro and in-vivo studies,and ultimately finding disease-modifying therapies for neurodegenerative disorders.

Application of next generation sequencing technology on contamination monitoring in microbiology laboratory

The surveillance and prevention of pathogenic microbiological contamination are the most important tasks of biosafety management in the lab.There is an urgent need to establish an effective and unbiased method to evaluate and monitor such contamination.This study aims to investigate the utility of next generation sequencing(NGS)method to detect possible contamination in the microbiology laboratory.Environmental samples were taken at multiple sites at the lab including the inner site of centrifuge rotor,the bench used for molecular biological tests,the benches of biosafety cabinets used for viral culture,clinical sample pre-treatment and nucleic acids extraction,by scrubbing the sites using sterile flocked swabs.The extracted total nucleic acids were used to construct the libraries for deep sequencing according to the protocol of Ion Torrent platform.At least 1G raw data was obtained for each sample.The reads of viruses and bacteria accounted for 0.01±0.02%,and 77.76±12.53%of total reads respectively.The viral sequences were likely to be derived from gene amplification products,the nucleic acids contaminated in fetal bovine serum.Reads from environmental microorganisms were also identified.Our results suggested that NGS method was capable of monitoring the nucleic acids contaminations from different sources in the lab,demonstrating its promising utility in monitoring and assessing the risk of potential laboratory contamination.The risk of contamination from reagents,remnant DNA and environment should be considered in data analysis and results interpretation.

Highly Aligned Ternary Nanofiber Matrices Loaded with MXene Expedite Regeneration of Volumetric Muscle Loss

Current therapeutic approaches for volumetric muscle loss(VML)face challenges due to limited graft availability and insufficient bioactivities.To overcome these limitations,tissue-engineered scaffolds have emerged as a promising alternative.In this study,we developed aligned ternary nanofibrous matrices comprised of poly(lactide-co-ε-caprolactone)integrated with collagen and Ti_(3)C_(2)T_(x)MXene nanoparticles(NPs)(PCM matrices),and explored their myogenic potential for skeletal muscle tissue regeneration.The PCM matrices demonstrated favorable physicochemical properties,including structural uniformity,alignment,microporosity,and hydrophilicity.In vitro assays revealed that the PCM matrices promoted cellular behaviors and myogenic differentiation of C2C12 myoblasts.Moreover,in vivo experiments demonstrated enhanced muscle remodeling and recovery in mice treated with PCM matrices following VML injury.Mechanistic insights from next-generation sequencing revealed that MXene NPs facilitated protein and ion availability within PCM matrices,leading to elevated intracellular Ca^(2+)levels in myoblasts through the activation of inducible nitric oxide synthase(i NOS)and serum/glucocorticoid regulated kinase 1(SGK1),ultimately promoting myogenic differentiation via the m TOR-AKT pathway.Additionally,upregulated i NOS and increased NO–contributed to myoblast proliferation and fiber fusion,thereby facilitating overall myoblast maturation.These findings underscore the potential of MXene NPs loaded within highly aligned matrices as therapeutic agents to promote skeletal muscle tissue recovery.

Confidence intervals for Markov chain transition probabilities based on next generation sequencing reads data

Background:Markov chains(MC)have been widely used to model molecular sequences.The estimations of MC transition matrix and confidence intervals of the transition probabilities from long sequence data have been intensively studied in the past decades.In next generation sequencing(NGS),a large amount of short reads are generated.These short reads can overlap and some regions of the genome may not be sequenced resulting in a new type of data.Based on NGS data,the transition probabilities of MC can be estimated by moment estimators.However,the classical asymptotic distribution theory for MC transition probability estimators based on long sequences is no longer valid.Methods:In this study,we present the asymptotic distributions of several statistics related to MC based on NGS data.We show that,after scaling by the effective coverage d defined in a previous study by the authors,these statistics based on NGS data approximate to the same distributions as the corresponding statistics for long sequences.Results:We apply the asymptotic properties of these statistics for finding the theoretical confidence regions for MC transition probabilities based on NGS short reads data.We validate our theoretical confidence intervals using both simulated data and real data sets,and compare the results with those by the parametric bootstrap method.Conclusions:We find that the asymptotic distributions of these statistics and the theoretical confidence intervals of transition probabilities based on NGS data given in this study are highly accurate,providing a powerful tool for NGS data analysis.

Comparison of the experimental methods in haplotype sequencing via next generation sequencing

Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy： （i） chromosomes separation, （ii） dilution pools, （iii） crosslinking and proximity ligation, （ix） long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future.

A Multiplex PCR-Based Next-Generation Sequencing Approach Has Detected a Common Large Deletion in STS Gene in a Patient with X-Linked Ichthyosis

Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by significant genetic and phenotypic heterogeneity. We developed a panel of 26 genes to be screened with the Ion Personal Genome Machine (PGM) for causative mutations relating to ichthyosis. Sequencing runs were obtained from a patient with ichthyosis using the Ion Torrent PGM and then processed with Ion Torrent Suite, Variant Caller, Coverage Analysis and wANNOVER tools. No causative mutations were found using Variant Caller and wANNOVER softwares, whereas the “Coverage Analysis” tool revealed a common large deletion in STS gene in a patient with X-linked ichthyosis. Identification of indels in Next Generation Sequencing (NGS) data is a veritable challenge. This study demonstrates the efficacy and effectiveness of using NGS approach to detect large deletions without resorting to specific algorithms for “indel” detection. Our results indicate that the NGS panel is a useful, rapid and cost-effective screening test for patients whose features are suggestive of a genetic etiology involving one of the genes embedded in the panel. It is an excellent alternative to Sanger sequencing as for costs, ease of analysis, and turnaround time.

Early adenocarcinoma mixed with a neuroendocrine carcinoma component arising in the gastroesophageal junction: A case report

BACKGROUND Early adenocarcinoma mixed with a neuroendocrine carcinoma(NEC)component arising in the gastroesophageal junctional(GEJ)region is rare and even rarer in young patients.Here,we report such a case in a 29-year-old Chinese man.CASE SUMMARY This patient presented to our hospital with a 3-mo history of dysphagia and regurgitation.Upper endoscopy revealed an elevated nodule in the distal esophagus 1.6 cm above the GEJ line,without Barrett’s esophagus or involvement of the gastric cardia.The nodule was completely resected by endoscopic submu-cosal dissection(ESD).Pathological examination confirmed diagnosis of intra-mucosal adenocarcinoma mixed with an NEC component,measuring 1.5 cm.Immunohistochemically,both adenocarcinoma and NEC components were positive for P53 with a Ki67 index of 90%;NEC was positive for synaptophysin and chromogranin.Next-generation sequencing of 196 genes demonstrated a novel germline mutation of the ERCC3 gene in the DNA repair pathway and a germline mutation of the RNF43 gene,a common gastric cancer driver gene,in addition to pathogenic somatic mutations in P53 and CHEK2 genes.The patient was alive without evidence of the disease 36 mo after ESD.CONCLUSION Early adenocarcinoma with an NEC component arising in the distal esophageal side of the GEJ region showed evidence of gastric origin.

Monogenic diabetes in children:An underdiagnosed and poorly managed clinical dilemma

Monogenic diabetes,constituting 1%-2%of global diabetes cases,arises from single gene defects with distinctive inheritance patterns.Despite over 50 associated genetic disorders,accurate diagnoses and management of monogenic diabetes remain inadequate,underscoring insufficient clinician awareness.The disease spectrum encompasses maturity-onset diabetes of the young(MODY),characterized by distinct genetic mutations affecting insulin secretion,and neonatal diabetes mellitus(NDM)-a heterogeneous group of severe hyperglycemic disorders in infants.Mitochondrial diabetes,autoimmune monogenic diabetes,genetic insulin resistance and lipodystrophy syndromes further diversify the monogenic diabetes landscape.A tailored approach based on phenotypic and biochemical factors to identify candidates for genetic screening is recommended for suspected cases of MODY.NDM diagnosis warrants immediate molecular genetic testing for infants under six months.Identifying these genetic defects presents a unique opportunity for precision medicine.Ongoing research aimed to develop cost-effective genetic testing methods and gene-based therapy can facilitate appropriate identification and optimize clinical outcomes.Identification and study of new genes offer a valuable opportunity to gain deeper insights into pancreatic cell biology and the pathogenic mechanisms underlying common forms of diabetes.The clinical review published in the recent issue of World Journal of Diabetes is such an attempt to fill-in our knowledge gap about this enigmatic disease.

Clinical metagenomic sequencing for rapid diagnosis of pneumonia and meningitis caused by Chlamydia psittaci

BACKGROUND Chlamydia psittaci(C.psittaci)is a gram-negative intracellular parasitic pathogenic bacterium that can infect avian and mammalian hosts,including humans.The detection of C.psittaci infections typically relies on traditional antigen-based immunoassays or serological testing that often lack sensitivity and/or specificity.Metagenomic next generation sequencing(mNGS)is an emerging tool for diagnosis.AIM To demonstrate that mNGS represents a valuable tool for rapid,sensitive,and accurate pathogen detection including C.psittaci infections.METHODS Four cases of psittacosis pneumonia and one case of pediatric psittacosis meningitis were diagnosed between December 2019 and May 2020 using mNGS at Changzhou Second People’s Hospital affiliated to Nanjing Medical University.Patients’clinical characteristics,manifestations,and treatment histories were retrospectively evaluated.RESULTS All five patients had a history of exposure to wild(psittacine or other birds)or domesticated birds(chickens).All patients had a high fever(>39℃)and three of them(60%)experienced organ insufficiency during the disease.The laboratory data showed normal to slightly increased leucocyte and neutrophil counts,and elevated procalcitonin levels in all five cases,and very high C-reactive protein levels in psittacosis pneumonia patients.mNGS identified a potential pathogen,C.psittaci,in patients’bronchoalveolar lavage fluid or cerebrospinal fluid.Computed tomography revealed lung air-space consolidation,pleural thickening,and effusion fluid buildup in psittacosis pneumonia cases,and an arachnoid cyst in the right temporal lobe of the pediatric psittacosis meningitis patient.All patients experienced complete recovery following the administration of targeted antichlamydia therapy.CONCLUSION This study not only demonstrated that mNGS represents a valuable tool for rapid,sensitive,and accurate pathogen detection,but also raised public health concerns over C.psittaci infections.

Clinical and genetic characteristics of retinoblastoma patients in a single center with four novel RB1 variants

AIM:To assess the clinical and genetic characteristics of children diagnosed with retinoblastoma(RB)at Gazi University Faculty of Medicine’s Department of Pediatric Oncology.METHODS:All cases diagnosed with RB and received treatment and follow-up in the Ophthalmology and Pediatric Oncology Department,October 2016 to May 2021 were evaluated retrospectively.The RB1 gene was analyzed by next-generation sequencing(NGS)technique in DNAs obtained from peripheral blood samples of the patients.RESULTS:This study included 53 cases with 67 RBaffected eyes during the study period.The mean age was 24.6(median:18.5,range:3–151)mo.There were 15(22.3%)Group D eyes and 39(58.2%)Group E eyes.The RB1 gene was sequenced by the NGS method in 19 patients.Heterozygous RB1:NM_000321.3:c.54_76del(p.Glu19AlafsTer4)variant was detected in a 15-month-old female with bilateral RB.Heterozygous RB1:NM_000321.3:c.1814+3A>T variant was detected in a 5.5-month-old male with bilateral RB.The intronic RB1:NM_000321.3:c.1332+4A>G variant was detected in patient 14,a 13-month-old male with unilateral RB.The RB1:NM_000321.3:c.575_576del(p.Lys192SerfsTer10)variant was found in an 18-month-old female with an allele frequency of 37%.These variants have not been reported in the literature and mutation databases.CONCLUSION:Four novel variants are described and one of them is found in two different patients.This data is crucial for assessing prognosis.It serves as a guide for estimating the long-term risk of secondary malignancy as well as the short-term risk of developing additional malignancies in the same eye and the other eye.

Characterization of cancer genomic heterogeneity by next-generation sequencing advances precision medicine in cancer treatment

Cancer is a heterogeneous disease with unique genomic and phenotypic features that differ between individual patients and even among individual tumor regions.In recent years,large-scale genomic studies and new next-generation sequencing technologies have uncovered more scientifc details about tumor heterogeneity,with significant implications for the choice of specific molecular biomarkers and clinical decision making Genomic heterogeneity signifcantly contributes to the generation of a diverse cell population during tumor development and progression,representing a determining factor for variation in tumor treatment response.It has been considered a prominent contributor to therapeutic failure,and increases the likelihood of resistance to future therapies in most common cancers.The understanding of molecular heterogeneity in cancer is a fundamental component of precision oncology,enabling the identification of genomic alteration of key genes and pathways that can be targeted therapeutically.Here,we review the emerging knowledge of tumor genomics and heterogeneity,as well as potential implications for precision medicine in cancer treatment and new therapeutic discoveries.An analysis and interpretation of the TCGA database was included.

Changes in the gut mycobiome in pediatric patients in relation to the clinical activity of Crohn's disease

BACKGROUND Numerous studies have shown that in Crohn’s disease(CD),the gut microbiota is of great importance in the induction and maintenance of inflammation in the gastrointestinal tract.Until recently,studies have focused almost exclusively on bacteria in the gut.Lately,more attention has been paid to the role of intestinal fungi.AIM To study the gut mycobiome analysis of pediatric patients with CD(in different stages of disease activity)compared to healthy children.METHODS Fecal samples were collected from patients:With active,newly diagnosed CD(n=50);active but previously diagnosed and treated CD(n=16);non-active CD and who were in clinical remission(n=39)and from healthy volunteers(n=40).Fungal DNA was isolated from the samples.Next,next generation sequencing(MiSeq,Illumina)was performed.The composition of mycobiota was correlated with clinical and blood parameters.RESULTS Candida spp.were overrepresented in CD patients,while in the control group,the most abundant genus was Saccharomyces.In CD patients,the percentage of Malassezia was almost twice that of the control(P<0.05).In active CD patients,we documented a higher abundance of Debaryomyces hansenii(D.hansenii)compared to the non-active CD and control(P<0.05)groups.Moreover,statistically significant changes in the abundance of Mycosphaerella,Rhodotorula,and Microidium were observed.The analyses at the species level and linear discriminant analysis showed that in each group it was possible to distinguish a specific species characteristic of a given patient population.Moreover,we have documented statistically significant correlations between:D.hansenii and patient age(negative);C.zeylanoides and patient age(positive);C.dubliniensis and calprotectin(positive);C.sake and calprotectin(positive);and C.tropicalis and pediatric CD activity index(PCDAI)(positive).CONCLUSION Mycobiome changes in CD patients,and the positive correlation of some species with calprotectin or PCDAI,give strong evidence that fungi may be of key importance in the development of CD.

Hotspots and frontiers of genetic research on pediatric cataracts from 2013 to 2022:a scientometric analysis

AIM:To explore the hotspots and frontiers of genetic research on pediatric cataracts.METHODS:Global publications from 2013 to 2022 related to genes in pediatric cataracts were extracted from the Web of Science Core Collection,and were analyzed in terms of the publication counts,countries,journals,authors,keywords,cited references,subject categories,and the underlying hotspots and frontiers.RESULTS:Totally 699 publications were included in the final analysis.The predominant actors were identified,with China(n=240)and PLoS One(n=33)being the most productive country and journal respectively.The research hotspots extracted from keywords were crystallin gene mutations,pathogenicity evaluation,phenotypes of ocular and neurodevelopmental abnormalities,genes encoding membrane proteins,and diagnosis of multisystemic disorders.The co-cited articles formed 10 clusters of research topics,including FYCO1(56 items),mutation screening(43 items),gap junction(29 items),the Warburg Micro syndrome(29 items),ephrin-A5(28 items),novel mutation(24 items),eye development and function(22 items),cholestanol(7 items),OCRL(6 items),and pathogenicity prediction(3 items).The research frontiers were FYCO1,ephrin-A5,and cholestanol.Cell biology showed the strongest bridging effects among different disciplines in the field(betweenness centrality=0.44).CONCLUSION:With the progress in next-generation sequencing and multidisciplinary collaboration,genetic research on pediatric cataracts broadens the knowledge scope of the crystalline lens,as well as other organs and systems,shedding light on the molecular mechanisms of systemic diseases.Cell biology may integrate multidisciplinary content to address cutting-edge issues in the field.

Co-infection of Chlamydia psittaci and Tropheryma whipplei: A case report

BACKGROUND The co-infection of Chlamydia psittaci(C.psittaci)and Tropheryma whipplei(T.whipplei)is unusual,and the detection of pathogenic microorganisms is particularly important for patients with severe diseases or poor experience in treatment.Early identification of pathogens can significantly improve the prognosis of the patients.Targeted next-generation sequencing(tNGS)is currently widely used in clinical practice for various infectious diseases,including respiratory infections,to achieve early,accurate,and rapid microbial diagnosis.CASE SUMMARY We report a case of a 40-year-old female patient with a history of contact with parrots who was diagnosed with C.psittaci and T.whipplei infection through bronchial lavage fluid targeted next generation sequencing.After moxifloxacin treatment,the patient's symptoms improved significantly,and the imaging changes were obviously resolved.CONCLUSION Coinfection with C.psittaci and T.whipplei is not common.In this case,timely and accurate identification of both pathogens was achieved using tNGS.Moreover,the efficacy of monotherapy with moxifloxacin was confirmed.