A case of tick-transmitted Q fever in Lishui,China diagnosed by next-generation sequencing

Q fever is a zoonotic disease caused by Coxiella burnetii. Most patients have non-specific symptoms at onset. In addition, routine diagnostic tests for C. burnetii are not sensitive, and the bacterium cannot grow in general culture medium. The diagnosis of Q fever therefore poses a challenge. This case study describes a man with a clear history of tick bite who had recurrent fever, pneumonia, and liver damage. Routine tests and bacterial cultures failed to clarify the pathogeny, but laboratory and imaging data suggested infection. After routine tests were exhausted, we detected the presence of C. burnetii in a whole blood sample using next-generation sequencing (NGS). To our knowledge, this is the first report of Q fever associated with Coxiella burnetii detected directly from blood samples in Lishui, China. NGS has revolutionized the diagnosis of infectious diseases, especially those caused by rare or newly discovered pathogens, and patient responses have finally proved its substantial benefits. NGS has important clinical significance for the early diagnosis of chronic Q fever. This proof-of-concept study is worthy of promotion in clinical practice.     

Detection of EGFR-SEPT14 fusion in cell-free DNA of a patient with advanced gastric cancer: A case report

BACKGROUNDGastric cancer is the fifth most diagnosed cancer worldwide and the third most common cause of cancer-related death. In recent decades, increasing application of next-generation sequencing has enabled detection of molecular aberrations, including fusions. In cases where tissue is difficult to obtain, cell-free DNA (cfDNA) is used for detecting mutations to identify the molecular profile of cancer. Here, we report a rare case of EGFR-SEPT14 fusion detected from cfDNA analysis in a patient with gastric cancer. CASE SUMMARYA 49-year-old female diagnosed with advanced gastric cancer in July 2019 received capecitabine and then combination chemotherapy of ramucirumab and paclitaxel, but ascites was detected. The therapy was switched to nivolumab, but disease progression was observed on a positron emission tomography/computed tomography scan in May 2020. Therapy was discontinued, and cfDNA next-generation sequencing was immediately evaluated. All genomic variants, including fusions, were analyzed from cfDNA. The following somatic alterations were detected from the patient’s cfDNA: an APC frameshift mutation (NM_000038.5:c.6579del, p.V2194fs) with variant allele frequency of 0.5%, an EGFR amplification with a copy number of 17.3, and an EGFR-SEPT14 fusion with variant allele frequency of 45.3%. The site of the fusion was exon 24 of EGFR fused to exon 10 of SEPT14. The fusion was in-frame and considered to be protooncogenic. Although the patient refused to continue therapy, we suggest that EGFR-targeted therapies be tried in such future cases. CONCLUSIONThe expanded applications of the cfDNA assay may open a new horizon in treatment of patients with advanced gastric cancer.     

Clonal diversity in KRAS mutant colorectal adenocarcinoma under treatment: Monitoring of cfDNA using reverse hybridization and DNA sequencing platforms

Biological heterogeneity is a key feature of malignancies that significantly contributes to disease progression and therapy resistance. Residual/relapsed tumor foci may represent genetically divergent subclones, which remain uncovered as repeated and multiple tumor sampling is usually limited. The analysis of circulating free DNA (cfDNA) from the peripheral blood plasma (also called a liquid biopsy, LB) is a new achievement that provides an effective tool for follow-up monitoring of cancer-related genetic status. The present study highlights the phenomenon of mutational variability observed in patients with metastatic KRAS mutant colorectal cancer (mCRC) during treatment with bevacizumab in combination in a longitudinal fashion.The prospective study included 490 mCRC patients evaluated between 2020 and 2022 in our institution. Out of the 211 KRAS mutant cases (43.06%) 12 tumors were identified with multiple KRAS gene variants (5.68%). Detailed follow-up investigations were possible in 3 of these patients including the genotyping of the primary and available metastatic tumors, and the peripheral blood cfDNA. cfDNA was collected from three different time points before and between cycles of combined treatment with bevacizumab chemotherapy. KRAS gene variants were identified using reverse-hybridization strips, and next-generation sequencing (NGS), and confirmed by conventional Sanger sequencing.Interestingly, surgery and multiple treatment cycles reorganized the mutational profiles in the selected cases. The effect of the treatments resulted either in the overrepresentation of one of the pre-existing gene variants or in the appearance of new KRAS variants absent in the primary sample, according to the plasma cfDNA findings. Besides the KRAS variants demonstrated by targeted analysis, NGS mutational profiling identified some additional pathogenic variants from the cfDNA samples (including NRAS and MET alterations).In conclusion, plasma cfDNA sampling enables the monitoring of mutational heterogeneity and subclonal dynamics of the actual metastatic tumor mass in mCRC. The pattern of molecular profile potentially reflects a differential drug response determining further progression.     

Severe pulmonary co-infection with varicella-zoster virus,Pneumocystis jirovecii and Cytomegalovirus: a case report

Pneumocystis jirovecii, Cytomegalovirus and varicella-zoster virus are all opportunistically infective pathogens, but pulmonary co-infection with these pathogens is rare. Herein, this case report describes a patient with autoimmune haemolytic anaemia treated with methylprednisolone and cyclosporine that presented with rapidly progressive severe respiratory failure. Analysis of microbial nucleic acid sequences in both blood and sputum using next-generation sequencing revealed pulmonary co-infection with Pneumocystis jirovecii, varicella-zoster virus, and possibly Cytomegalovirus. After timely targeted and supportive treatments, the patient recovered. This case report highlights the imaging features of co-infection with these pathogens, the importance of next-generation sequencing for early diagnosis in immunosuppressed patients, and the effects of corticosteroid therapy.     

Early diagnosis of a newborn with tuberous sclerosis caused by a genetic mutation

ObjectiveTuberous sclerosis (TSC) is an autosomal dominant disorder, often detected during childhood. We present the results of genetic testing in a newborn with suspected TSC.MethodsA newborn with no specific clinical manifestations of TSC showed evidence of TSC on magnetic resonance imaging and echocardiography. Next-generation sequencing (NGS) and multiple ligation-dependent probe amplification (MLPA) of the TSC1 and TSC2 gene exons were carried out to confirm the diagnosis.ResultsThe results of MLPA were negative, but NGS showed a heterozygous mutation in the TSC1 gene comprising insertion of a T residue at c.2165 (exon 17) to c.2166 (exon 17), indicating a loss of function mutation. These results were verified by Sanger sequencing. This genetic change was present in the newborn but the parental genotypes were wild-type, indicating a de novo mutation.ConclusionsIn this case, a case of TSC caused by a heterozygous mutation in the TSC1 gene was confirmed by NGS sequencing. This indicates the suitability of genetic testing for the early diagnosis of clinically rare and difficult-to-diagnose diseases, to guide clinical treatment.     

Chemotherapy-associated clonal hematopoiesis mutations should be taken seriously in plasma cell-free DNA KRAS/NRAS/BRAF genotyping for metastatic colorectal cancer

BackgroundGenotyping of plasma cell-free DNA (cfDNA) is an increasingly important method to assess the tumor mutation status in colorectal cancer (CRC) patients. Clonal hematopoiesis (CH) releases non-tumor somatic mutations into blood, causing false positive results in cfDNA-based tumor genotyping. It is still not clear if CH should be examined in all CRC patients undergoing cfDNA analysis.MethodsWe analyzed cfDNA KRAS, NRAS and BRAF genotypes in 236 metastatic CRC patients, who had matched tissue genotyping results, by next-generation sequencing using plasma cfDNA. The cfDNA-only mutations with allele frequencies (AFs) < 5% were highly suspicious for being CH-derived mutations. The origins of cfDNA mutations were confirmed by droplet digital polymerase chain reaction (ddPCR) using paired peripheral blood cells (PBCs) and CH-derived mutations were finally determined. One patient with a CH-derived mutation was followed up and the subpopulation of blood cells, in which CH was present, was investigated.ResultsThree CH-derived mutations, KRAS Q61H, KRAS G12D and KRAS G12V, were identified in the patient cohort. All three patients harboring corresponding CH-derived mutations had a prior chemotherapy history. The CH-derived KRAS G12V mutation in a patient was found only present in lymphocytes and persisting under treatment. For all cfDNA mutations, the CH-derived ones were clustered in the patients with < 5% mutation AF and prior chemotherapy.ConclusionThe prevalence of CH in CRC patients was limited, and prior chemotherapy was a contributing factor of CH. It is recommended for patients with < 5% mutation AF and prior chemotherapy to have genotyping analysis of their PBCs following plasma cfDNA genotyping.     

Cross‐platform comparison of next‐generation sequencing and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for detecting KRAS/NRAS/BRAF/PIK3CA mutations in cfDNA from metastatic colorectal cancer patients

BackgroundExamining tumor KRAS/NRAS/BRAF/PIK3CA status in metastatic colorectal cancer (mCRC) is essential for treatment selection and prognosis evaluation. Cell‐free DNA (cfDNA) in plasma is a feasible source for tumor gene analysis.MethodsIn this study, we recruited mCRC patients and analyzed their KRAS/NRAS/BRAF/PIK3CA status in cfDNA using two platforms, next‐generation sequencing (NGS) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). The performance between the two platforms and the concordance rate between cfDNA and tissue were analyzed. The relationship between cfDNA‐related variables and clinical variables was also assessed. Tumor mutations in cfDNA from patients receiving continuous treatments were monitored in the follow‐ups.ResultsNext‐generation sequencing and MALDI‐TOF had similar specificity (100.0% vs. 99.3%) and negative predictive value (99.9% vs. 99.4%), whereas NGS had higher sensitivity (97.1% vs. 85.3% of MALDI‐TOF) and positive predictive value (100% vs. 82.9% of MALDI‐TOF). The overall concordance rate of NGS and MALDI‐TOF was 98.6%. For the reportable types of mutations in both cfDNA and tissue, the concordance rate was 96.1%. Among 28 tissue‐positive patients, the allele frequencies of tumor mutations in cfDNA were higher in patients with primary tumor burden (p = 0.0141). Both CEA and CA 19‐9 were positively correlated with cfDNA concentration (r = 0.3278 and r = 0.3992). The allele frequencies of tumor mutations changed with disease progression.ConclusionsNext‐generation sequencing showed slightly better performance in detecting cfDNA mutations and was more suitable for clinical practice. cfDNA‐related variables reflected the tumor status and showed a promising potential in monitoring disease progression.     

Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

IntroductionTo evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia.MethodsIn this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses.ResultsOf the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times.ConclusionOur NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.     

宏基因二代测序技术对脊柱感染病原学诊断的价值

目的:探讨宏基因二代测序(metagenomic next-generation sequencing,mNGS)技术对脊柱感染病原学诊断的价值。方法:回顾性分析2018年1月至2019年12月复旦大学附属中山医院收治的24例疑似脊柱感染患者的病例资料,采用常规微生物培养法和mNGS技术检测脊柱组织标本。结果:mNGS总体检测阳性率高于常规培养法(62.5%vs 35.0%),mNGS平均耗时36～48 h,明显短于常规培养法的平均天数21.8 d。脊柱感染主要检出病原体为结核分枝杆菌、金黄色葡萄球菌、大肠埃希菌。结论:mNGS能够快速检测脊柱感染病原体,为临床早期精准治疗提供重要的病原学依据。     

Performance evaluation of an amplicon-based next-generation sequencing panel for BRCA1 and BRCA2 variant detection

BackgroundAs next‐generation sequencing (NGS) technology matures, various amplicon‐based NGS tests for BRCA1/2 genotyping have been introduced. This study was designed to evaluate an NGS test using a newly released amplicon‐based panel, AmpliSeq for Illumina BRCA Panel (AmpliSeq panel), for detection of clinically significant BRCA variants, and to compare it to another amplicon‐based NGS test confirmed by Sanger sequencing.MethodsWe reviewed BRCA test results done by NGS using the TruSeq Custom Amplicon kit from patients suspected of hereditary breast/ovarian cancer syndrome (HBOC) in 2018. Of those, 96 residual samples with 100 clinically significant variants were included in this study using predefined criteria: 100 variants were distributed throughout the BRCA1 and BRCA2 genes. All target variants were confirmed by Sanger sequencing. Duplicate NGS testing of these samples was performed using the AmpliSeq panel, and the concordance of results from the two amplicon‐based NGS tests was assessed.ResultsNinety‐nine of 100 variants were detected in duplicate BRCA1/2 genotyping using the AmpliSeq panel (sensitivity, 99%; specificity, 100%). In the discordant case, one variant (BRCA1 c.3627dupA) was found only in repeat 1, but not in repeat 2. Automated nomenclature of all variants, except for two indel variants, was in consensus with Human Genome Variation Society nomenclature.ConclusionOur findings confirm that the analytic performance of the AmpliSeq panel is satisfactory, with high sensitivity and specificity.     

第二代测序技术及其在急性髓系白血病和骨髓增生异常综合征中的运用

第2代测序技术（next-generation sequencing,NGS）悄然取代经典的Sanger测序技术,成为科研人员发掘人类肿瘤疾病遗传学秘密的最实用、最可靠的方法。随着技术的不断更新及完善,进行全基因组测序不再遥不可及。近年来,部分科学家将此项技术运用于血液系统恶性肿瘤的研究,积极推动急性髓系白血病、骨髓增生异常综合征的全基因组测序的进程,期待从源头上找出部分血液系统恶性肿瘤的致病机理。本文就第2代测序技术及全基因组测序、外显子基因组测序和转录基因组测序在急性髓系白血病、骨髓增生异常综合征两种疾病中的运用进行综述。     

Metachronous pulmonary and pancreatic metastases arising from sigmoid colon cancer: A case report

BACKGROUNDMetachronous pulmonary and pancreatic metastases from colorectal cancer are rare. The diagnosis of pancreatic metastases is difficult and predominantly relies on computed tomography, pathology and immunohistochemistry. Here, we describe the use of next-generation sequencing (NGS) for determination of the origin of metastasis and prognostic prediction of colorectal cancer.CASE SUMMARYA 59-year-old man was diagnosed with sigmoid adenocarcinoma stage IIA (T3N0M0) and underwent surgery in April 2014, followed by XELOX adjuvant chemotherapy. The patient developed pulmonary metastasis in the right upper lung and underwent surgery in May 2016 without further adjuvant chemotherapy. In May 2018, pancreatic metastasis was found and he underwent pancreaticoduodenectomy. After surgery, he was treated with adjuvant S-1 chemotherapy from June 2018 to March 2019. Histopathological review of the specimens from all three lesions indicated consistent patterns characteristic of colon cancer. Concordant gene mutation profiles were observed across the three lesions that included oncogenic driver mutations most frequently seen in colon cancer (e.g., APC, TP53, KRAS and FBXW7). Blood circulating tumor (ct)DNA before adjuvant chemotherapy was undetectable with NGS, suggesting a favorable response to chemotherapy. The patient was alive and well at the latest follow-up visit, achieving a disease-free survival of 17 mo.CONCLUSIONThe genetic profiles of primary tumor, metastases and ctDNA may have clinical value in auxiliary diagnosis, prognosis and therapeutic decision-making.     

Primary myelofibrosis with concurrent CALR and MPL mutations: A case report

BACKGROUNDPrimary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by recurrent mutations in the JAK2, CALR, and MPL genes. The CALR and MPL co-mutation is very rare. To our knowledge, no more than five cases have been reported. Here, we report a case of PMF in which a CALR and MPL co-mutation was detected by next-generation sequencing (NGS) technology, and a literature review was performed.CASE SUMMARYA 73-year-old woman was admitted to our hospital in 2018 due to abdominal distension. The patient had splenomegaly, lymphadenopathy, leukopenia, anemia, and immature granulocytes in peripheral blood. There were dacrocytes and atypical megakaryocytes in bone marrow, and megakaryocytic proliferation was very active, accompanied by reticulin fibrosis grade 2. By NGS analysis of the bone marrow sample, we detected mutations in CALR, MPL, and PIK3RI, while JAK2 V617F and BCR-ABL were negative. Therefore, the patient was diagnosed with PMF and received oral ruxolitinib. However, the spleen and hematologic responses were poor. We review the literature, analyze previous reports of the mutation sites in our patient and differences between our patient and other reported cases of co-mutated CALR and MPL genes, and discuss the reason why the CALR and MPL co-mutations are rare and possible mechanisms and their impact on the prognosis of patients.CONCLUSIONCALR and MPL mutations can be concurrent in MPN, but they are rare. The use of NGS may help to identify more patients with co-mutated CALR and MPL genes. This will help to further explore the mechanism and its impact on these patients to develop appropriate treatment strategies.     

Next generation sequencing applications for cardiovascular disease

The Human Genome Project (HGP), as the primary sequencing of the human genome, lasted more than one decade to be completed using the traditional Sanger’s method. At present, next-generation sequencing (NGS) technology could provide the genome sequence data in hours. NGS has also decreased the expense of sequencing; therefore, nowadays it is possible to carry out both whole-genome (WGS) and whole-exome sequencing (WES) for the variations detection in patients with rare genetic diseases as well as complex disorders such as common cardiovascular diseases (CVDs). Finding new variants may contribute to establishing a risk profile for the pathology process of diseases. Here, recent applications of NGS in cardiovascular medicine are discussed; both Mendelian disorders of the cardiovascular system and complex genetic CVDs including inherited cardiomyopathy, channelopathies, stroke, coronary artery disease (CAD) and are considered. We also state some future use of NGS in clinical practice for increasing our information about the CVDs genetics and the limitations of this new technology.

• Key messages

• Traditional Sanger’s method was the mainstay for Human Genome Project (HGP); Sanger sequencing has high fidelity but is slow and costly as compared to next generation methods.

• Within cardiovascular medicine, NGS has been shown to be successful in identifying novel causative mutations and in the diagnosis of Mendelian diseases which are caused by a single variant in a single gene.

• NGS has provided the opportunity to perform parallel analysis of a great number of genes in an unbiased approach (i.e. without knowing the underlying biological mechanism) which probably contribute to advance our knowledge regarding the pathology of complex diseases such as CVD.

     

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement–negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.The detection of recurrent chromosomal rearrangements by cytogenetics was one of the earliest clinical molecular oncology assays and continues to play a major role in cancer diagnosis and prognosis.^1,2 Although translocations in the clinical laboratory are generally detected by cytogenetics, fluorescence in situ hybridization (FISH), or RT-PCR, studies have demonstrated that they may also be detected by next-generation sequencing (NGS) of DNA or RNA.^3–5 DNA-level translocations can be detected in particular areas of interest by first performing hybrid capture enrichment to target one or both partner genes in a translocation, followed by NGS.^4,6 NGS-based translocation detection has several advantages over conventional clinical laboratory methods, such as the ability to precisely define the breakpoint region, detect cryptic rearrangements and unknown partner genes, and run in parallel with gene mutation detection.Chromosomal rearrangements are detected in the clinical laboratory by routine cytogenetics, FISH, or RT-PCR; however, these methods have limitations. Cytogenetic studies, including chromosome analysis and metaphase FISH, require actively dividing cells, which can be especially difficult to obtain from solid tumors. In addition, chromosome analysis is of limited resolution, particularly in oncology specimens, and is therefore insensitive to cryptic and complex rearrangements.^5,7,8 Some rearrangements can be assayed via RNA-based RT-PCR methods, but this approach is less useful for translocations with a large number of partner genes or those with potentially diverse breakpoints.^9,10 FISH is among the most commonly used laboratory methods for the detection of chromosomal rearrangements and offers high sensitivity and the ability to test routine interphase, formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, FISH relies on highly trained individuals to score rearrangements by fluorescent microcopy and is an inherently low-resolution method that may be confounded by complex, multiway rearrangements and may require numerous probes to fully elucidate translocation partners for promiscuous genes, such as KMT2A.^5,10 Finally, FISH results are generally difficult to validate by orthogonal methods, outside less sensitive cytogenetic assays.Two of the most commonly tested translocations in the clinical laboratory are for rearrangements of the anaplastic lymphoma kinase gene, ALK, in non–small cell lung cancer and of the mixed-lineage leukemia gene, KMT2A (formerly known as MLL), in acute leukemia. The EML4-ALK fusion results from an inversion event on chromosome 2p that generally causes an in-frame fusion of EML4 exons 1 to 13 to ALK exons 20 to 29, producing an aberrant fusion gene with constitutive kinase activity, sensitive to crizotinib.^11–14 The occurrence of ALK fusions and other common lung cancer gene mutations in KRAS and EGFR are generally considered to be mutually exclusive, arguing that these tumors represent a distinct subset of lung cancers.¹⁵ Although not pharmacologically targetable, KMT2A rearrangements are of diagnostic and prognostic significance in acute leukemias, including both acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).^16,17 KMT2A rearrangements can be readily detected by FISH using break-apart probes; however, elucidation of the translocation partner gene may be difficult because >100 have been identified.^10,18NGS has had a tremendous effect on cancer discovery and is now becoming routine in the clinical molecular oncology laboratory.^3,19–21 NGS allows for the cost-effective, simultaneous evaluation of numerous sequence variants as part of focused clinical oncology panels or whole exomes. We and other groups have previously found that a range of DNA variants, including translocations, insertions or deletions, and copy number variants, can be detected from targeted NGS data and that it is possible to identify DNA-level breakpoints with single-nucleotide precision.^4,22,23 However, to be useful in the clinical setting, a thorough evaluation of the sensitivity and specificity of structural variation (SV) detection by NGS compared with standard methods is required. Given that numerous potential translocations can be evaluated by NGS simultaneously as part of a larger NGS cancer panel, for little to no additional cost, such methods could provide a significant savings for laboratories that perform multiple single-gene tests and multiple FISH assays on oncology specimens.We present a comprehensive evaluation of targeted translocation detection by NGS in the clinical laboratory by comparing four publicly available translocation detection tools (including the laboratory derived ClusterFAST) on targeted NGS data from 13 cases with ALK or KMT2A rearrangements (six lung carcinomas and one anaplastic large cell carcinoma with ALK rearrangements; six leukemias with KMT2A rearrangements) and 77 cancers negative for ALK and KMT2A rearrangements by FISH. We found that translocations can be reliably detected at the DNA level by targeted NGS panels and that such methods offer sensitivity and specificity similar to that of routine FISH with the advantage of single-nucleotide breakpoint resolution. Further, we examine approaches to designing capture probes for targeted NGS evaluation, evaluate the minimal coverage levels necessary to detect translocations, and explore methods to reduce false-positive translocation reports.     

下一代测序技术在分子诊断中的应用

DNA测序是破译人类疾病的一种强大技术,尤其在癌症方面。飞速发展的下一代测序（next—generation sequencing,NGS）极大降低了测序成本,并且实现了高通量,这使我们可以获得整个基因组的序列,以及那些临床上确诊病人的全部基因组信息。然而下一代测序技术带来诸多益处的同时也带来了挑战,那就是怎样使这个技术在临床诊断中成为常规手段。本文就目前NGS的几大技术平台原理,在临床诊断中的应用,以及目前面临的挑战等进行综述。     

Clinical characteristics and ABCC2 genotype in Dubin-Johnson syndrome: A case report and review of the literature

BACKGROUNDDubin-Johnson syndrome (DJS) is a benign autosomal recessive liver disease involving mutations of the ABCC2 gene. It is characterized by chronic or intermittent conjugated hyperbilirubinemia, with chronic idiopathic jaundice as the main clinical manifestation. Genetic alterations of the ABCC2 gene are commonly used for diagnosing DJS; however, the causative ABCC2 point mutation in Chinese patients remains unknown. Research on ABCC2 mutations in Chinese DJS patients is extremely rare, and the diagnosis of DJS remains limited. The routine analysis of ABCC2 mutations is helpful for the diagnosis of DJS. Here, we report the clinical characteristics and ABCC2 genotype of an adult female DJS patient. This article is to expound the discovery of more potentially pathogenic ABCC2 variants will that contribute to DJS identification.CASE SUMMARYThis study investigated a woman referred for DJS and involved clinical and genetic analyses. ABCC2 mutations were identified by next-generation sequencing (NGS). The patient showed intermittent jaundice and conjugated hyper-bilirubinemia. Histopathological examinations were consistent with the typical phenotype of DJS. Genetic diagnostic analysis revealed an ABCC2 genotype exhibiting a pathogenic variant, namely c.2443C>T (p.Arg815*), which has not been reported previously in the domestic or foreign literature.CONCLUSIONPathogenic ABCC2 mutations play an important role in the diagnosis of DJS, especially in patients with atypical presentations. Currently, NGS is used in the routine analysis of DJS cases and such tests of further cases will better illuminate the relationship between various genotypes and phenotypes of DJS.     

Severe thrombocytopenia and jaundice associated with Lemierre's syndrome:A case report

Dear editor,Lemierre’s syndrome was first described in detail by AndréLemierre in 1936 as infectious thrombophlebitis of the internal jugular vein(IJV).[1]In the current case,the pathogen was confirmed by next-generation sequencing(NGS).After proper use of antibiotics,the patient recovered and was discharged.     

第二代测序技术及其在急性髓系白血病和骨髓增生异常综合征中的运用

第2代测序技术(next-generation sequencing,NGS)悄然取代经典的Sanger测序技术,成为科研人员发掘人类肿瘤疾病遗传学秘密的最实用、最可靠的方法.随着技术的不断更新及完善,进行全基因组测序不再遥不可及.近年来,部分科学家将此项技术运用于血液系统恶性肿瘤的研究,积极推动急性髓系白血病、骨髓增生异常综合征的全基因组测序的进程,期待从源头上找出部分血液系统恶性肿瘤的致病机理.本文就第2代测序技术及全基因组测序、外显子基因组测序和转录基因组测序在急性髓系白血病、骨髓增生异常综合征两种疾病中的运用进行综述.     

An update on liquid biopsy analysis for diagnostic and monitoring applications in non-small cell lung cancer

Introduction: Collection of tumor samples is not always feasible in non-small cell lung cancer (NSCLC) patients, and circulating free DNA (cfDNA) extracted from blood represents a viable alternative. Different sensitive platforms have been developed for genetic cfDNA testing, some of which are already in clinical use. However, several difficulties remain, particularly the lack of standardization of these methodologies.

Areas covered: Here, the authors present a review of the literature to update the applicability of cfDNA for diagnosis and monitoring of NSCLC patients.

Expert commentary: Detection of somatic alterations in cfDNA is already in use in clinical practice and provides valuable information for patient management. Monitoring baseline alterations and emergence of resistance mutations is one of the most important clinical applications and can be used to non-invasively track disease evolution. Today, different technologies are available for cfDNA analysis, including whole-genome or exome sequencing and targeted methods that focus on a selection of genes of interest in a specific disease. In the case of Next Generation Sequencing (NGS) approaches, in depth coverage of candidate mutation loci can be achieved by selecting a limited number of targeted genes.