Investigation of the molecular genetic causes of non-syndromic primary ovarian ınsufficiency by next generation sequencing analysis

ABSTRACT Objective: The aim of this study is to investigate the molecular genetic causes of non-syndromic primary ovarian insufficiency (POI) cases with the gene panel based on next generation sequencing analysis and to establish the relationship between genotype and phenotype. Subjects and methods: Twenty three cases aged 14-40 years followed up with POI were included. Patients with a karyotype of 46, XX, primary or secondary amenorrhea before the age of 40, with elevated FSH (>40 IU/mL) and low AMH levels (<0.03 ng/mL) were included in the study. Molecular genetic analyzes were performed by the next generation sequencing analysis method targeted with the TruSightTM Exome panel. Results: Median age of the cases was 17.8 (14.0-24.3) years, and 12 (52%) cases admitted before the age of 18. Fifteen (65%) patients had consanguineous parents. In 2 (8.6%) cases, variants detected were in genes that have been previously proven to cause POI. One was homozygous variant in FIGLA gene and the other was homozygous variant in PSMC3IP gene. Heterozygous variants were detected in PROK2, WDR11 and CHD7 associated with hypogonadotropic hypogonadism, but these variants are insufficient to contribute to the POI phenotype. Conclusion: Genetic panels based on next generation sequencing analysis technologies can be used to determine the molecular genetic diagnosis of POI, which has a highly heterogeneous genetic basis.

Silage quality and bacterial diversity of silages inoculated with Listeria monocytogenes and Lacticaseibacillus paracasei_6714

Introduction: King grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum purpuphoides) and pineapple peel (Ananas comosus) silages are food alternatives for livestock in conditions of feed shortage. Objective: To describe the dynamics of the microbiota present in king grass and pineapple silage during the fermentation process using next generation sequencing (NGS) and to evaluate the protective effect of Lacticaseibacillus paracasei_6714 as a silage inoculum against Listeria monocytogenes. Methods: We used an unrestricted randomized design to characterize the microbiota present in silages made from king grass harvested 70 days after regrowth and pineapple peel. We inoculated mixtures of grass and peel with L. paracasei_6714 or L. monocytogenes, or both, with a non-inoculated treatment as control. The nutritional and fermentative profile was evaluated after 30 days. After 15 and 30 days of fermentation, we used 16S rRNA analysis to determine the dynamics and diversity of the microbiota in the inoculated and control silages. Result: Dry matter content and digestibility did not differ significantly; however, there were differences in crude protein, pH and organic acids. We obtained 4432 amplicon sequence variants of Proteobacteria, Firmicutes, Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes and Patescibacteria. The relative abundance of each phylum varied depending on the material and fermentation period. Phylum similarity was over 70 % (but not greater than 50 % with Bray-Curtis at the species level). Conclusion: These bacterial communities seem to have an important role during silage fermentation. Proper management of silage processing can reduce or eliminate pathogenic bacteria.

Introducción: Los ensilajes del pasto king grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum purpuphoides) y cáscaras de piña (Ananas comosus) son alternativas de alimento para ganado en condiciones de escasez alimentaria. Objetivo: Describir las dinámicas de la microbiota presente en los ensilajes de king grass y piña durante el proceso de fermentación usando secuenciación de próxima generación (NGS) y evaluar el efecto de protección de Lacticaseibacillus paracasei_6714 como inoculante de ensilaje ante Listeria monocytogenes. Métodos: Usamos un diseño aleatorio no restringido para caracterizar la microbiota presente en ensilajes de king Grass cosechados 70 días después de rebrote y de cáscaras de piña. Inoculamos mezclas de pasto y cáscara con L. paracasei_6714 o L. monocytogenes, o ambos, con un tratamiento control sin inocular. El perfil nutricional y de fermentación fue evaluado luego de 30 días. Después de 15 y 30 días de fermentación, usamos un análisis de para determinar la dinámicas y diversidad de la microbiota en los ensilajes inoculados y control. Resultados: Los contenidos de materia seca y digestibilidad, no difirieron significativamente; sin embargo, hubo diferencias en proteína cruda, pH y ácidos orgánicos. Obtuvimos 4 432 secuencias variantes de amplicon de Proteobacteria, Firmicutes, Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes y de Patescibacteria. La abundancia relativa de cada filo vario dependiendo del material y periodo de fermentación. Similitudes de filo fueron mayores al 70 % (pero no mayor que 50 % con Bray-Curtis a nivel de especie). Conclusión: Estas comunidades bacterianas parecen cumplir un papel importante durante la fermentación del ensilaje. Un manejo apropiado del proceso de ensilaje puede reducir o eliminar baterías patogénicas.

Clinicopathological and molecular analyses of uterine carcinosarcomas using next-generation sequencing: A single-center experience

Background: Uterine carcinosarcomas (UCS) constitute 3–4% of all uterine malignancies and 16% of deaths caused due to uterine neoplasms. Aim: In this study, we aimed to perform DNA-based mutation analysis in 12 genes (KRAS, NRAS, EGFR, C-KIT, BRAF, PDGFRA, ALK, ERBB2, ERBB3, ESR1, RAF1, PIK3CA) to determine the molecular subtypes of UCS using next-generation sequencing (NGS) in patients with aggressive UCS and poor prognosis. We aimed to compare the results of our analysis with clinicopathological data to contribute to the development of targeted therapy approaches related to the molecular changes of UCS. Materials and Methods: In this study, we included 12 cases diagnosed with uterine carcinosarcomas and examined the changes in oncogenes that play a role in UCS pathogenesis. For the analysis of mutation, the clinicopathological data were compared with the variations in the DNA-based gene panel consisting of 12 genes and 1237 variants in the UCS using the NGS method. Results: EGFR mutation was found in 91.7% of the cases, mutation in 41.7%, PDGFRA mutation in 25%, KRAS and PIK3CA mutation in 16.7%, and C-KIT mutation in 8.3% of the cases. Although no statistical significance was found between the detected mutation and clinicopathological data, it was concluded that PDGFRA mutation might be associated with advanced-stage disease development. Conclusion: This study's findings regarding different molecular types of UCS and information on oncogenesis of UCS can provide inferences for targeted therapies in the future by identifying targetable mutations representing early oncogenic events and thereby contribute toward further studies on this subject.

Implementación asistencial de estudios genómicos aplicados al diagnóstico molecular / Implementation of genomic studies applied to molecular diagnostics

El Hospital Garrahan ha sido pionero en el diagnóstico molecular de patologías pediátricas en Argentina. Los avances tecnológicos de las últimas décadas en el área de la biología molecular, sentaron las bases para la optimización y ampliación del diagnóstico molecular a partir de la secuenciación masiva en paralelo de múltiples genes. El presente trabajo describe el proceso de implementación de los estudios de secuenciación de nueva generación y el desarrollo de la Unidad de Genómica en un hospital público pediátrico de alta complejidad, así como su impacto en las capacidades diagnósticas de enfermedades poco frecuentes de origen genético. La creación del Grupo Interdisciplinario de Estudios Genómicos constituyó la vía institucional para la toma de decisiones que implican la implementación de nuevos estudios genómicos y el establecimiento de prioridades diagnósticas, extendiendo la disponibilidad del diagnóstico molecular a más disciplinas. La Unidad de Genómica trabaja en diseñar las estrategias que permitan la mayor optimización de los recursos con los que cuenta el hospital, teniendo en cuenta el equipamiento disponible, las prioridades establecidas y la frecuencia de las distintas patologías. Se demuestra el salto significativo operado en nuestras capacidades diagnósticas, tanto en la variedad de enfermedades como en el número de genes analizados, habiendo estudiado a la fecha alrededor de 2.000 pacientes, muchos de los cuales ven de este modo finalizada su odisea diagnóstica. Los estudios de NGS se han convertido en una herramienta de la práctica diaria para la atención de un número importante de pacientes de nuestro hospital. Continuaremos trabajando para ampliar su aplicación a la mayor cantidad de patologías, a través de los mecanismos institucionales ya existentes (AU)

The Garrahan Hospital has been a pioneer in the molecular diagnosis of pediatric diseases in Argentina. The technological advances of the last decades in the area of molecular biology have laid the foundations for the optimization and expansion of molecular diagnostics through massive parallel sequencing of multiple genes. This study describes the process of implementation of next-generation sequencing studies and the development of the Genomics Unit in a public pediatric tertiary hospital, and its impact on the capacity to diagnose rare diseases of genetic origin. The creation of the Interdisciplinary Group of Genomic Studies constituted the institutional pathway for decision-making involving the implementation of new genomic studies and the establishment of diagnostic priorities, extending the availability of molecular diagnostics to additional disciplines. The Genomics Unit is working to design strategies that allow for optimization of the resources available to the hospital, taking into account the equipment available, the priorities established, and the frequency of the different diseases. It demonstrates the significant leap in our diagnostic capabilities, both in the variety of diseases and in the number of genes analyzed. To date, around 2,000 patients have been studies, many of whom have thus completed their diagnostic odyssey. NGS studies have become a tool in daily practice for the care of a significant number of patients in our hospital. We will continue working to expand its application to as many diseases as possible, through the existing institutional mechanisms (AU)

Next-generation sequencing-based genetic testing and phenotype correlation in retinitis pigmentosa patients from India

Purpose: Inherited retinal dystrophies (IRD) are a heterogeneous group of retinal diseases leading to progressive loss of photoreceptors through apoptosis. Retinitis pigmentosa (RP) is considered the most common form of IRD. Panel?based testing in RP has proven effective in identifying the causative genetic mutations in 70% and 80% of the patients. This is a retrospective, observational, single?center study of 107 RP patients who had undergone next?generation sequencing?based targeted gene panel testing for IRD genes. These patients were inspected for common phenotypic features to arrive at meaningful genotype–phenotype correlation. Methods: Patients underwent complete ophthalmic examination, and blood was collected from the proband for DNA extraction after documenting the pedigree. Targeted Next Generation Sequencing (NGS) was done by panel?based testing for IRD genes followed by co?segregation analysis wherever applicable. Results: Of the 107 patients, 72 patients had pathogenic mutations. The mean age of onset of symptoms was 14 ± 12 years (range: 5–55). Mean (Best Corrected Visual Acuity) BCVA was 6/48 (0.9 logMAR) (range 0.0–3.0). At presentation, over one?third of eyes had BCVA worse than 6/60 (<1 logMAR). Phenotype analysis with the gene defects showed overlapping features, such as peripheral well?defined chorioretinal atrophic patches in patients with CERKL, PROM1, and RPE65 gene mutations and large macular lesions in patients with RDH12 and CRX gene mutations, respectively. Nummular or clump?like pigmentation was noted in CRB1, TTC8, PDE6A, and PDE6B. Conclusion: NGS?based genetic testing can help clinicians to diagnose RP more accurately, and phenotypic correlations can also help in better patient counselling with respect to prognosis and guidance regarding ongoing newer gene?based therapies.

Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India

Background & objectives: Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Methods: Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. Results: EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Interpretation & conclusions: Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

Neonatal ureaplasma meningitis: a report of 2 cases and literature review / 中国新生儿科杂志

Objective:To study the clinical features and treatment strategy of neonatal ureaplasma meningitis.Methods:During 2021, the clinical data of 2 neonates with ureaplasma meningitis treated in Children's Hospital of Hunan Province were retrospectively analyzed. Literature on this subject were searched in the following databases: CNKI, Wanfang Database, Chinese Medical Journal Full-Text Database, CQVIP database, SinoMed, PubMed, Embase and Web of Science (up to March 2022). The key words included “infant”, “neonate”, “newborn”, “ureaplasma”, “mycoplasma urealytium”, “meningitis”, “central nervous system infection”, “brain”. The clinical data, treatment and prognosis of patients from the literature were summarized.Results:Case 1, female, gestational age(GA) 33 +3 weeks, intracranial hemorrhage (ICH) and ventricular dilatation were found on 2 d after birth. The cerebrospinal fluid (CSF) routine and biochemistry tests indicated meningitis, but the CSF culture was negative. No improvement after antibiotic treatment. CSF metagenomic next-generation sequencing (mNGS) and 23S rRNA showed Ureaplasma urealyticum on 30 d after birth. The patient was treated with doxycycline (DOX) for 21 d until mNGS turned negative and DOX was discontinued. However, the disease recurred 23 d later and erythromycin was added with DOX as combined therapy. The patient was followed up until 6 months without neurodevelopmental disabilities. Case 2, male, GA 26 weeks, ICH and ventricular dilatation were found on 10 d after birth. The CSF routine and biochemistry tests indicated meningitis, but the CSF culture was negative. No improvement after antibiotic treatment. CSF mNGS and 23S rRNA showed Ureaplasma parvum. The patient received erythromycin therapy for 32 d and had normal neurodevelopment at 5 months. According to the literature, 43 cases were reported including the 2 cases descirbed above, 17 cases were full-term infants and 26 cases were preterm infants. The median CSF leukocytes, glucose and proteins were 566 cells/mm 3, 0.2 mmol/L and 2.2 g/L. 27 cases were diagnosed based on CSF culture, 6 cases using mNGS, 4 cases with both CSF culture and PCR method and 6 cases with other methods. Macrolides alone were used in 14 cases, macrolides combined with another antibiotic were used in 8 cases, non-macrolide antibiotics were used in 9 cases and 12 cases didn't receive any anti-ureaplasma therapy. All 17 term infants survived, however, 8 cases with hydrocephalus. Among the 26 preterm infants, 8 patients died, 18 patients had periventricular-intraventricular hemorrhage and 15 patients had hydrocephalus. Conclusions:Neonatal ureaplasma meningitis has significantly lower CSF glucose level with hydrocephalus as the common complication. For intracranial infections of unknown etiology and no response to treatment, mNGS is helpful in determining the pathogen.Neonatal ureaplasma meningitis should be treated with macrolides alone or as add-on therapy.

Clinical feature and etiological analysis of 101 neonates with central nervous system infection / 中国小儿急救医学

Objective:To analyze the clinical features and etiological results of neonatal central nervous system(CNS) infection and provide basis for optimization of pathogen detection strategy for CNS infection.Methods:We collected the clinical and laboratory data of hospitalized neonates with clinical diagnosis of CNS infection in the neonatal department at Hebei Provincial Children′s Hospital, from January 1, 2020 to August 31, 2021.The clinical manifestations of the enrolled neonates, as well as the cerebrospinal fluid(CSF)pathogens detected by conventional and molecular biological detection techniques were analyzed.Laboratory characteristics of different kinds of pathogen were compared.Results:A total of 101 eligible neonates were enrolled.The median gestational age was 38.8(36.2, 39.6)weeks, with a prematurity rate 26.7%.There were 68 boys.The median age of onset was 9(2, 14)days.Blood culture was positive in 19(18.8%) cases, including 17 cases of bacteria and two cases of fungus.Positive findings were found in CSF specimens of 33(32.7%)cases by various methods including 13 bacteria, 19 viruses and one fungi.Streptococcus group B and Escherichia coli were the first two bacteria in CSF.Enterovirus was the most common virus in CSF.In terms of detection methods of CSF pathogens, seven cases(7/101, 6.9%) were detected by CSF culture, two cases(2/21, 9.5%)by smear, 22 cases(22/45, 48.9%)by single-virus targeted/multiplex polymerase chain reaction and four cases(4/7, 57.1%)by metagenomic next-generation sequencing.The CSF white blood cell counts, protein levels and blood C-reactive protein levels were higher in the cases with bacteria/fungi detection from CNS infection than in those with virus detection( P<0.05). Almost all neonates(98/101, 97.0%)were clinically cured or significantly improved before discharge.Two neonates were discharged against medical advice and one neonate was transferred to the other hospital after clinical improvement. Conclusion:Combined use of conventional and molecular biological detection techniques can significantly improve the etiological positive rate of neonatal CNS infection.Viral infection is not rare in the neonatal population.Our study demonstrated the spectrum of organism causing neonatal CNS infection, which provided a basis for the optimization of pathogen detection strategy.

Clinical significance of next-generation sequencing-based IGH/IGK gene rearrangement analysis in the diagnosis of minimal residual disease of children with acute B-cell lymphoblastic leukemia / 中华实用儿科临床杂志

Objective:To assess the clinical significance of next-generation sequencing (NGS)-based IGH/ IGK gene rearrangement analysis versus flow cytometry (FCM) in diagnosing minimal residual disease (MRD) of children with acute B-cell lymphoblastic leukemia (B-ALL). Methods:Clinical data, NGS-MRD and FCM-MRD findings at the initial diagnosis and after induction chemotherapy of 85 children diagnosed as B-ALL in Children′s Hospital of Nanjing Medical University from July 2019 to July 2021, were retrospectively analyzed.The sensitivity of the two methods, and the positive rate were compared by χ2 test or Fisher′ s test.The correlation was identified by Spearman correlation analysis. Results:Dominant clone sequences were detected in all children at the initial diagnosis by NGS, while selection markers were identified by FCM in 75(88.2%) patients.Positive MRD rate detected by NGS-MRD was significantly higher than that of FCM-MRD at the same time point after induction chemotherapy[31.8%(27/85) vs.9.4%(8/85), P<0.001]. Compared with those of FCM-MRD, NGS-MRD had good sensitivity (100.0%), specificity (75.3%) and negative predictive value (100.0%), and the positive predictive value was 29.6%.MRD results detected by NGS were consistent with that of FCM ( r=0.569, P<0.001). By July 27, 2022, 2 patients with NGS-MRD (+ )FCM-MRD (-)relapsed during maintenance chemotherapy. Conclusions:NGS is highly consistent with FCM in the detection of MRD in children with B-ALL, which is more sensitive.The combination of NGS-MRD and FCM-MRD benefits more in monitoring MRD in children with B-ALL after induction chemotherapy.

Advances in international newborn screening / 中华实用儿科临床杂志

Newborn screening is an important national public health policy and measure to reduce birth defects and improve the quality of China′s birth population.In the early 1960s, Dr.Robert Guthrie of the United States invented the first newborn screening test, namely, semi-quantitative determination of phenylalanine in dry blood filter paper for screening phenylketonuria.In the 1990s, tandem mass spectrometry (MS/MS) began to be applied to the screening of genetic metabolic diseases in newborns.This technology enabled the detection of multiple diseases by one test, and increased the types of diseases detected.In the past 10 years, with the development of screening technology, the invention of new drugs, the improvement of treatment methods, and especially the application of new technologies such as newborn genetic screening, the source of mutations can be identified at the molecular level.Moreover, newborn screening is extended to patients who are not candidates for MS/MS.Many genetic diseases are able to be screened and diagnosed early.Effective management and quality control of newborn disease screening are prerequisites for improving the quality and accuracy of results.Secondary and multi-level detection strategies, different biochemical or biochemical genetic testing methods, and the integration of targeted and non-targeted multi-omics data have a wide range of applications and great clinical value.

Application of high-throughput next-generation sequencing in diagnosis and treatment of myeloid hematologic malignancies in the era of precision medicine / 白血病·淋巴瘤

With the wide application of high-throughput next-generation sequencing (NGS) and other molecular genetic detection technologies, researchers have a more and more in-depth understanding of the pathogenesis of hematologic malignancies, especially of the myeloid hematologic malignancies, which makes the diagnosis and treatment of myeloid hematologic malignancies into an era of precision medicine. At the 64th American Society of Hematology (ASH) Annual Meeting in 2022, there were a series of new progresses regarding the application of NGS in the diagnosis and classification, risk stratification, treatment guidance, and minimal residual disease monitoring of myeloid hematologic malignancies. This article focuses on the progress of NGS application in acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms.

Clinical Application of the Next Generation Sequencing for Molecular Classification in Endometrial Carcinomas / 中山大学学报(医学科学版)

ObjectiveTo explore the clinical application of molecular classification in endometrial cancers with the next generation sequencing （NGS）. MethodsTotally 112 cases of endometrial carcinoma diagnosed by pathology in The Sun Yat-sen University Cancer Center were collected. All of them were tested by hybridized-capture second-generation sequencing based on 1，021 gene panel. The molecular variation spectrum of each subtype and its relationship between the clinicopathological features were analyzed. ResultsThe cases were distributed as follows： 8 （7.1%） POLE mutation， 34 （30.4%） mismatch repair deficient， 26 （23.2%） TP53 mutation， 44 （39.3%） non-specific molecular profile. The median tumor mutation burden was respectively 252.0， 38.4， 5.8 and 5.4 Muts/Mb. There were no significantly differences among four subtypes in clinicopathological features such as age， histological grade， lymph node metastasis and clinical stage. PTEN （75.5%）， PIK3CA （66.7%）， ARID1A （55.9%）， TP53 （40.2%）， NF1 （29.4%） were the most common mutations in endometrial cancers. ConclusionsThe utilization of NGS in endometrial cancers can simultaneously identify molecular subgroups， screen Lynch syndrome and obtain molecular variation spectrum， which can provide guidance for immunotherapy and targeted therapy， contribute to further accumulation and exploration of molecular genetic characteristics.

Clinical Characteristics and Diagnostic Methods of Pulmonary Mucormycosis / 中山大学学报(医学科学版)

ObjectiveTo summarize the clinical features and prognosis of pulmonary mucormycosis （PM） in southern China， and to explore the diagnostic value of metagenomic next generation sequencing （mNGS） in PM. MethodsThe clinical manifestations， diagnosis， treatment and prognosis of patients diagnosed with PM in The First Affiliated Hospital of Sun Yat-sen University from January 1， 2019 to January 31， 2022 who had undergone mNGS detection in lung tissue or alveolar lavage fluid were collected retrospectively. A total of 14 patients with PM were included， including 4 patients with confirmed diagnosis and 10 patients with clinical diagnosis. ResultsAll patients had underlying medical conditions， with hematological malignancies and diabetes being the most common. The most common symptoms were fever （n = 10）， cough （n = 9） and shortness of breath （n = 9）. Consolidation was the most common sign of chest CT， followed by mass， mostly with cavity. On laboratory tests， decreased CD4+T lymphocytes， elevated CD8+T lymphocytes， and decreased CD4+/CD8+ ratio， and presentation with pleural effusion indicate poor prognosis. The positive rate of mNGS diagnosis was 78.5%， which was significantly higher than that of histopathology （50%）， fungus rapid fluorescence staining （61.5%） and fungal culture （23.1%） of bronchoalveolar lavage fluid. ConclusionsPulmonary mucormycosis is more likely to occur in patients with underlying diseases or who are immunocompromised. The clinical manifestations lack specificity. The low CD4/CD8 ratio and presentation of pleural effusion on CT imaging indicate poor prognosis of patients. mNGS is a rapid， convenient and sensitive method for the diagnosis of PM， which has advantages in the diagnosis of pulmonary mucormycosis.

Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC / 医学前沿

Anaplastic lymphoma kinase (ALK) is the most common fusion gene involved in non-small cell lung cancer (NSCLC), and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors (ALK-TKIs). However, the clinical efficacy is highly variable. Pre-existing intratumoral heterogeneity (ITH) has been proven to contribute to the poor treatment response and the resistance to targeted therapies. In this work, we investigated whether the variant allele frequencies (VAFs) of ALK fusions can help assess ITH and predict targeted therapy efficacy. Through the application of next-generation sequencing (NGS), 7.2% (326/4548) of patients were detected to be ALK positive. On the basis of the adjusted VAF (adjVAF, VAF normalization for tumor purity) of four different threshold values (adjVAF < 50%, 40%, 30%, or 20%), the association of ALK subclonality with crizotinib efficacy was assessed. Nonetheless, no statistical association was observed between median progression-free survival (PFS) and ALK subclonality assessed by adjVAF, and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib. Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.

Clinical and genetic diagnosis for 26 paitents with hereditary spherocytosis / 中南大学学报(医学版)

OBJECTIVES@#Hereditary spherocytosis (HS) is the most common hereditary defect of the red cell membrane, mainly characterized by anemia, jaundice, and splenomegaly. Due to the atypical clinical manifestations and negative family history of some patients, as well as the low sensitivity and specificity of traditional laboratory examinations, it is easy for it to escape diagnosis or be misdiagnosed. At present, it has been confirmed that the mutation of ANK1, SPTB, SPTA1, SLC4A1 and EPB42 genes can cause the deletion of their corresponding coding proteins, and thus lead to the defect of erythrocyte membrane. This study aims to analyze the feasibility and clinical application value of HS gene diagnosis.@*METHODS@#Data of 26 patients from Hunan, China with HS admitted to the Department of Hematology, Second Xiangya Hospital of Central South University from January 2018 to September 2021 were retrospectively collected, and their clinical manifestations and results of laboratory examinations were analyzed. Next-generation sequencing (NGS) combined with Sanger sequencing were applied. The mutation of HS pathogenic gene and the variation of uridine diphosphate-glucuronosyl transferase 1 family polypeptide A1 (UGT1A1), a key enzyme in the regulation of bilirubin metabolism, were detected. The results of pathogenic gene variations were interpreted pathogenic gene variations in accordance with the Standards and guidelines for the interpretation of sequence variants published by the American College of Medical Genetics and Genomics (ACMG). The clinical characteristics of patients with different gene variants were analyzed, and the clinical diagnosis and genetic diagnosis were compared.@*RESULTS@#Among the 26 patients with HS, there were 23 cases of anemia, 25 cases of jaundice, 24 cases of splenomegaly, and 14 cases of cholelithiasis. There were 16 cases with family history and 10 cases without family history. The results of HS mutation test were positive in 25 cases and negative in 1 case. A total of 18 heterozygous mutations of HS pathogenic genes were detected in 19 families, among which 14 were pathogenic, 1 was likely pathogenic and 3 were of unknown significance. SPTB mutations (12) and ANK1 mutations (4) were the most common. The main variation types were nonsense mutation (9). There were no significant differences in peripheral blood cell parameters and hemolysis indicators between the SPTB mutant group and the ANK1 mutant group (all P>0.05). The rate of splenectomy in ANK1 mutation group was higher than that in SPTB mutation group, and the difference was statistically significant (χ2=6.970, P=0.014). There were no significant differences in peripheral blood cell parameters and hemolysis indicators among different mutation types (nonsense mutation, frameshift mutation, splice site mutation and missense mutation) (all P>0.05). Among the 18 clinically confirmedpatients, there were 17 cases whose diagnosis is consistent with the genetic diagnosis. Eight patients were clinically suspected, and all of them were confirmed by detection of HS gene mutation. Twenty-four patients with HS underwent UGT1A1 mutation detection, among which 5 patients carried UGT1A1 mutation resulting in a decrease in enzyme activity, and 19 patients had normal enzyme activity. The level of total bilirubin (TBIL) in the group with reduced enzyme activity was higher than that in the group with normal enzyme activity, and the difference was statistically significant (U=22, P=0.038).@*CONCLUSIONS@#Most patients with HS have anemia, jaundice and splenomegaly, often accompanied by cholelithiasis. SPTB and ANK1 mutations are the most common mutations in HS pathogenic genes among patients in Hunan, China, and there was no significant correlation between genotype and clinical phenotype. Genetic diagnosis is highly consistent with clinical diagnosis. The decrease of UGT1A1 enzyme activity can lead to the aggravation of jaundice in HS patients. Clinical combined gene diagnosis is beneficial for the rapid and precision diagnosis of HS. The detection of UGT1A1 enzyme activity related gene variation plays an important role in evaluation of HS jaundice.

Analysis of Gene Recombination between HLA-B and -DRB1, HLA-DQB1 and -DPB1 Loci / 中国实验血液学杂志

OBJECTIVE@#To investigate the recombinations within the human leukocyte antigen (HLA) region in two families.@*METHODS@#Genomic DNA was extracted from the peripheral blood specimens of the different family members. HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci were genotyped using polymerase chain reaction-sequence specific oligonucleotide probing technique (PCR-SSO) and next-generation sequencing technique. HLA haplotype was determined by genetic analysis of the pedigree.@*RESULTS@#The haplotypes of HLA-A*11:01~C*03:04~B*13:01~DRB1*12:02~DQB1*03:01~DPB1*05:01:01G and HLA-A*03:01~C*04:01~B*35:03~DRB1*12:01~DQB1*03:01~DPB1*04:01:01G in the family 1 were recombined between HLA-B and HLA-DRB1 loci, which formed the haplotype of HLA-A*11:01~C*03:04~B*13:01~DRB1* 12:01~DQB1*03:01~DPB1*04:01:01G. The haplotypes of HLA-A *02:06~C*03:03~B*35:01~DRB1*08:02~DQB1*04:02~ DPB1*13:01:01G and HLA-A *11:01~C*07:02~B*38:02~DRB1*15:02~DQB1*05:01~DPB1*05:01:01G in the family 2 were recombined between HLA-DQB1 and HLA-DPB1 loci, which formed the haplotype of HLA-A*02:06~C*03:03~B*35:01~ DRB1*08:02~DQB1*04:02~DPB1*05:01:01G.@*CONCLUSION@#The gene recombination events between HLA-B and -DRB1, HLA-DQB1 and -DPB1 loci were found respectively in two Chinese Han families.

Research Progress of Next Generation Sequencing in Acute Leukemia --Review / 中国实验血液学杂志

With the advent of precision medicine, next-generation sequencing (NGS) is playing an increasingly important role in clinical oncology diagnosis and treatment with its advantages of high sensitivity, high accuracy, high efficiency and operability. NGS reveals the genetic characteristics of acute leukemia(AL) patients by screening for specific disease-causing genes to identify occult as well as complex genetic mutations in patients with AL, leading to early diagnosis and targeted drug therapy for AL patients, as well as to predict disease recurrence by detecting mnimal residual disease (MRD) and analyzing mutated genes to determine patient prognosis. NGS plays an increasingly important role in the diagnosis, treatment and prognosis assessment in AL, providing a direction for the pursuit of precision medicine. This paper reviews the research progress of NGS in AL.

Mutational Spectrum and Prognosis Analysis of Young Patients with Diffuse Large B-Cell Lymphoma Based on Next-Generation Sequencing / 中国实验血液学杂志

OBJECTIVE@#To investigate the mutational spectrum in young patients with diffuse large B-cell lymphoma (DLBCL) based on next generation sequencing (NGS), and to provide a basis for in-depth understanding of the molecular biological characteristics and accurate prognosis of young DLBCL.@*METHODS@#From March 2009 to March 2021, 68 young DLBCL patients with complete initial diagnosis data from the Department of Hematology, The People's Hospital Xinjiang Uygur Autonomous Region were retrospectively analyzed, and their paraffin-embedded tissues were subjected to targeted sequencing analysis by NGS technology (including 475 Target genes), and the differences in gene mutation profiles and signaling pathways between high-risk patients with aaIPI ≥2 and low-intermediate risk patients with aaIPI <2 were compared.@*RESULTS@#A total of 44 high-frequency mutation genes were detected in 68 young DLBCL patients. By comparing the high-frequency mutation genes in aaIPI high-risk group and low-intermediate risk group, it was found that CARD11 mutation in aaIPI high-risk group was significantly higher than that in low-intermediate risk group (P =0.002), while MGA mutation (P =0.037) only appeared in the aaIPI high-risk group, and SPEN mutation (P =0.004) only appeared in the aaIPI low-intermediate risk group. The high-frequency mutation genes and clinical indicators of the aaIPI high-risk group were included in the survival analysis, and the results showed that TP53 (P =0.009, P =0.027), POU2AF1 (P =0.003, P =0.006) and CCND3 (P =0.040, P =0.014) genes mutations were associated with worse PFS and OS, while B2M was associated with better PFS (P =0.014) and OS (P =0.013). Multivariate COX regression analysis showed that the TP53, POU2AF1 and CCND3 were independent risk factors for PFS（P ＝0.021，P =0.005，P =0.020） and OS（P ＝0.042，P =0.010，P =0.013）.@*CONCLUSION@#The aaIPI staging combination with molecular biology markers is more conducive to accurately judging the prognosis of young DLBCL patients. TP53, POU2AF1 and CCND3 mutations predict worse survival in the patients with the aaIPI high-risk group.

Value of metagenomic next-generation sequencing in children with hematological malignancies complicated with infections / 中国当代儿科杂志

OBJECTIVES@#To explore the value of metagenomic next-generation sequencing (mNGS) in the pathogen identification in children with hematological malignancies complicated with infections.@*METHODS@#A retrospective analysis was conducted on clinical data and pathogenic test results of 43 children with hematological malignancies who underwent microbial culture and mNGS due to infections in the Third Xiangya Hospital of Central South University between June 2020 and July 2022. Differences in detection rates and characteristics of pathogenic microorganisms detected by mNGS and microbial culture were compared.@*RESULTS@#A total of 54 specimens were examined, and the overall detection rate of pathogen by mNGS (80%, 43/54) was significantly higher than that by microbial culture (30%, 16/54) (P<0.001). The most commonly detected infection type by mNGS was viral infection, followed by fungal infection combined viral infection, while that by microbial culture was bacterial infection, followed by fungal infection. The detection rate of fungi by mNGS (33%, 18/54) was higher than that by microbial culture (6%, 3/54) (P<0.001). The detection rate of two or more pathogenic microorganisms by mNGS was higher at 48% compared to microbial culture at 9% (P<0.05). The detection rate of two or more types of pathogenic microorganisms by mNGS was also significantly higher at 33% compared to microbial culture at 2% (P<0.05). The most commonly detected bacteria and fungi by mNGS were Pseudomonas aeruginosa and Candida tropicalis, respectively, in peripheral blood, while Streptococcus pneumoniae and Pneumocystis jirovecii were most commonly detected in bronchoalveolar lavage fluid. Treatment adjustments based on mNGS results were beneficial for 35% (15/43) of the cases.@*CONCLUSIONS@#mNGS has a higher detection rate than microbial culture and has obvious advantages in diagnosing mixed and fungal infections, making it a useful supplementary diagnostic method to microbial culture.

Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP / 中国实验血液学杂志

OBJECTIVE@#To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method.@*METHODS@#All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology.@*RESULTS@#A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database.@*CONCLUSION@#The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.