¿Qué hay de nuevo en el diagnóstico de amiloidosis? / What is new in diagnosis of amyloidosis?

La amiloidosis siempre ha representado un desafío diagnóstico. En el año 2020, el Grupo de Estudio de Amiloidosis (GEA), confeccionó la Guía de Práctica Clínica para el Diagnóstico de Amiloidosis. Nuevas líneas de investigación se han desarrollado posteriormente. Esta revisión narrativa tiene como intención explorar el estado del arte en el diagnóstico de la amiloidosis. En pacientes con amiloidosis se recomienda la tipificación de la proteína mediante espectrometría de masa, técnica de difícil ejecución por requerir de microdisectores láser para la preparación de la muestra. Algunas publicaciones recientes proponen otros métodos para obtener la muestra de amiloide que se va a analizar, permitiendo prescindir de la microdisección. Por otra parte, en pacientes con Amiloidosis ATTR confirmada, la recomendación de secuenciar el gen amiloidogénico se encontraba destinada a los casos sospechosos de ATTR hereditaria (ATTRv,), pero actualmente esta se ha extendido a todos los pacientes sin importar la edad. En lo que respecta a los estudios complementarios orientados al diagnóstico de compromiso cardíaco, se ha propuesto el uso de la inteligencia artificial para su interpretación, permitiendo la detección temprana de la enfermedad y el correcto diagnóstico diferencial. Para el diagnóstico de neuropatía, las últimas publicaciones proponen el uso de la cadena ligera de neurofilamento sérica, que también podría resultar un indicador útil para seguimiento. Finalmente, con referencia a la amiloidosis AL, la comunidad científica se encuentra interesada en definir qué características determinan el carácter amiloidogénico de las cadenas livianas. La N-glicosilación de dichas proteínas impresiona ser uno de los determinantes en cuestión. (AU)

Amyloidosis has always represented a diagnostic challenge. In 2020, the Amyloidosis Study Group (ASG) developed the "Clinical Practice Guideline for the Diagnosis of Amyloidosis". New lines of research have subsequently emerged. This narrative review aims to explore the state of the art in the diagnosis of amyloidosis diagnosis. In patients with amyloidosis, protein typing by mass spectrometry is recommended, a technique hard to perform because it requires laser microdissection for sample preparation. Recent publications propose other methods to obtain the amyloid sample to be analyzed, making it possible to dispense with microdissection. On the other hand, in patients with confirmed TTR amyloidosis (aTTR), the recommendation to sequence the amyloidogenic gene was intended for suspected cases of hereditary aTTR but has now been extended to all patients regardless of age. (AU)

Hallazgos incidentales en medicina genómica / Incidental Findings in Genomic Medicine

Las técnicas de Biología Molecular de última generación, como es la secuenciación masiva en paralelo o NGS (Next Generation Sequencing), permite obtener gran cantidad de información genómica, la cual muchas veces va más allá de la detección de una variante patogénica en un gen que explique la patología (hallazgo primario). Es así como surgió desde hace años la discusión internacional respecto a la decisión a tomar frente a los hallazgos secundarios accionables, es decir, aquellos hallazgos de variantes clasificadas como patogénicas o probablemente patogénicas que no están relacionadas con el fenotipo del paciente, pero que tiene alguna medida preventiva o tratamiento posible y, por lo tanto, podría ser de utilidad para la salud del paciente. Luego de revisar la bibliografía internacional y debatir entre los expertos del Hospital de Pediatría Garrahan, se logró establecer una política institucional y reforzar el hecho de que se trata de una disciplina multidisciplinaria. Así, fue posible definir que solo se atenderá las cuestiones relacionadas con la edad pediátrica, dejando para un tratamiento posterior aquellas variantes detectadas en genes que sean accionables en edad adulta. En el Hospital Garrahan, ha sido posible definir claramente cómo proceder frente a los hallazgos secundarios, al adaptar el consentimiento informado a esta necesidad, definiendo cuándo serán informados, y sabiendo que serán buscados intencionalmente en los genes clínicamente accionables enlistados en la última publicación del American College of Medical Genetics and Genomics, siempre y cuando el paciente/padre/tutor lo consienta (AU)

The latest generation of molecular biology techniques, including massive parallel sequencing or NGS (Next Generation Sequencing), allows us to obtain a whealth of genomic information, which often goes beyond the detection of a pathogenic variant in a gene that explains the pathology (primary finding). As a result, an international discussion has arisen over the years regarding the decision-making concerning actionable secondary findings, it means, those findings of variants classified as pathogenic or probably pathogenic that are not related to the patient's phenotype, but which have some possible preventive measure or treatment and, therefore, could be useful for the patient's health. After reviewing the international literature and discussing among the experts of the Hospital de Pediatría Garrahan, an institutional policy was established and the concept that this is a multidisciplinary discipline was reinforced. Consequently, it has been defined that only issues related to children will be addressed, reserving those variants detected in genes that are actionable in adulthood for later treatment. At Garrahan Hospital, we were able to clearly define how to proceed with secondary findings by adapting the informed consent to this need, defining when they will be reported, and knowing that they will be intentionally searched for in the clinically actionable genes listed in the latest publication of the American College of Medical Genetics and Genomics, as long as the patient/parent/guardian consents (AU)

Uso de PCR digital para definir terapéuticas específicas y seguimiento en enfermedades autoinflamatorias / Use of digital PCR to define specific therapeutics and follow-up in autoinflammatory diseases

Las enfermedades autoinflamatorias (AIDs) son un grupo heterogéneo de desórdenes monogénicos o poligénicos, con características de disregulación inmune innata y/o adaptativa, cuyo mecanismo central es la autoinflamación pero también pueden presentarse con autoinmunidad e inmunodeficiencia. En estos últimos años el desarrollo de las tecnologías de secuenciación masiva han provocado una explosión en el descubrimiento de nuevos genes responsables de AIDs monogénicas. Esto remarca la importancia de implementar este tipo de estudios para llegar a un diagnóstico definitivo sobre todo en este grupo de patologías genéticamente muy diversas donde los fenotipos clínicos se solapan. Sin embargo, dada la presencia de variantes de significación incierta (VUS), los resultados pueden no ser concluyentes planteándose la necesidad de desarrollar pruebas funcionales para determinar la patogenicidad de dichas variantes genéticas. En nuestro grupo de trabajo estamos aplicando la PCR digital en gotas (ddPCR), una técnica cuantitativa de 3era generación altamente sensible, especifica y reproducible que no necesita de curvas de calibración, para desarrollar pruebas funcionales que permitan no sólo reclasificar variantes VUS para lograr diagnósticos definitivos sino también estudiar los mecanismos responsables de las principales AIDs que permitan una estratificación de las terapéuticas especificas a aplicar y de esta manera poder contribuir al diagnóstico, tratamiento y seguimiento de nuestros pacientes en forma personalizada. (AU)

Autoinflammatory diseases (AIDs) are a heterogeneous group of monogenic or polygenic disorders, with characteristics of inborn and/or adaptive immune dysregulation, whose central mechanism is autoinflammation but may also present with autoimmunity and immunodeficiency. In recent years the development of massive sequencing technologies has led to an exponential increase in the discovery of new genes responsible for monogenic AIDs. This emphasizes the importance of the implementation of this type of studies to make a definitive diagnosis, especially in this group of genetically very diverse diseases with overlapping clinical phenotypes. However, given the presence of variants of uncertain significance (VUS), the results may not be conclusive, raising the need to develop functional tests to determine the pathogenicity of these genetic variants. In our working group we are applying droplet digital PCR (ddPCR), a highly sensitive, specific and reproducible third generation quantitative technique that does not require calibration curves, to develop functional tests that allow not only to reclassify VUS variants to achieve definitive diagnoses but also to study the mechanisms responsible for the main AIDs that allow for the stratification of specific treatments to be used and thereby contribute to the individualized diagnosis, treatment, and follow-up of our patients (AU)

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)

Muerte súbita y secuenciación de siguiente generación. Revisión bibliográfica / Sudden death and next generation sequencing. A review

Introducción: la muerte súbita se trata de un evento fatal e imprevisible. Realizada la autopsia y estudios complementarios, en ausencia de otros hallazgos que expliquen la causa de muerte, se clasifica como muerte súbita inexplicada. Siendo recomendable en estos casos realizar análisis genéticos, especialmente con metodologías de secuenciación de siguiente generación, las que permiten explicar un porcentaje importante de estos casos. Objetivo: analizar las publicaciones más relevantes sobre secuenciación de siguiente generación, aplicada a la autopsia molecular, para determinar aquellas muertes súbitas inexplicables relacionadas a cardiomiopatías y canalopatías. Metodología: se realizó la búsqueda en PubMed del Instituto Nacional de Salud usando palabras clave en inglés y español: NGS, muerte súbita, autopsia molecular y sus combinaciones. Además, se realizaron búsquedas en OMIN y ClinVar relacionada a las diferentes afecciones cardiacas relacionadas a muerte súbita. Los criterios de inclusión: artículos completos en español e inglés de libre acceso, con antigüedad máxima de diez años, realizados en cualquier área geográfica y que trataran sobre la temática. Resultados: para secuenciación de siguiente generación, muerte súbita se encontraron más de 22000 y 65000 publicaciones, respectivamente. En cambio, al combinar las palabras clave se recuperaron 74 trabajos, según los criterios de inclusión y objetivo del trabajo se revisaron 67 artículos. La aplicación de las plataformas de secuenciación en la investigación de casos de muerte súbita tomo auge en el 2014 y en poco tiempo, demostraron su versatilidad para el análisis de una gran cantidad de genes simultáneamente, de forma rápida y a bajo costo. Conclusiones: las patologías asociadas a muerte súbita son múltiples, complejas y pueden generar fenotipos variables que dificultan el análisis genético de las mismas. Las plataformas de secuenciación de siguiente generación son sumamente útiles en los casos de muerte súbita inexplicada, además permiten la identificación de variantes genéticas en familiares para la implementación de medidas preventivas...(AU)

Community structure of endophytic fungi in roots and leaves of Fagopyrum mill and Avena sativa in a Chinese northern cold region

In order to explore the endophytic fungi of Fagopyrum Mill and Avena sativa, Illumina Miseq high-throughput sequencing was used to analyze the community structure and diversity of endophytic fungi in leaves and roots of buckwheat and oat at the mature stage. The results of community structure showed that there were 205 operational taxonomic units (OTUs) in buckwheat roots and 181 OTUs in buckwheat leaves based on 97% sequence similarity level. There were 152 OTUs and 127 OTUs in the root and the leaf of oat, respectively. At the phylum level, Ascomycota and Basidiomycota were the dominant endophytic fungi in buckwheat roots and leaves, while Ascomycota was the dominant endophytic fungus in oat roots and leaves. Alpha diversity analysis showed that the Ace index, Chao index and Shannon index of buckwheat roots were higher than that of buckwheat leaves, and the three indices of oat roots were also higher than that of oat leaves, indicating that the richness and diversity of endophytic fungi community in roots were higher than that in leaves. Biomarkers were found by significant difference analysis in buckwheat and oat. The endophytic functional groups of buckwheat and oat were mainly distributed in Pathotroph and Saprotroph. The results of this study laid a foundation for fully exploiting the dominant endophytic fungal resources of buckwheat and oat and further developing microbial fertilizers.

Application of deep mutational scanning technology in protein research / 生物工程学报

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.

Circulating Exosomal LncRNAs as Novel Diagnostic Predictors of Severity and Sites of White Matter Hyperintensities / 生物医学与环境科学（英文）

OBJECTIVE@#Exosomal long noncoding RNAs (lncRNAs) are the key to diagnosing and treating various diseases. This study aimed to investigate the diagnostic value of plasma exosomal lncRNAs in white matter hyperintensities (WMH).@*METHODS@#We used high-throughput sequencing to determine the differential expression (DE) profiles of lncRNAs in plasma exosomes from WMH patients and controls. The sequencing results were verified in a validation cohort using qRT-PCR. The diagnostic potential of candidate exosomal lncRNAs was proven by binary logistic analysis and receiver operating characteristic (ROC) curves. The diagnostic value of DE exo-lncRNAs was determined by the area under the curve (AUC). The WMH group was then divided into subgroups according to the Fazekas scale and white matter lesion site, and the correlation of DE exo-lncRNAs in the subgroup was evaluated.@*RESULTS@#In our results, four DE exo-lncRNAs were identified, and ROC curve analysis revealed that exo-lnc_011797 and exo-lnc_004326 exhibited diagnostic efficacy for WMH. Furthermore, WMH subgroup analysis showed exo-lnc_011797 expression was significantly increased in Fazekas 3 patients and was significantly elevated in patients with paraventricular matter hyperintensities.@*CONCLUSION@#Plasma exosomal lncRNAs have potential diagnostic value in WMH. Moreover, exo-lnc_011797 is considered to be a predictor of the severity and location of WMH.

High-Throughput Sequencing Technology for Detection of Gene Mutations in Myeloid Malignancies and Its Clinical Prognostic Significance / 中国实验血液学杂志

OBJECTIVE@#To detect the gene mutations in patients with myeloid malignancies by high-throughput sequencing and explore the correlation between gene mutations and prognosis.@*METHODS@#A retrospective analysis was performed on 56 patients with myeloid malignancies who were hospitalized in the department of hematology, Peking University International Hospital from January 2020 to May 2021. The genetic mutations of the patients were detected by next-generation sequencing technology, and the correlation between the genetic mutations and prognosis of myeloid malignancies was analyzed.@*RESULTS@#In 56 patients, the number of mutated genes detected in a single patient is 0-9, with a median of 3. Sequencing results showed that the most common mutated genes were RUNX1(21.4%), TET2(17.9%), DNMT3A(17.9%), TP53(14.3%) and ASXL1(14.3%), among which the most common mutations occurred in the signaling pathway-related genes (23.3%) and the transcription factor genes (18.3%). 84% of the patients carried multiple mutated genes (≥2), and correlation analysis showed there were obvious co-occurring mutations between WT1 and FLT3, NPM1 and FLT3-ITD, and MYC and FLT3. TP53 mutation was more common in MDS patients.The overall survival time of patients with NRAS mutation was significantly shortened (P =0.049). The prognosis of patients with TP53 mutation was poor compared with those without TP53 mutation, but the difference wasn't statistically significant (P =0.08).@*CONCLUSION@#The application of next-generation sequencing technology is of great significance in myeloid malignancies, which is helpful to better understand the pathogenesis of the disease, to judge the prognosis and to find possible therapeutic targets.

Application of metagenomic next-generation sequencing of bronchoalveolar lavage fluid in the diagnosis and treatment of refractory pneumonia in children / 中国当代儿科杂志

OBJECTIVES@#To investigate the clinical application of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in the etiological diagnosis and treatment of refractory pneumonia (RTP) in children.@*METHODS@#A retrospective analysis was performed on 160 children with RTP who were admitted to the Department of Pediatric Internal Medicine, Maternal and Child Health Hospital of Inner Mongolia Autonomous Region, from January 2020 to March 2023. According to whether mNGS was performed, they were divided into two groups: mNGS (n=80) and traditional testing (n=80). All children received the tests of inflammatory markers and pathogen tests after admission. Traditional pathogenicity tests included microbial culture (sputum specimen collected by suction tube), nucleic acid detection of respiratory pathogens, and serological test (mycoplasma, tuberculosis, and fungi). For the mNGS group, BALF specimens were collected after bronchoscopy and were sent to the laboratory for mNGS and microbial culture. The two groups were analyzed and compared in terms of the detection of pathogens and treatment.@*RESULTS@#Compared with the traditional testing group, the mNGS group had a significantly higher detection rate of pathogens (92% vs 58%, P<0.05), with more types of pathogens and a higher diagnostic rate of mixed infections. Compared with the traditional testing group, the mNGS group had a significantly higher treatment response rate and a significantly lower incidence rate of complications during hospitalization (P<0.05). Treatment was adjusted for 68 children in the mNGS group according to the results of mNGS, with a treatment response rate of 96% (65/68) after adjustment.@*CONCLUSIONS@#Compared with traditional pathogen tests, BALF mNGS can significantly improve the detection rate of pathogens and find some rare pathogens. In clinical practice, when encountering bottlenecks during the diagnosis and treatment of children with RTP, it is advisable to promptly perform the mNGS to identify the pathogens.

Traceability of Geographic Origin Using Human Skin and Oral Microbiota / 法医学杂志

OBJECTIVES@#To explore the possibility of using human skin and oral microorganisms to estimate the geographic origin of an individual through the sequencing analysis of bacterial 16S rRNA gene.@*METHODS@#Microbial DNA was extracted from the palm and oral microorganisms of the Han population in Shanghai and Chifeng, Inner Mongolia, and the composition and diversity of the microbiota were analyzed by full-length 16S rRNA gene sequencing. Then, differential species were screened and a geographic location prediction model was constructed.@*RESULTS@#The compositions of palm and oral microorganisms between Shanghai and Chifeng samples were both different. The abundance and uniformity of palm side skin microorganisms were higher in Chifeng samples than in Shanghai samples, while there was no significant difference in oral microorganisms. Permutational multivariate analysis of variance (PERMANOVA) confirmed that th e β -diversity between the samples from the two places were statistically significant, and the coefficients of determination (R2) for skin and oral samples were 0.129 and 0.102, respectively. Through principal co-ordinates analysis (PCoA), the samples from the two places could be preliminarily distinguished. The predictive model had the accuracies of 0.90 and 0.83 for the geographic origin using the skin and oral samples, respectively.@*CONCLUSIONS@#There are differences in the compositions of palm and oral microbiota between Han populations in Shanghai and Chifeng. The prediction model constructed by the random forest algorithm can trace the unknown individuals from the above two places.

Diagnosis of a case with Hermansky-Pudlak syndrome type 5 through high-throughput sequencing and a literature review / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical and genetic characteristics of a patient with Hermansky-Pudlak syndrome type 5 (HPS-5).@*METHODS@#A child with HPS-5 who had attended the Children's Hospital Affiliated to Shandong University on October 3, 2019 was selected as the study subject. Clinical data of the child were collected. Genetic variant was analyzed through high-throughput sequencing. A literature review was also carried out.@*RESULTS@#The child, a 1-year-and-5-month-old girl, had nystagmus since childhood, lost of retinal pigmentation by fundus examination and easy bruising. High-throughput sequencing revealed that she has harbored compound heterozygous variants of the HPS5 gene, namely c.1562_1563delAA (p.F521Sfs*27) and c.1404C>A (p.C468X), which were inherited from his father and mother, respectively. Based on the guidelines from the American College for Medical Genetics and Genomics (ACMG), both variants were predicted to be pathogenic (PVS+PM2_Supporting+PM3+PP4). Among 18 previously reported HPS-5 patients, all had had eye problems, and most of them had tendency for bleeding. Eight cases had carried compound heterozygous variants of the HPS5 gene, 8 carried homozygous variants, 2 carried double homozygous variants, and most of them were null mutations.@*CONCLUSION@#The c.1562_1563delAA(p.F521Sfs*27) and c.1404C>A (p.C468X) compound heterozygous variants of the HPS5 gene probably underlay the HPS-5 in this child. High-throughput sequencing has provided an important tool for the diagnosis. HSP-5 patients usually have typical ocular albinism and/or oculocutaneous albinism and tendency of bleeding, which are commonly caused by compound heterozygous and homozygous variants of the HPS5 gene, though serious complications have been rare.

Analysis of CLCN4 gene variant in a child with Raynaud-Claes syndrome / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical phenotype and genetic variant in a child with Raynaud-Claes syndrome (RCS).@*METHODS@#A child who was diagnosed with RCS at the Children's Hospital Affiliated to Zhengzhou University for delayed language and motor development in August 2022 was selected as the study subject. Clinical data of the child were collected, and potential genetic variant was detected by next-generation sequencing and Sanger sequencing. The pathogenicity of the candidate variant was analyzed.@*RESULTS@#The child, a 4-year-and-4-month-old male, has manifested global developmental delay, speech disorders, special facial features and behavioral abnormalities. Genetic testing revealed that he has harbored a hemizygous c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene, which was not detected in either of his parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as pathogenic (PVS1+PS2+PM2_Supporting).@*CONCLUSION@#The c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene probably underlay the PCS in this child. Above finding has expanded the mutational spectrum of the CLCN4 gene and enabled genetic counseling and prenatal diagnosis for his family.

Analysis of clinical characteristics and genetic variants in a child with Isolated sulfite oxidase deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a child with Isolated sulfite oxidase deficiency (ISOD).@*METHODS@#The child and her parents were subjected to targeted capture and next-generation sequencing. Pathogenicity of candidate variants was assessed based on the guidelines from the American College of Medical Genetics and Genomics (ACMG).@*RESULTS@#The child was found to harbor compound heterozygous variants of the SUOX gene, namely c.1200C>G (p.Tyr400*) and c.1406_1421delCCTGGCAGGTGGCTAA (p.Thr469Serfs*20), which were inherited from her mother and father, respectively. The c.1200C>G was a known pathogenic variant, while the c.1406_1421delCCTGGCAGGTGGCTAA was unreported previously and predicted to be a pathogenic variant (PVS1+PM2_Supporting +PM3) based on the guidelines from the American College of Medical Genetics and Genomics.@*CONCLUSION@#The compound c.1200C>G and c.1406_1421delCCTGGCAGGTGGCTAA variants of the SUOX gene probably underlay the pathogenesis of ISOD in this child. Above finding has expanded the spectrum of SUOX gene variants and provided molecular evidence for the clinical diagnosis and genetic counseling for this pedigree.

UhpTE350Q mutation along with the presence of fosA6/5 genes in the genome probably contributes to inherent fosfomycin resistance of Klebsiella pneumoniae / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism underlying inherent fosfomycin resistance of Klebsiella pneumoniae (K. pneumoniae).@*METHODS@#The draft genomic sequences of 14 clinical hypervirulent/hypermucoviscous K. pneumoniae (HvKP/ HmKP) isolates were obtained using the next-generation sequencing technology. The genomic sequences were analyzed using the Resistance Gene Identifier (RGI) software for predicting the resistome based on homology and SNP models in the Comprehensive Antibiotic Resistance Database (CARD) and for identification of the presence of phosphomycin resistancerelated genes uhpt and fosA and their mutations in the bacterial genomes. The results were verified by analyzing a total of 521 full-length genomic sequences of K. pneumonia strains obtained from GenBank.@*RESULTS@#All the 14 clinical isolates of HvKP/ HmKP carried hexose phosphate transporter (UhpT) gene mutation, in which the glutamic acid was mutated to glutamine at 350aa (UhpTE350Q mutation); the presence of fosA6 gene was detected in 12 (85.71%) of the isolates and fosA5 gene was detected in the other 2 (14.29%) isolates. Analysis of the genomic sequences of 521 K. pneumonia strains from GenBank showed that 508 (97.50%) strains carried UhpTE350Q mutation, 439 (84.26%) strains harbored fosA6, and 80 (15.36%) strains harbored fosA5; 507 (97.31%) strains were found to have both UhpTE350Q mutation and fosA6/5 genes in the genome. Only 12 (2.30%) strains carried fosA6/5 genes without UhpTE350Q mutation; 1 (0.19%) strain had only UhpTE350Q mutation without fosA6/5 genes, and another strain contained neither UhpTE350Q mutation nor fosA6/5 genes.@*CONCLUSION@#UhpTE350Q mutation with the presence of fosA6/5 genes are ubiquitous in K. pneumonia genomes, indicating a possible intrinsic mechanism of fosfomycin resistance in the bacterium to limit the use of fosfomycin against infections caused by K. pneumoniae, especially the multi-resistant HvKP/HmKP strains.

Genomics of next generation sequencing in pediatric B-acute lymphoblastic leukemia and its impact on minimal residual disease / 中华儿科杂志

Objective: To describe the gene mutation profile of newly diagnosed pediatric B-acute lymphoblastic leukemia (B-ALL) and analyze its effect on minimal residual disease (MRD). Methods: A total of 506 newly diagnosed B-ALL children treated in Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from September 2018 to July 2021 were enrolled in this retrospective cohort study. The enrolled children were divided into MRD ≥1.00% group and <1.00% group according to MRD results on the 19th day since chemotherapy, and MRD ≥0.01% group and <0.01% group according to MRD results on the 46th day. Clinical characteristics and gene mutations of two groups were compared. Comparisons between groups were performed with chi-square test or Fisher's exact test. Independent risk factors of MRD results on the 19th day and the 46th day were analyzed by Logistic regression model. Results: Among all 506 patients, there were 318 males and 188 females. On the 19th day, there were 114 patients in the MRD ≥1.00% group and 392 patients in the MRD <1.00% group. On the 46th day, there were 76 patients in the MRD ≥0.01% group and 430 patients in the MRD <0.01% group. A total of 187 gene mutations were detected in 487 (96.2%) of 506 children. The most common gene mutations were signal transduction-related KRAS gene mutations in 111 cases (22.8%) and NRAS gene mutations in 99 cases (20.3%). Multivariate analysis showed that PTPN11 (OR=1.92, 95%CI 1.00-3.63), KMT2A (OR=3.51, 95%CI 1.07-11.50) gene mutations and TEL-AML1 (OR=0.48, 95%CI 0.27-0.87), BCR-ABL1 (OR=0.27, 95%CI 0.08-0.92) fusion genes and age >10 years (OR=1.91, 95%CI 1.12-3.24) were independent influencing factors for MRD ≥1.00% on the 19th day. BCORL1 (OR=2.96, 95%CI 1.18-7.44), JAK2 (OR=2.99, 95%CI 1.07-8.42) and JAK3 (OR=4.83, 95%CI 1.50-15.60) gene mutations and TEL-AML1 (OR=0.43, 95%CI 0.21-0.87) fusion gene were independent influencing factors for MRD ≥0.01% on the 46th day. Conclusions: Children with B-ALL are prone to genetic mutations, with abnormalities in the RAS signaling pathway being the most common. Signal transduction related PTPN11, JAK2 and JAK3 gene mutations, epigenetic related KMT2A gene mutation and transcription factor related BCORL1 gene mutation are independent risk factors for MRD.

Comparative study of next generation sequencing and immunohistochemistry on molecular classification of endometrial carcinoma / 中华病理学杂志

Objective: To investigate the differences in molecular classification of endometrial carcinoma (EC) between various technical methods and to explore molecular classification schemes suitable for Chinese population. Methods: The study used a comprehensive scheme of next generation sequencing (NGS) and immunohistochemistry for molecular classification of 254 EC cases that were obtained at Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China from April 2021 to March 2022. According to the recommended threshold of Sanger sequencing which was approximate-20% variant allele fraction (VAF), NGS data were extracted to simulate the results of Sanger sequencing. Results: The 254 EC patients had a mean age of 51 years (range, 24 to 89 years). Combination of POLE (9-14 exons), TP53 total exons and microsatellite instability (MSI) detection was a better single scheme than NGS alone, while combination of MSI fragment analysis and conventional immunohistochemistry was the best solution and seemed best aligned with TCGA data and recent studies. POLE ultramuted type, mismatch repair defect type, TP53 mutant type and non-specific molecular characteristic type accounted for 11.4% (29/254), 31.5% (80/254), 22.4% (57/254) and 34.6% (88/254) of the cases, respectively. If Sanger sequencing was adopted for POLE and TP53 detection, the frequencies of these EC types were 9.1% (23/254), 31.5% (80/254), 12.9% (33/254) and 46.6% (118/254), respectively, with greatly increasing non-specific molecular characteristics cases. If POLE was detected by Sanger sequencing and others by immunohistochemistry, they were 9.1% (23/254), 42.2% (92/218), 13.8% (35/254) and 40.9% (105/254), respectively, with increasing the false positive rates of the mismatch repair defect group. Conclusions: Small and medium-sized NGS panels with MSI detection is a better solution than NGS alone. Sanger sequencing is currently available for POLE mutation detection, which is not sensitive enough for TP53 mutation detection, and seems equivalent to the efficiency of TP53 by immunohistochemistry. Further optimization of small and medium-sized NGS panels covering MSI detection and POLE and TP53 full exons may be the best choice for the future to meet national conditions.

Mutation characteristics of osteosarcoma: a single center study of 64 cases using next-generation sequencing / 中华病理学杂志

Objective: To investigate the distribution and characteristics of gene mutations in osteosarcoma, and to analyze the frequency and types of detectable mutations, and to identify potential targets for individualized treatment of osteosarcoma. Methods: The fresh tissue or paraffin-embedded tissue samples of 64 cases of osteosarcoma that were surgically resected or biopsied and then subject to next generation sequencing, were collected from Beijing Jishuitan Hospital, China from November 2018 to December 2021. The tumor DNA was extracted to detect the somatic and germline mutations using targeted sequencing technology. Results: Among the 64 patients, 41 were males and 23 were females. The patient age ranged from 6 to 65 years with a median age of 17 years, including 36 children (under 18 years old) and 28 adults. There were 52 cases of conventional osteosarcoma, 3 cases of telangiectatic osteosarcoma, 7 cases of secondary osteosarcoma, and 2 cases of parosteosarcoma. The detection rate of gene mutations was overall 84.4% (54/64). There were 324 variations in 180 mutated genes, including 125 genes with copy number variations, 109 single nucleotide variants, 83 insertions or deletions, and 7 gene fusions. The most common mutated genes were TP53, VEGFA, CCND3, ATRX, MYC, RB1, PTEN, GLI1, CDK4 and PTPRD. Among them, TP53 had the highest mutation rate (21/64, 32.8%), single nucleotide variant was the main mutation type (14/23, 60.9%), and 2 cases carried the TP53 germline mutation. VEGFA and CCND3 showed copy number amplification simultaneously in 7 cases. Conclusions: The high-frequency mutation of TP53 suggests that it plays an important role in the pathogenesis and development of osteosarcoma. VEGFA, CCND3 and ATRX are mutated genes in osteosarcoma and worthy of further studies. Combination of pathologic diagnosis and next generation sequencing with clinical practice can guide individualized treatment for patients with refractory, recurrent and metastatic osteosarcoma.

Application progress of high-throughput sequencing in antiphospholipid syndrome / 中华预防医学杂志

Antiphospholipid syndrome (APS) is characterized by arterial and venous thrombosis and(or) morbid pregnancy, accompanied by persistent antiphospholipid antibody (aPL) positivity. However, due to the complex pathogenesis of APS and the large individual differences in the expression of aPL profiles of patients, the problem of APS diagnosis, prognosis judgment and risk assessment may not be solved only from antibody level. It is necessary to use new technologies and multiple dimensions to explore novel APS biomarkers. The application of next generation sequencing (NGS) technology in diseases with high incidence of somatic mutations, such as genetic diseases and tumors, has been very mature. Thus, gradually understanding the research and application progress of APS by NGS technology from genome, transcriptome, epigenome and other aspects is meaningful. This article reviews the related research of NGS technology in APS, and provide more reference for the deep understanding of the APS-related screening markers and disease pathogenesis.

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.