Postoperative infection after anterior cruciate ligament reconstruction caused by Finegoldia magna: a case report / 中华骨科杂志

One case of knee infection after anterior cruciate ligament reconstruction caused by the gram-positive anaerobic bacterium Finegoldia magna was reported. The patient was admitted to hospital due to fever and knee joint swelling and pain after anterior cruciate ligament reconstruction. Through medical history, physical examination, imaging examination and next-generation sequencing, it was confirmed that the infection was caused by Finegoldia magna. Through literature review, 37 literatures on infectious diseases caused by Finegoldia magna was retrieved and analyzed, and the identification points of anaerobic bacteria, the application of second-generation sequencing technology and the treatment status of infection after anterior cruciate ligament reconstruction were reviewed. The incidence of infection after arthroscopic anterior cruciate ligament reconstruction is low, while anaerobic infection is even more rare and difficult to culture. The next-generation sequencing can be used to assist the diagnosis. On the basis of giving priority to the preservation of the reconstructed ligament, the combined use of arthroscopic debridement, irrigation and sensitive antibiotics is the main treatment method.

Gain-of-function STAT1 mutation and visceral leishmaniasis

ABSTRACT Gain-of-function mutations in the STAT1 gene have been initially associated with chronic mucocutaneous candidiasis. However, further research has shown that STAT1 GOF variants may increase susceptibility to infection by other intracellular pathogens. This report describes the first case of disseminated leishmaniasis associated with a STAT1 GOF mutation in a pediatric patient who did not have chronic mucocutaneous candidiasis. The patient was a four-year-old boy presenting with fever, severe asthenia, hepatosplenomegaly, pancytopenia, and liver failure. Bone marrow aspirate revealed hemophagocytosis and Leishmania parasites. Treatment consisted primarily of liposomal amphotericin B, as per the Hemophagocytic Lymphohistiocytosis 2004 protocol. After eight weeks of treatment, the patient did not improve and was submitted to diagnostic splenectomy. Activated macrophages and nodular spleen necrosis secondary to the visceral leishmaniasis were detected. Unfortunately, the patient died in the second week after splenectomy due to overwhelming systemic infection. DNA sequencing revealed a pathogenic (p. R274Q) GOF mutation in STAT1.

Fibroblast growth factor receptor 3 gene (FGFR3) mutations in high-grade muscle-invasive urothelial bladder cancer in a Brazilian population: evaluation and prevalence

ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Value of detection of cell-free fetal DNA in maternal peripheral blood for Down's syndrome screening / 中国基层医药

Objective:To investigate the value of detection of cell-free fetal DNA in maternal peripheral blood for Down's syndrome screening.Methods:A total of 1667 pregnant women who were at a higher risk of having a baby with Down's syndrome who received Down's syndrome screening in the First People's Hospital of Datong between January 2020 and March 2021 were prospectively analyzed. After detection of cell-free fetal DNA in maternal peripheral blood, pregnant women who were at a higher risk of having a baby with Down's syndrome decided whether to accept amniocentesis for fetal karyotype. Then follow-up was performed for collecting related information. Finally, detection results of cell-free fetal DNA in maternal peripheral blood, fetal karyotype results and pregnancy outcomes were analyzed.Results:The positive predictive value of detecting cell-free fetal DNA in maternal peripheral blood for trisomy 21, trisomy 18, and trisomy 13 and chromosome abnormality were 100.0%, 100.0%, 0.0% and 66.7%, respectively. The sensitivity and total specificity of detecting cell-free fetal DNA in maternal peripheral blood were 100.0% and 99.8%, respectively. The false positive rate of detecting cell-free fetal DNA in maternal peripheral blood for trisomy 13 and chromosome abnormality was 0.12% and 0.06%, respectively.Conclusion:A high degree of coincidence between detection results of cell-free fetal DNA in maternal peripheral blood and fetal karyotype results can be used as a prenatal screening for Down's syndrome. This has certain guiding significance for invasive prenatal diagnosis through amniocentesis-based fetal karyotype analysis.

Analysis of pathogenic genes in two cases of Sj?gren-Larsson syndrome / 中华皮肤科杂志

Objective:To detect pathogenic mutations in 2 patients with Sj?gren-Larsson syndrome.Methods:Clinical data were collected from 2 children with Sj?gren-Larsson syndrome, who were diagnosed and treated in Department of Dermatology, Capital Institute of Pediatrics, and genetic testing was performed to clarify their pathogenic mutations.Results:Both the 2 patients presented with typical ichthyosis-like skin manifestations, accompanied by a certain degree of intellectual disorder and growth, development and motor retardation. A previously reported homozygous mutation c.1157A>G was identified in the ALDH3A2 gene in case 1; compound heterozygous mutations c.1157A>G and c.1309A>T were identified in the ALDH3A2 gene in case 2, which were considered as novel pathogenic mutations.Conclusion:Genetic testing should be performed as early as possible in patients with suspected Sj?gren-Larsson syndrome, in order to facilitate early definite diagnosis.

A novel mutation of SPAST gene in a hereditary spastic paraplegia type 4 family / 中华内科杂志

Objective:To clarify the pathogenicity and further explore the association between genotype and clinical phenotype of this variant, analyzing a novel variation of SPAST gene in hereditary spastic paraplegia (HSP) family from Changzhi city, Shanxi Province.Methods:A family with HSP was tracked and collected in Neurology Department of Heping Hospital Affiliated to Changzhi Medical College in October 2019. Peripheral venous blood of 2 ml was extracted from the proband and 8 other members of the family, genomic DNA was extracted from the blood samples, and the genes of spastic paraplegia were screened by next-generation sequencing (NGS). HGMD, 1000G, OMIM databases and PolyPhen2, SIFT and other software were used for bioinformatics analysis of suspected mutations. Multiplex ligation-dependent probe amplification (MLPA) was used to further screen for total deletions/duplications in patients who remained negative after targeting NGS, and Sanger sequencing was performed to verify the suspected pathogenic mutation sites in the family to determine co-isolation of the mutation sites in the family members. Finally, it is necessary to refer to the latest version of The American College of Medical Genetics and Genomics (ACMG) sequence variation interpretation guidelines to interpret the mutation sites to determine pathogenicity.Results:The HSP family consist 47 members of 4 generations and 10 patients, with onset ages ranging from 2 to 44 years. The proband′s daughter only showed positive bilateral Babbitt signs on physical examination, and the rest of the patients showed spasticity and weakness of lower limbs with varying severity on this basis. Preliminary screening by next-generation sequencing technology showed that the proband had frame-shift variation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) and missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg). Then, Sanger sequencing was used for in-family verification, which showed SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was detected in the affected members include father, brother, son and daughter, and not detected in the unaffected normal members, the proband′s wife, mother, sister and sister-in-law. However, the unaffected of mother detected missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg), while the remaining members did not detect this variation. The results of MLPA showed that no large fragment variation was found.Conclusions:The genetic pattern of the HSP family was autosomal dominant, and the clinical characteristics were consistent with hereditary spastic paraplegia type 4 (SPG4). Co-segregation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was found in the HSP family and was the pathogenicity cause of this SPG4 family, and it was a newly discovered mutation locus.

Fundamentos y aplicaciones biomédicas de las principales tecnologías de secuenciación: una revisión de literatura / Biomedical Foundations and Applications of Major Sequencing Technologies: A Literature Review / Fundamentos e aplicações biomédicas das principais tecnologias de sequenciamento: uma re-visão da literatura

Introducción: el objetivo de la secuenciación es determinar la composición de los nucleótidos presentes en el ADN o el ARN. Desde la finalización del proyecto genoma humano, surgieron diversas tecnologías de secuenciación rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio y Oxford Nanopore, más precisas y costoeficientes, que permiten desarrollar proyectos a gran escala y estudiar genes y genomas, la composición de microbiomas, enfermedades metabólicas y enfermedades genéticas que afectan a la población. Objetivo: describir los fundamentos de los métodos de secuenciación de ADN y sus aplicaciones en las ciencias biomédicas. Métodos: revisión descriptiva de las principales estrategias de secuenciación de ADN de primera, segunda y tercera generación y su aplicación en el entorno biomédico, a partir de la búsqueda de artículos en bases de datos electró-nicas especializadas en investigación científica. Se encontraron 118 documentos, de los cuales se excluyeron 6, por no cumplir con los criterios de inclusión, y se seleccionaron 112, por cumplir con todos los requisitos. Conclusiones:el surgimiento de los métodos de secuenciación de siguiente generación arroja una gran canti-dad de datos, incluidos genomas secuenciados completamente de varias especies, con un rendimiento extenso, tiempos reducidos y costoeficiencia, que lleva a la completa transformación de las ciencias de la vida y logra un progreso sin precedentes en el análisis de genomas, la evaluación de la ecología microbiana y el diagnóstico de enfermedades.

Introduction: The purpose of sequencing is to determine the composition of the nucleotides present in DNA or RNA. Since the completion of the human genome project, several sequencing technologies such as Roche 454, SOLiD, Illumina, Ion Torrent, PacBio and Oxford Nanopore have emerged as tools for rapid sequencing, with greater precision and cost-efficiency, allowing the development of lar-ge-scale projects and the study of genes and genomes, along with the composition of microbiomes and the study of metabolic and genetic diseases that affect the population. Objective: To describe the foundations of the methods of DNA sequencing and their applications in the biomedical sciences. Methods: Descriptive review of the main strategies of first, second and third generation DNA sequencing and their application in the biomedical environment. This review was carried out by searching articles in electronic databases specialized in scientific research. A total of 118 papers were found, of which 6 were excluded as they did not meet the inclusion criteria and 112 were selected as meeting all the requirements. Conclusions: The emergence of next-generation sequencing methods yielding a wealth of data, including fully sequenced genomes of various species, with extensive throughput, reduced time and cost-effec-tiveness that has led to the complete transformation of the life sciences, achieving unprecedented progress in genome analysis, assessment of microbial ecology and disease diagnosis

Introdução: o objetivo do sequenciamento é determinar a composição dos nucleotídeos presentes no DNA ou RNA. Desde a conclusão do projeto do genoma humano, surgiram diversas tecnologias de sequenciamento rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio e Oxford Nanopore, mais precisas e econômicas, que permitem o desenvolvimento de projetos de grande escala e estudo de genes e genomas, composição de microbiomas, doenças metabólicas e genéticas que afetam a popula-ção. Objetivo: descrever os fundamentos dos métodos de sequenciamento de DNA e suas aplicações nas ciências biomédicas. Métodos: revisão descritiva das principais estratégias de sequenciamento de DNA de primeira, segunda e terceira geração e sua aplicação no ambiente biomédico, a partir da busca de artigos em bases de dados eletrônicas especializadas em pesquisa científica. Foram encontrados 118 documen-tos, dos quais 6 foram excluídos por não atenderem aos critérios de inclusão e 112 fo-ram seleciona-dos por atenderem a todos os requerimentos. Conclusões: o surgimento de métodos de sequenciamento de próxima geração rende uma riqueza de dados, incluindo genomas totalmente se-quenciados de várias espécies, com produção extensa, tempos reduzidos e eficiência de custo, levando à transformação completa das ciências da vida e alcançando um progresso sem precedentes no genoma análise, avaliação de ecologia microbiana e diagnóstico de doenças.

Application of circulating free DNA in the diagnosis and treatment of hepatocellular carcinoma / 临床肝胆病杂志

Hepatocellular carcinoma has a low early diagnostic rate, and there is a lack of highly sensitive and specific tumor markers. In recent years, fluid biopsy technique, represented by circulating free DNA (cfDNA), has become an auxiliary method for the diagnosis of cancer and has attracted more and more attention due to its advantages of noninvasiveness, convenience, and repeatability. With reference to the recent studies in China and foreign countries, this article summarizes and analyzes the advances in cfDNA in the diagnosis and treatment of hepatocellular carcinoma from the aspects of biological characteristics, detection techniques, and clinical application, so as to provide a basis for clinical diagnosis and treatment.

Advances in the clinical application of high-throughput sequencing in detecting microbiota in cirrhotic ascites, serum, and feces / 临床肝胆病杂志

Liver cirrhosis is a chronic progressive liver disease, and patients with liver cirrhosis are highly susceptible to infection. The changes in microbiota in ascites, serum, and feces are associated with the progression and complications of liver cirrhosis. However, effective bacterial culture cannot be performed for most patients due to low bacterial abundance in ascites and blood, and thus it is difficult to conduct a comprehensive analysis. The high-throughput and high-speed advantages of next-generation sequencing technique can be used for pathogen biology and clinical diagnosis, including pathogen detection and identification, strain typing, and microbiome studies, to help clinicians optimize the application of antimicrobials. At present, the next-generation sequencing technique gradually becomes mature, making it possible to analyze the whole genome of microbiota.

Infectious endocarditis by Bartonella species: report of two cases / Endocarditis infecciosa por Bartonella: descripción de dos casos en Chile

ABSTRACT Infectious endocarditis (IE) by Bartonella species is an emerging problem worldwide. We report two cases of native valve Bartonella-associated IE events, both affecting adult male patients with a history of alcohol abuse and a low socioeconomic status. Admissions were due to pancytopenia and bleeding in one case and embolic stroke in the other. Blood cultures were negative and IgG indirect immunofluorescence assays (IFA) were positive for B. henselae/B. quintana in high titers (1/16,384-1/16,384, and 1/32,768 -1/16,384, respectively). Cases were classified as definitive IE events according to modified Duke criteria due to the presence of valve vegetations with at least three minor criteria. One patient required aortic mechanical valve replacement and survived, and the other died after a massive hemorrhagic transformation of his stroke. PCR amplification and sequencing of the 16S ribosomal bacterial DNA from a valve tissue sample obtained at surgery in the patient who survived, confirmed B. quintana as the etiological agent. Bartonella-associated IE is an emerging problem in Chile, present in disadvantaged populations. It should be suspected in patients with culture-negative IE. IFA does not discriminate between B. henselae and B. quintana infection, but high titers suggest IE. Complementary PCR techniques may help to elucidate the final causative agent.

La endocarditis infecciosa(EI) asociada a Bartonella es un problema emergente a nivel mundial. Publicamos los 2 primeros casos de EI en válvula nativa asociados a Bartonella en Chile, los que afectaron a pacientes masculinos con historia de consumo de alcohol y bajos ingresos. La hospitalización fue provocada por pancitopenia y hemorragias en un caso y por un evento cerebrovascular en el otro. Se solicitó serología para Bartonella por inmunofluorescencia indirecta (IFI) para ampliar el estudio ante hemocultivos negativos y en ambos casos se reportaron resultados intensamente positivos para B. henselae y B. quintana1/16.384-1/16.384 y 1/32.768 -1/16.384, respectivamente). Los casos se clasificaron como eventos definitivos de EI según los criterios modificados de Duke debido a la presencia de vegetaciones valvulares con al menos 3 criterios menores. Un paciente requirió reemplazo valvular aórtico y sobrevivió, y el otro falleció tras una transformación hemorrágica masiva del infarto cerebral. La amplificación del ADN ribosomal 16S por RCP y posterior secuenciación de una muestra de tejido valvular confirmó la presencia de B. quintana. La EI por Bartonella sp. es un problema emergente en Chile, probablemente asociada a poblaciones desfavorecidas, la que debe ser sospechada en pacientes con cultivos negativos. La IFI no permite discriminar infecciones por B. henselae o B. quintana pero los títulos altos sugieren EI. Técnicas complementarias por RCP pueden ayudar a dilucidar el diagnóstico.

JAK2-mutated acute myeloid leukemia: comparison of next-generation sequencing (NGS) and single nucleotide polymorphism array (SNPa) findings between two cases

JAK2 mutations are rare in de novo acute myeloid leukemia (AML), and JAK2-mutated acute myeloid leukemia (AML) patients usually have a previous history of myeloproliferative neoplasms (MPNs). Current advances in laboratory techniques, such as single nucleotide polymorphism array (SNPa) and next-generation sequencing (NGS), have facilitated new insight into the molecular basis of hematologic diseases. Herein, we present two cases of JAK2-mutated AML in which both SNPa and NGS methods added valuable information. Both cases had leukemogenic collaboration, namely, copy-neutral loss of heterozygosity (CN-LOH), detected on chromosome 9. One of the cases exhibited both JAK2 and IDH2 mutations, most likely having originated as an MPN with leukemic transformation, while the other case was classified as a de novo AML with JAK2, CEBPA, and FLT3 mutations.

Clinical significance of next-generation sequencing technology for monitoring molecular minimal residual disease in acute myeloid leukemia / 白血病·淋巴瘤

Objective@#To investigate the value of next-generation sequencing (NGS) technology in the prognosis monitoring and treatment guidance for molecular minimal residual disease (MRD) in acute myeloid leukemia (AML) patients with complete remission (CR).@*Methods@#The clinical data of 68 AML (non-acute promyelocytic leukemia) patients who received gene mutation spectrum by using NGS technology at initial diagnosis and in CR phase in Tangdu Hospital of Air Force Military Medical University from January 2016 to July 2018 were retrospectively analyzed. The recurrence and survival of both molecular MRD positive group and negative group were analyzed and compared, and the value of NGS technology and multiparameter flow cytometry (MFC) were also analyzed in MRD monitoring.@*Results@#There were 39 males (57.4%) and 29 females (42.6%) in 68 patients, and the median age was 52 years old (8-82 years old). Molecular MRD positive group included 38 patients, while negative group included 30 patients. Residual mutation gene type in CR phase was most frequently detected in epigenetic regulator gene mutations, such as ASXL1, TET2, DNMT3A and IDH1/IDH2. Statistical analysis showed that the 2-year cumulative recurrence rate (CIR) in the molecular MRD positive group was higher than that in the molecular MRD negative group (86.8% vs. 51.3%; χ2 = 9.249, P = 0.002); the 2-year relapse-free survival (RFS) rate in the molecular MRD positive group was lower than that in the molecular MRD negative group (13.2% vs. 48.7%; χ2 = 9.249, P = 0.002); the 2-year overall survival (OS) rate in the molecular MRD positive group was lower than that in the molecular MRD negative group (58.0% vs. 100%; χ2 = 4.122, P = 0.042). Up to follow-up date, 3 patients with molecular MRD positive and 1 patient with molecular MRD negative who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) were still in disease-free survival. The results of monitoring MRD showed high consistency (76.7%, 33/43) in NGS and MFC. Compared with the other groups, the patients with both positive NGS and MFC had a higher relapse rate, and the difference was statistically significantly (P < 0.05).@*Conclusions@#Molecular MRD of AML patients is detected by using NGS technology, which could be used to predict the relapse and survival, suggesting that molecular MRD may guide post-remission treatment regimens and the determination of allo-HSCT indications.

Application of low-depth whole-genome sequencing for copy number variations in genetic diagnosis of X-linked ichthyosis due to STS gene deletion / 中华皮肤科杂志

Objective@#To evaluate the application value and significance of low-depth whole-genome sequencing for copy number variations (CNV-Seq) in the genetic diagnosis and prenatal diagnosis of X-linked ichthyosis (XLI) due to STS gene deletion.@*Methods@#Clinical data were collected from 3 616 subjects who received CNV-Seq, and single-gene test results were collected from 7 patients or pedigrees with ichthyosis in The First Affiliated Hospital of Zhengzhou University in 2018. The 3 616 samples included 2 891 prenatal samples from pregnant women (most were amniotic fluid samples, some fetal villus samples, very few umbilical blood samples) and 725 peripheral blood samples from other subjects. Genomic DNA was extracted from amniocytes or peripheral blood, and then subjected to CNV-Seq. Quantitative PCR (qPCR) and single nucleotide polymorphism (SNP) -comparative genomic hybridization (CGH) array were performed to verify the detected CNVs. Pathogenicity of the CNVs was analyzed according to the database of genomic variants (DGV) , database of genomic variation and phenotype in humans using ensembl resources (DECIPHER) , clinical genome resource (ClinGen) and online Mendelian inheritance in man (OMIM) .@*Results@#Of the 3 616 subjects receiving CNV-Seq, Xp22.31 deletion was identified in prenatal samples from 6 pregnant women, including 5 male and 1 female fetuses. The deleted fragment of Xp22.31 covered the XLI region containing the major gene STS. The parental CNV-Seq showed that the Xp22.31 deletion was spontaneous mutation in 2 of the 6 fetuses, and inherited from the parents in the other 4 fetuses. qPCR confirmed that the female fetus was a carrier of a complete heterozygous deletion of the STS gene, and there was a complete deletion of the STS gene in the other 5 male fetuses. SNP-CGH array also confirmed that the female fetus was heterozygous Xp22.31 deletion carrier, which was consistent with the CNV-Seq results. Ichthyosis gene panel sequencing in the 7 patients with ichthyosis showed 1 with harlequin ichthyosis, 2 with ichthyosis vulgaris, 3 with XLI, and no causative mutation in 1. CNV-Seq confirmed that Xp22.31 deletion existed in the above 2 patients with XLI due to STS gene deletion. Moreover, Xp22.31 duplication was found in 16 out of 3 616 subjects receiving CNV-Seq, but they were all individuals or fetuses with normal phenotype.@*Conclusions@#CNV-Seq is a stable and reliable method for screening whole-genome CNVs, and can be applied to genetic diagnosis and prenatal diagnosis of XLI due to STS gene deletion. The deletion of Xp22.31 fragment containing the STS gene can cause XLI, and the duplication of the same region is highly likely to be the polymorphic variation.

Clinical significance of next‐generation sequencing technology for monitoring molecular minimal residual disease in acute myeloid leukemia / 白血病·淋巴瘤

Objective To investigate the value of next-generation sequencing (NGS) technology in the prognosis monitoring and treatment guidance for molecular minimal residual disease (MRD) in acute myeloid leukemia (AML) patients with complete remission (CR). Methods The clinical data of 68 AML (non-acute promyelocytic leukemia) patients who received gene mutation spectrum by using NGS technology at initial diagnosis and in CR phase in Tangdu Hospital of Air Force Military Medical University from January 2016 to July 2018 were retrospectively analyzed. The recurrence and survival of both molecular MRD positive group and negative group were analyzed and compared, and the value of NGS technology and multiparameter flow cytometry (MFC) were also analyzed in MRD monitoring. Results There were 39 males (57.4% ) and 29 females (42.6%) in 68 patients, and the median age was 52 years old (8-82 years old). Molecular MRD positive group included 38 patients, while negative group included 30 patients. Residual mutation gene type in CR phase was most frequently detected in epigenetic regulator gene mutations, such as ASXL1, TET2, DNMT3A and IDH1/IDH2. Statistical analysis showed that the 2-year cumulative recurrence rate (CIR) in the molecular MRD positive group was higher than that in the molecular MRD negative group (86.8% vs. 51.3%;χ 2＝ 9.249, P＝ 0.002); the 2-year relapse-free survival (RFS) rate in the molecular MRD positive group was lower than that in the molecular MRD negative group (13.2% vs. 48.7%; χ2＝ 9.249, P＝ 0.002); the 2-year overall survival (OS) rate in the molecular MRD positive group was lower than that in the molecular MRD negative group (58.0% vs. 100%; χ 2 ＝ 4.122, P＝ 0.042). Up to follow-up date, 3 patients with molecular MRD positive and 1 patient with molecular MRD negative who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) were still in disease-free survival. The results of monitoring MRD showed high consistency (76.7%, 33/43) in NGS and MFC. Compared with the other groups, the patients with both positive NGS and MFC had a higher relapse rate, and the difference was statistically significantly (P < 0.05). Conclusions Molecular MRD of AML patients is detected by using NGS technology, which could be used to predict the relapse and survival, suggesting that molecular MRD may guide post-remission treatment regimens and the determination of allo-HSCT indications.

Association of ABCA1 rare nonsynonymous variants with primary open angle glaucoma / 中华实验眼科杂志

Objective To explore the rare nonsynonymous variants of ABCA1 gene in primary open angle glaucoma (POAG).Methods A prospective cohort study was carried out.Three hundred and ninety-eight POAG patients and 198 healthy controls matched in age and gender were recruited from March 2017 to March 2018 in Eye and Ear Nose Throat (ENT) Hospital of Fudan University.The periphery blood of 2-5 ml from all the subjects was collected for extraction of DNA,and rare variant analysis of the ABCA1 gene was conducted by whole exome sequencing (WES) data of these subjects.The study protocol was approved by Ethic Committee of Eye and Ear Nose Throat Hospital of Fudan University and Sichuan Provincial People's Hospital (No.2016-32-1,and written informed consent was obtained from each subject prior to entering the study cohort.Results A total of 21 rare nonsynonymous variants (minor allele frequency MAF＜0.O1) were detected in the coding regions of ABCA1 gene in 27 subjects of the 398 POAG,with the detection rate of 6.8％.Among them,c.4310C＞A (p.Thr1437Asn),c.3772G＞T(p.Asp1258Tyr),c.775A＞G (p.Lys259Glu) and c.1507_1508insGAGGT (p.Glu503GlyfsX7) were four novel variants.In the 198 healthy controls,five rare nonsynonymous variants were detected in the ABCA1 gene from five subjects respectively,with the detection rate of 2.5％,the detection rate of nonsynonymous in POAG group was higher than that in healthy control group,showing a significant difference (x2=4.72,P =0.03,OR =2.81).Conclusions Rare nonsynonymous variants in ABCA1 is associated with the pathogenesis of POAG.These variants can enrich the variation spectrum of ABCA1.

Congenital Analbuminemia in a Korean Male Diagnosed with Single Nucleotide Polymorphism in the ALB Gene: The First Case Reported in Korea

Congenital analbuminemia (CAA) is an autosomal recessive disease characterized by extremely low serum levels of albumin. CAA is caused by various homozygous or heterozygous mutations of the ALB gene. Patients often exhibit no clinical symptoms, aside from rare accompanying conditions, such as fatigue, ankle edema, and hypotension. This case report describes the case of a 28-year-old asymptomatic Korean male referred to our center with hypocalcemia, vitamin D deficiency, and hypoalbuminemia who was diagnosed with CAA. To determine the cause of hypoalbuminemia in the patient, laboratory tests, radiological examination, and DNA sequencing were performed. The patient was confirmed to not exhibit any other clinical conditions that can induce hypoalbuminemia and was diagnosed with CAA using DNA sequencing. The present case of CAA is the first to be reported in Korea.

Analysis of CSF1PO and D18S51 Loci Based on Ion Torrent PGM™ Platform / 法医学杂志

OBJECTIVES@#To analyse and detect CSF1PO and D18S51 loci by next generation sequencing （NGS） technology for the study on their sequence polymorphism.@*METHODS@#The peripheral blood samples were collected from 165 unrelated individuals of Chinese Han population. DNA samples were obtained by QIAamp DNA Mini kit. The library was constructed by Ion Plus Fragment Library. DNA sequencing analysis was performed on Ion Torrent PGM™ Platform. The newfound alleles were verified by Sanger sequencing. Data were analysed by Torrent Suite™ v5.0.2 and Integrative Genomics Viewer for the genotype identification and frequency count. The data were analysed statistically by PowerState v12.@*RESULTS@#The length and sequence polymorphisms of CSF1PO and D18S51 loci were simultaneously obtained by NGS technology. A new genotype was found on CSF1PO locus, and two new genotypes on D18S51 locus. Sanger sequencing was used to verify the newfound alleles found by NGS technology, and the results of verification showed consistency.@*CONCLUSIONS@#The structure of core repeats on CSF1PO and D18S51 loci was detected by NGS in this study for the improvement of the identifying performance of locus.

Detection of circulating tumor DNA in epidermal growth factor receptor-TKI relapsed non-small cell lung cancer patients using next-generation sequencing and an analysis of the resistant mechanisms / 中华病理学杂志

Objective@#Next-generation sequencing (NGS) was performed on circulating tumor DNA (ctDNA) samples from tyrosine kinase inhibitor (TKI)-naïve non-small cell lung cancers (NSCLC) and TKI-relapsed NSCLC to investigate the clinical value.@*Methods@#A total of 381 plasma samples from patients who were diagnosed with lung cancer in Cancer Hospital Chinese Academy of Medical Sciences from March 2017 to May 2018 were enrolled in the study. NGS was performed using a custom-designed panel that covers 10 lung cancer-related driven genes. Paired plasma-tissue samples from 39 patients were collected to analyses the sensitivity and specificity of detecting driver gene mutations using ctDNA. NGS was also performed on plasma samples from TKI-relapsed patients to identify TKI resistance mechanisms.@*Results@#Thirty-nine plasma samples collected from 39 NSCLC patients (including 21 female and 18 male) with corresponding tissue biopsies were analyzed for the sensitivity and specificity. The average age was 56 years (range 29 to 82 years). A high concordance of 84.62% (33/39) was observed between ctDNA and tissue biopsies. Compared with tissue biopsies, NGS sensitivity for ctDNA was 82.14% and specificity was 90.91%.Among these 39 patients, 34 were advanced stage patients (III-IV stage). The concordance, sensitivity, and specificity for ctDNA among the advanced stage patients were 88.24% (30/34), 86.36% (29/34) and 91.67% (31/34), respectively. Among the 381 plasma samples [including 231 TKI-naïve patients and 150 epithelial growth factor receptor(EGFR)-TKI relapsed patients], EGFR mutation was the most common driver gene among the 221 TKI-naïve lung adenocarcinoma patients (32.58%, 72/221). For 133 patients who progressed after first-generation EGFR-TKI, T790M was found to be the most frequent resistant mechanism (39.10%, 52/133), as well as bypass activation (3.01%, 4/133; such as MET amplification and ERBB2 amplification). Among those first-generation EGFR-TKI relapsed patients with EGFR sensitive mutations, T790M was detected in 53.06% (52/98). For the 17 patients who progressed after third-generation EGFR-TKI, C797S was found to be the most common resistant mechanism (4/17).@*Conclusions@#The concordance, sensitivity and specificity between ctDNA and tissue biopsies are acceptable. ctDNA analysis provides valuable information for lung cancer patients′ targeted treatment, especially for patients not fitted for biopsies.

Impact of postnatal exposure to antibiotics on intestinal microbiome in preterm infants / 中华围产医学杂志

Objective To assess the impact of postnatal exposure to antibiotics on intestinal microbiome in preterm infants with 16S rDNA sequencing technology.Methods This study was conducted on 19 preterm infants admitted to the neonatal intensive care unit (NICU) at Tongji Hospital immediately after birth from September 2015 to February 2016.Two groups were set up according to the duration of antibiotic exposure (＜3 d,n=10;＞7 d,n=9).Fecal samples were collected from each infant within the first day and 2 or 3 weeks after bitrth.High-throughput sequencer (Hiseq 2500) was used for sequencing,from which information on composition and abundance of species,phylogenetic evolution and bacterial community diversity was obtained.Intergroup differences was analyzed with independent samples t-test or Fisher's exact test.Results (1) No statistically significant difference was found in general information about the infants between the two groups.(2) The intestinal flora in preterm infants was mainly composed of Lactococcus,Enterococcus and Bacillus for both groups before antibiotic treatment (36.41％,23.40％ and 14.98％).The proportions of Lactococcus and Bacillus were decreased significantly (1.73％ and 1.25％,P＜0.01) with Enterococcus becoming the predomiant genus (16.73％) after antibiotic treatment,while the relative proportions of Staphylococcus,Clostridium and Bifidobacterium were raised.(3) The Shannon index was decreased after antibiotic exposure [(2.34±0.84) vs (1.06±0.96) in ＜3 d group,and (2.64± 1.04) vs (0.35±0.36) in ＞7 d group,both P＜0.05],and the other three Alpha diversity indexes,including observed species,Chaol and PD whole tree indexes,were also decreased within each group (all P＜0.05).(4) Bacterial assemblages showed high beta diversity in both groups before the usage of antibiotics,but antibiotic therapy reduced the diversity.(5) Anoism analysis showed significant differences in the composition of intestinal flora within each group before and after antibiotic exposure (R=0.555and 0.733,both P=0.001),but no difference was found between the two groups after antibiotic exposure (R=0.060,P=0.138).Conclusions Antibiotic exposure,even short-term (＜3 d) administration,may significantly change the distribution of intestinal microbiota in preterm infants.Prolonged usage of antibiotics could have detrimental influence on intestinal flora.Therefore,for preterm babies,prescription of antibiotics should be cautious,even short-term empirical usage.

Application of next generation sequencing technology in the detection of pathogenic bacteria in synovial fluid of prosthetic joint infection / 中华骨科杂志

Objective To investigate the role of next generation sequencing technology in the detection of pathogenic bacteria in synovial fluid of prosthetic joint infection.Methods Nine samples of synovial fluid specimens of prosthetic joint infection patients with positive microbial culture from October,1 2016 to April 1,2017 were collected.Each specimen (200 μl) was used for next generation sequencing.Total DNA was extracted from synovial fluid samples.The collected DNA samples were amplified by PCR in the V4 region of 16S rDNA gene.The amplified products were sequenced using the Illumina Miseq platform,2× 250 bp double-end sequencing strategy.The sequencing results were compared with the SILVA database to analyze the types of bacteria and relative abundance in the DNA samples.A total of 200 μl sterile double-distilled deionized water was used as control.Results Nine cases of microbial culture positive prosthetic joint infection synovial fluid DNA samples were sequenced by 16S rDNA amplicon sequencing and yielded 3 132 415 high-quality reads and 3 752 operational taxonomic units (OTU).At the level of bacteria,a total of 9 different bacterial gates were detected on 9 DNA samples.At the level of bacteria,34 different bacteria were detected by 16S rDNA amplicon sequencing.Each DNA sample was detected by 16S rDNA amplicon sequencing and the bacterial genus was identical to that of laboratory culture.16S rDNA amplicon sequencing detected more species of bacteria [6(3,9.5)] than bacterial cultures [(1.0(1.0,1.0)].There was statistically significant difference in the number of bacteria detected in the same specimen between the 16S rDNA amplicon sequencing and the laboratory culture (Z=2.533,P=0.011).Among them,the dominant bacterial population (highest abundance) detected by 16S rDNA amplicon sequencing in four DNA samples was consistent with the results of laboratory culture.Conclusion In the prosthetic joint infection,the 16S rDNA amplicon sequencing technology can accurately detect pathogens that are consistent with the laboratory culture,and can detect other bacteria outside the laboratory culture.This technology can provide the basis for clinical diagnosis and antibiotic selection.