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INTRODUCTION: The colorectal serrated pathway involves precursor lesions known as sessile serrated lesions (SSL) and traditional serrated adenomas (TSA). Mutations in BRAF or KRAS are crucial early events in this pathway. Additional genetic and epigenetic changes contribute to the progression of these lesions into high-grade lesions and, eventually, invasive carcinoma. METHODS: We employed digital spatial profiling to investigate the transcriptional changes associated with SSL and TSA. The genes identified are confirmed by immunohistochemical (IHC) staining. Colorectal cancer (CRC) cell lines with CEACAM6 overexpression and knockdown were established to study the roles of CEACAM6 on tumorigenesis of CRC. RESULTS: Ten genes were upregulated in SSL and TSA, and seven were upregulated in both types of lesions. IHC staining confirmed overexpression of CEACAM6, LCN2, KRT19, and lysozyme in SSL and TSA. CEACAM6 expression is an early event in the serrated pathway but a late event in the conventional pathway. Using cell line models, we confirmed that CEACAM6 promotes CRC cells' proliferation, migration, and invasion abilities. CONCLUSION: These results highlight that the transcriptional changes in the early stages of tumorigenesis exhibit relative uniformity. Identifying these early events may hold significant promise in elucidating the mechanisms behind tumor initiation.
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AIMS: The microbial profiles of peri-implantitis and periodontitis (PT) are inconclusive. The controversies mainly arise from the differences in sampling sites, targeted gene fragment, and microbiome analysis techniques. The objective of this study was to explore the microbiomes of peri-implantitis (PI), control implants (CI), PT and control teeth (CT), and the microbial change of PI after nonsurgical treatment (PIAT). METHODS: Twenty-two patients diagnosed with both PT and peri-implantitis were recruited. Clinical periodontal parameters and radiographic bone levels were recorded. In each patient, the subgingival and submucosal plaque samples were collected from sites with PI, CI, PT, CT, and PIAT. Microbiome diversity was analyzed by high-throughput amplicon sequencing using full-length of 16S rRNA gene by next generation sequencing. RESULTS: The 16S rRNA gene sequencing analysis revealed 512 OTUs in oral microbiome and 377 OTUs reached strain levels. The PI and PT groups possessed their own unique core microbiome. Treponema denticola was predominant in PI with probing depth of 8-10 mm. Interestingly, Thermovirga lienii DSM 17291 and Dialister invisus DSM 15470 were found to associate with PI. Nonsurgical treatment for peri-implantitis did not significantly alter the microbiome, except Rothia aeria. CONCLUSION: Our study suggests Treponemas species may play a pivotal role in peri-implantitis. Nonsurgical treatment did not exert a major influence on the peri-implantitis microbiome in short-term follow-up. PT and peri-implantitis possess the unique microbiome profiles, and different therapeutic strategies may be suggested in the future.
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Microbiota , Periimplantitis , Periodontitis , ARN Ribosómico 16S , Humanos , Periimplantitis/microbiología , Periimplantitis/terapia , ARN Ribosómico 16S/análisis , Masculino , Femenino , Persona de Mediana Edad , Periodontitis/microbiología , Periodontitis/terapia , Secuenciación de Nucleótidos de Alto Rendimiento , Anciano , AdultoRESUMEN
OBJECTIVES: To determine how advanced genetic analysis methods may help in clinical diagnosis. STUDY DESIGN: We report a combined genetic diagnosis approach for patients with clinical suspicion of genetic liver diseases in a tertiary referral center, using tools either tier 1: Sanger sequencing on SLC2SA13, ATP8B1, ABCB11, ABCB4, and JAG1 genes, tier 2: panel-based next generation sequencing (NGS), or tier 3: whole-exome sequencing (WES) analysis. RESULTS: In a total of 374 patients undergoing genetic analysis, 175 patients received tier 1 Sanger sequencing based on phenotypic suspicion, and pathogenic variants were identified in 38 patients (21.7%). Tier 2 included 216 patients (39 of tier 1-negative patients) who received panel-based NGS, and pathogenic variants were identified in 60 (27.8%). In tier 3, 41 patients received WES analysis, and 20 (48.8%) obtained genetic diagnosis. Pathogenic variants were detected in 6 of 19 (31.6%) who tested negative in tier 2, and a greater detection rate in 14 of 22 (63.6%) patients with deteriorating/multiorgan disease receiving one-step WES (P = .041). The overall disease spectrum is comprised of 35 genetic defects; 90% of genes belong to the functional categories of small molecule metabolism, ciliopathy, bile duct development, and membrane transport. Only 13 (37%) genetic diseases were detected in more than 2 families. A hypothetical approach using a small panel-based NGS can serve as the first tier with diagnostic yield of 27.8% (98/352). CONCLUSIONS: NGS based genetic test using a combined panel-WES approach is efficient for the diagnosis of the highly diverse genetic liver diseases.
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Pruebas Genéticas , Hepatopatías , Humanos , Secuenciación del Exoma , Hepatopatías/diagnóstico , Hepatopatías/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MutaciónRESUMEN
A genetic hallmark of malignant germ cell tumours (GCTs) is isochromosome 12p, but oncogenes located in 12p that are specifically expressed in GCT have not yet been identified. SIN3-HDAC complex-associated factor (SINHCAF) is a subunit of the Sin3/histone deacetylase (HDAC) complex, and it defines a Sin3a-Hdac complex variant that is required for the self-renewal of mouse embryonic stem cells. This study demonstrated that SINHCAF is expressed in a vast majority of malignant GCTs and is rarely expressed in somatic malignancy. Fluorescence in situ hybridisation revealed SINHCAF amplification in malignant GCTs. SINHCAF silencing using shRNA reduced anchorage-dependent cell proliferation and tumoursphere formation and inhibited tumour cell migration and invasion in GCT cell lines. Moreover, in the GCT cell line NTERA2/D1, SINHCAF silencing inhibited the expression of genes associated with embryonic stem cells and induced the expression of genes associated with neuronal and white fat cell differentiation. Compared with somatic cell lines, GCT cell lines were more susceptible to HDAC inhibitor treatment. Thus, we identified SINHCAF to be a potential oncogene located in the amplicon of chromosome 12p and showed that SINHCAF was specifically expressed in malignant GCTs. HDAC inhibitor treatment may counteract the oncogenic activity of SINHCAF and is a promising therapeutic approach for GCTs. © 2022 The Pathological Society of Great Britain and Ireland.
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Ensamble y Desensamble de Cromatina , Histona Desacetilasas , Neoplasias de Células Germinales y Embrionarias , Humanos , Masculino , Ensamble y Desensamble de Cromatina/genética , Cromosomas Humanos Par 12/genética , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Hibridación Fluorescente in Situ , Neoplasias de Células Germinales y Embrionarias/genética , OncogenesRESUMEN
BACKGROUNDS: Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. OBJECTIVE: This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. METHODS: Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. RESULTS: Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. CONCLUSION: Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.
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Actinobacteria , Pérdida de Hueso Alveolar , Placa Dental , Periodontitis , Ratones , Animales , Microtomografía por Rayos X/efectos adversos , Pérdida de Hueso Alveolar/patología , Periodontitis/metabolismo , Bacterias , Placa Dental/complicacionesRESUMEN
OBJECTIVE: To test the application of a target enrichment next-generation sequencing (NGS) jaundice panel in genetic diagnosis of pediatric liver diseases. STUDY DESIGN: We developed a capture-based target enrichment NGS jaundice panel containing 42 known disease-causing genes associated with jaundice or cholestasis and 10 pathway-related genes. During 2015-2017, 102 pediatric patients with various forms of cholestasis or idiopathic liver diseases were tested, including patients with initial diagnosis of cholestasis in infancy, progressive familial intrahepatic cholestasis, syndromic cholestasis, Wilson disease, and others. RESULTS: Of the 102 patients, 137 mutations/variants in 44 different genes were identified in 84 patients. The genetic disease diagnosis rate was 33 of 102 (32.4%). A total of 79 of 102 (77.5%) of patients had at least 1 heterozygous genetic variation. Those with progressive intrahepatic cholestasis or syndromic cholestasis in infancy had a diagnostic rate of 62.5%. Disease-causing mutations, including ATP8B1, ABCB11, ABCB4, ABCC2, TJP2, NR1H4 (FXR), JAG1, AKR1D1, CYP7B1, PKHD1, ATP7B, and SLC25A13, were identified. Nine patients had unpredicted genetic diagnosis with atypical phenotype or novel mutations in the investigational genes. We propose an NGS diagnosis classification categorizing patients into high (n = 24), moderate (n = 9), or weak (n = 25) levels of genotype-phenotype correlations to facilitate patient management. CONCLUSIONS: This panel enabled high-throughput detection of genetic variants and disease diagnosis in patients with a long list of candidate causative genes. A NGS report with diagnosis classification may aid clinicians in data interpretation and patient management.
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Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Colestasis Intrahepática/diagnóstico , ADN/genética , Mutación , Receptores Citoplasmáticos y Nucleares/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Preescolar , Colestasis Intrahepática/genética , Colestasis Intrahepática/metabolismo , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Receptores Citoplasmáticos y Nucleares/metabolismo , Estudios RetrospectivosRESUMEN
STUDY QUESTION: Can the use of whole-exome sequencing (WES) together with single nucleotide polymorphism (SNP) array help to identify novel causative genes of isolated Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome? SUMMARY ANSWER: OR4M2 (olfactory receptor, family 4, subfamily M, member 2) and PDE11A (phosphodiesterase 11A) gene loss-of-function variants as well as deletions at 15q11.2, 19q13.31, 1p36.21, and 1q44 were identified as possible commonly altered regions in patients with type 1 MRKH. WHAT IS KNOWN ALREADY: The isolated form of Müllerian aplasia is the most common subtype of MRKH syndrome, which invariably leads to difficulties producing offspring in affected women. However, there is little information currently available to allow for genetic testing and counseling to be performed for those affected by this syndrome. STUDY DESIGN, SIZE AND DURATION: This was a case-series genetic study. A total of seven consecutive unrelated women with type 1 MRKH were enrolled. The enrollment and experimental procedures were performed over a 2-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole exome-targeted next-generation sequencing and SNP array (Affymetrix Genome-Wide Human SNP Array 6.0) were performed on the first five unrelated women with type 1 MRKH syndrome. The data were combined, and the '3-hit principal' was applied on a genome-wide scale to search for the common causative genes. Quantitative PCR (qPCR) and Sanger sequencing were used to validate the identified genomic copy number losses and variants. Replication tests using direct Sanger sequencing and qPCR were performed on the remaining two women with type 1 MRKH syndrome to support the credibility of the potential candidate genes and deletions. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 3443 damaging variants based on WES were shown to intersect with 1336 copy number variations (deletions) derived from the SNP array. Four highly recurrent deletions at 15q11.2 (80%), 19q13.31 (40%), 1p36.21 (40%) and 1q44 (40%) were identified in the first five women with type 1 MRKH syndrome and were considered to be novel candidate aberrations. A previously reported 1q21.1 deletion was also recurrent in two of the first five women with type 1 MRKH syndrome. The 1q44 and 19q13.31 deletions were present in at least one of the two additional patients. Damaging variants were detected in HNRNPCL1 (heterogeneous nuclear ribonucleoprotein C-like 1), OR2T2 (olfactory receptor, family 2, subfamily T, member 2), OR4M2, ZNF816 (zinc finger protein 816), and PDE11A in several of the initial five patients. Among these, the damaging variants of OR4M2 (located at 15q11.2) and PDE11A were found in at least one of the two additional patients with type 1 MRKH. LIMITATIONS, REASONS FOR CAUTION: In this study, we only searched for the deletions or damaging variants causing loss-of-function of genes in at least three of the initial five patients (3-hit criteria). Therefore, the study was designed to only detect common causative genes. Genomic duplications and/or rare individual mutations that may have also contributed to MRKH syndrome were not investigated. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrated the feasibility of the use of combined data from both WES and SNP arrays for the identification of possible common causative genetic aberrations in patients with type 1 MRKH syndrome on a genome-wide scale. Further validation of our found causative genes is required before applying on genetic testing and counseling. STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the National Science Council of Taiwan (NSC98-2314-B002-105-MY3 and NSC 100-2314-B002-027-MY3). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
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Trastornos del Desarrollo Sexual 46, XX/genética , Anomalías Congénitas/genética , Exoma/genética , Conductos Paramesonéfricos/anomalías , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Adulto , Femenino , Eliminación de Gen , HumanosRESUMEN
Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.
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Fenotipo Secretor Asociado a la Senescencia , Fiebre Tifoidea , Humanos , Fiebre Tifoidea/metabolismo , Mutágenos/metabolismo , Senescencia Celular/fisiología , Mitocondrias/metabolismo , ADN Mitocondrial/metabolismo , Salmonella , FenotipoRESUMEN
Background and Aim: Alagille syndrome (ALGS) is a multisystem disorder with variable clinical courses. This study investigated the clinical and genetic features of ALGS patients with different outcomes and analyzed the liver pathology at liver transplantation (LT) compared with that in biliary atresia (BA). Methods: We report the clinical characteristics, outcomes, and genetic mutations of 25 children with ALGS followed for a median of 7.3 years. Patients were classified into (i) jaundice-free (JF) group (resolving jaundice after 2 years of age); (ii) progressive disease (PD) group (persistent jaundice or progressive cholestasis). In addition, we analyzed the explant liver in 10 ALGS patients compared with 20 age-matched BA patients at the time of LT. Results: Nine patients (36%) in the JF group had a favorable outcome, with longer native liver survival than patients with PD (n = 16, P < 0.001). Fourteen of the PD group patients received LT or died. We identified 18 different JAG1 mutations in 22 patients. Three unrelated probands in the JF group had the same de novo mutation in JAG1, c.2122-2125delCAGT. Compared with BA children, ALGS patients had lower METAVIR scores in liver pathology, higher serum albumin levels, and lower weight-for-age z-scores when receiving LT. Conclusion: One-third of ALGS patients had JF and a favorable course. Children with ALGS presenting with persistent jaundice beyond 2 years of age should be cautioned for poor prognosis. ALGS patients tend to have a lesser extent of cirrhosis, and more growth problems than BA patients at the time of LT.
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A kinesin family member 5b (KIF5B)-MET proto-oncogene, receptor tyrosine kinase (MET) rearrangement was reported in patients with lung adenocarcinoma but its oncogenic function was not fully evaluated. We used one-step reverse transcription-polymerase chain reaction for RNA samples to screen for the KIF5B-MET fusion in 206 lung adenocarcinoma and 28 pulmonary sarcomatoid carcinoma patients. Genomic breakpoints of KIF5B-MET were determined by targeted next-generation sequencing. Soft agar colony formation assays, proliferation assays, and a xenograft mouse model were used to investigate its oncogenic activity. In addition, specific MET inhibitors were administered to evaluate their anti-tumor activities. A KIF5B-MET fusion variant in a patient with a mixed-type adenocarcinoma and sarcomatoid tumor was identified, and another case was found in a pulmonary sarcomatoid carcinoma patient. Both cases carried the same chimeric gene, a fusion between exons 1-24 of KIF5B and exons 15-21 of MET. KIF5B-MET-overexpressing cells exhibited significantly increased proliferation and colony-forming ability. Xenograft tumors harboring the fusion gene demonstrated significantly elevated tumor growth. Ectopic expression of the fusion gene stimulated the phosphorylation of KIF5B-MET as well as downstream STAT3, AKT, and ERK1/2 signaling pathways. The MET inhibitors significantly repressed cell proliferation; phosphorylation of downstream STAT3, AKT, and ERK1/2; and xenograft tumorigenicity. In conclusion, the KIF5B-MET variant was demonstrated to have an oncogenic function in cancer cells. These findings have immediate clinical implications for the targeted therapy of subgroups of non-small cell lung cancer patients.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Variación Genética/genética , Cinesinas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas c-met/genética , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Animales , Carcinogénesis/genética , Línea Celular , Proliferación Celular/genética , Exones/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proto-Oncogenes Mas , Translocación Genética/genéticaRESUMEN
Massively parallel sequencing (MPS) technology enables the simultaneous analysis of a huge number of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels). MPS also enables the detection of the alleles of minor contributors in a highly unbalanced DNA mixture. In this study, we established a 1204-marker panel optimized for MPS consisting of 987 autosomal markers (964 SNPs and 23 indels), 27 X-chromosome SNPs, 61 Y-chromosome markers (56 SNPs and 5 indels), and 129 mitochondrial SNPs. The DNA samples of six unrelated individuals (two men and four women), 26 nondegraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99), and eight highly artificially degraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99) were analyzed through MPS by using the panel. A scoring system was developed to determine the minor contributors in DNA mixtures based on the genotypes identified using MPS. The genotypes of the 1204 markers were successfully profiled through MPS by using the custom-designed panel. The efficiency of MPS for analyzing these highly degraded samples was lower than that for analyzing nondegraded samples. All minor contributors in the 26 nondegraded and 8 degraded DNA mixtures were accurately assigned using this scoring system based on 964 autosomal SNPs. An association between the observed reads ratio and theoretical ratio of the minor component was noted for nondegraded mixtures. In conclusion, we established a 1204-marker individual identification panel for MPS that successfully analyzed autosomal, X-chromosome, Y-chromosome, and mitochondrial SNPs and indels simultaneously. In combination with the newly developed scoring system, the panel can accurately identify minor contributors in nondegraded and highly degraded DNA mixtures.
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Dermatoglifia del ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación INDEL , Polimorfismo de Nucleótido Simple , Cromosomas Humanos X , Cromosomas Humanos Y , Degradación Necrótica del ADN , Sondas de ADN , Femenino , Marcadores Genéticos , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Análisis de Secuencia de ADNRESUMEN
Conventional cytogenetics can categorize patients with acute myeloid leukemia (AML) into favorable, intermediate, and unfavorable-risk groups; however, patients with intermediate-risk cytogenetics represent the major population with variable outcomes. Because molecular profiling can assist with AML prognosis and next-generation sequencing allows simultaneous sequencing of many target genes, we analyzed 260 genes in 112 patients with de novo AML who received standard treatment. Multivariate analysis showed that karyotypes and mutation status of TET2, PHF6, KIT, and NPM1mutation /FLT3- internal tandem duplication (ITD)negative were independent prognostic factors for the entire cohort. Among patients with intermediate-risk cytogenetics, patients with mutations in CEBPAdouble mutation , IDH2, and NPM1 in the absence of FLT3-ITD were associated with improved Overall survival (OS), similar to those with favorable-risk cytogenetics; patients with mutations in TET2, RUNX1, ASXL1, and DNMT3A were associated with reduced OS, similar to those with unfavorable-risk cytogenetics. We concluded that integration of cytogenetic and molecular profiling improves prognostic stratification of patients into three groups with more distinct prognoses (P < 0.001) and significantly reduces the number of patients classified as intermediate risk. In addition, our study demonstrates that next-generation sequencing (NGS)-based multi-gene sequencing is clinically applicable in establishing an accurate risk stratification system for guiding therapeutic decisions.
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Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Mieloide Aguda/genética , Mutación , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Anciano , Niño , Análisis Citogenético , Femenino , Redes Reguladoras de Genes , Humanos , Masculino , Persona de Mediana Edad , Nucleofosmina , Pronóstico , Análisis de Supervivencia , Adulto JovenRESUMEN
In this study, the complete mitogenome sequence of Northwestern Pacific 2 (NWP2) cryptic species of flathead mullet, Mugil cephalus (Teleostei: Mugilidae) has been amplified by long-range PCR and sequenced by next-generation sequencing method. The assembled mitogenome, consisting of 16,686 bp, had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a non-coding control region of D-loop. D-loop was 909 bp length and was located between tRNA-Pro and tRNA-Phe. The overall base composition of NWP2 M. cephalus was 28.4% for A, 29.8% for C, 26.5% for T and 15.3% for G. The complete mitogenome may provide essential and important DNA molecular data for further phylogenetic and evolutionary analysis for flathead mullet species complex.
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Genoma Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Smegmamorpha/genética , Animales , Filogenia , ARN Ribosómico/genética , ARN de Transferencia/genética , Smegmamorpha/clasificaciónRESUMEN
INTRODUCTION: Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD). However, only few cases carrying LRRK2 mutations have been reported in Taiwanese PD patients. METHODS: We used targeted next generation sequencing (NGS), covering 24 candidate genes involved in neurodegenerative disorders, to analyze 40 probands with familial PD, and 10 patients with mixed neurodegenerative disorders. Sanger sequencing of the identified mutation in the first set of the study was performed in additional 270 PD patients, including 139 familial PD and 131 early-onset PD (onset age less than 50 years old), and 300 age/gender matched control subjects. RESULTS: We found a missense variant, p.I2012T, in the LRRK2 gene in one sporadic patient having early-onset frontotemporal dementia with parkinsonism and dystonia. Sanger sequencing this substitution in additional 270 PD patients in the second set of the study revealed two additional variant carriers: one having autosomal-dominant familial PD, and one with sporadic PD. The p.I2012T substitution was absent in 300 normal control subjects. Analyzing family members of the proband with p.I2012T revealed co-segregation of the variant and parkinsonism. Clinical presentations, levodopa responses, and Tc99mTRODAT-SPECT imaging findings of this index family were similar to idiopathic PD. CONCLUSIONS: Our results revealed clinical heterogeneity of the LRRK2 p.I2012T substitution, and demonstrated the use of targeted NGS for genetic diagnosis in multiplex families with PD or mixed neurodegenerative disorders.
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Predisposición Genética a la Enfermedad , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , Enfermedades Neurodegenerativas/genética , Trastornos Parkinsonianos/genética , Adulto , Edad de Inicio , Anciano , Encéfalo/diagnóstico por imagen , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Heterocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/diagnóstico por imagen , Trastornos Parkinsonianos/diagnóstico por imagen , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Since BRCA mutations are only responsible for 10-20% of cases of breast cancer in patients with early-onset or a family history and since next-generation sequencing technology allows the simultaneous sequencing of a large number of target genes, testing for multiple cancer-predisposing genes is now being considered, but its significance in clinical practice remains unclear. We then developed a sequencing panel containing 68 genes that had cancer risk association for patients with early-onset or familial breast cancer. A total of 133 patients were enrolled and 30 (22.6%) were found to carry germline deleterious mutations, 9 in BRCA1, 11 in BRCA2, 2 in RAD50, 2 in TP53 and one each in ATM, BRIP1, FANCI, MSH2, MUTYH, and RAD51C. Triple-negative breast cancer (TNBC) was associated with the highest mutation rate (45.5%, p = 0.025). Seven of the 9 BRCA1 mutations and the single FANCI mutation were in the TNBC group; 9 of the 11 BRCA2, 1 of the 2 RAD50 as well as BRIP1, MSH2, MUTYH, and RAD51C mutations were in the hormone receptor (HR)(+)Her2(-) group, and the other RAD50, ATM, and TP53 mutations were in the HR(+)Her2(+) group. Mutation carriers were considered as high-risk to develop malignancy and advised to receive cancer screening. Screening protocols of non-BRCA genes were based on their biologic functions; for example, patients carrying RAD51C mutation received a screening protocol similar to that for BRCA, since BRCA and RAD51C are both involved in homologous recombination. In conclusion, we consider that multiple gene sequencing in cancer risk assessment is clinically valuable.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Medición de Riesgo , Adulto , Edad de Inicio , Anciano , Neoplasias de la Mama/clasificación , Femenino , Genes Supresores de Tumor , Pruebas Genéticas , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chromosome 4q exhibits high frequency of allelic loss in hepatocellular carcinoma (HCC). This study aimed to elucidate the interaction of the frequent aberrant mRNA expression of alpha-fetoprotein (AFP), osteopontin (OPN) and a novel short isoform of annexin A10 (ANXA10S) at 4q in the tumor progression among 294 patients who received surgical resection of unifocal primary HCC. AFP overexpression, OPN overexpression and ANXA10S down-regulation correlated with high-grade and high-stage tumors, early tumor recurrence (all P<0.0001), and lower 10-year survival (all P=0.000001). The AFP overexpression correlated with OPN overexpression (P=0.0026) and ANXA10S down-regulation (P=0.00001), while OPN overexpression correlated with ANXA10S down-regulation (P=0.00001). Pair-wise combinations revealed interactive effects between these genetic variants for tumor grade, tumor stage and early recurrence (all P<0.0001). HCCs with more genetic aberrations had more frequent high tumor grade, portal vein invasion (stage IIIB-IV) and early recurrence (all P<0.0001). The 10-year survival rate for HCCs with all three genetic alterations was the lowest (7%), followed by those with two (22%) or one event (29%), and the highest for those without these changes (43%), P=0.000001. The prognostic stratification using these molecular factors was similar to that of histopathological staging. These three genetic alterations also helped to identify different subgroups of patients of stage II HCC but with different prognosis (P=0.015). In conclusion, the aberrant expressions of AFP, OPN and ANXA10S cooperatively contribute to tumor progression and poor prognosis, and are useful for molecular staging of HCC and the subclassification of stage II HCC without vascular invasion.
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Anexinas/biosíntesis , Carcinoma Hepatocelular/genética , Cromosomas Humanos Par 4 , Perfilación de la Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Sialoglicoproteínas/biosíntesis , alfa-Fetoproteínas/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Anexinas/genética , Secuencia de Bases , Progresión de la Enfermedad , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Osteopontina , Pronóstico , Estudios Retrospectivos , Sialoglicoproteínas/genética , Análisis de Supervivencia , Regulación hacia Arriba , alfa-Fetoproteínas/genéticaRESUMEN
OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP(ND251)) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP(ND251), as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.
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Esclerosis Amiotrófica Lateral/metabolismo , Autofagia/genética , Proteínas del Ojo/metabolismo , Mutación/genética , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Autofagia/fisiología , Sitios de Unión , Proteínas de Ciclo Celular , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Ojo/genética , Humanos , Proteínas de Transporte de Membrana , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica/genética , Pliegue de ProteínaRESUMEN
Several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are characterized by inclusion bodies formed by TDP-43 (TDP). We established cell and transgenic Drosophila models expressing TDP carboxyl terminal fragment (ND251 and ND207), which developed aggregates recapitulating important features of TDP inclusions in ALS/FTLD-U, including hyperphosphorylation at previously reported serine(403,404,409,410) residues, polyubiquitination and colocalization with optineurin. These models were used to address the pathogenic role of hyperphosphorylation in ALS/FTLD-U. We demonstrated that hyperphosphorylation and ubiquitination occurred temporally later than aggregation in cells. Expression of CK2α which phosphorylated TDP decreased the aggregation propensity of ND251 or ND207; this effect could be blocked by CK2 inhibitor DMAT. Mutation of serines(379,403,404,409,410) to alanines (S5A) to eliminate phosphorylation increased the aggregation propensity and number of aggregates of TDP, but mutation to aspartic acids (S5D) or glutamic acids (S5E) to simulate hyperphosphorylation had the opposite effect. Functionally, ND251 or ND207 aggregates decreased the number of neurites of Neuro2a cells induced by retinoic acid or number of cells by MTT assay. S5A mutation aggravated, but S5E mutation alleviated these cytotoxic effects of aggregates. Finally, ND251 or ND251S5A developed aggregates in neurons, and salivary gland of transgenic Drosophila, but ND251S5E did not. Taken together, our data indicate that hyperphosphorylation may represent a compensatory defense mechanism to stop or prevent pathogenic TDP from aggregation. Therefore, enhancement of phosphorylation may serve as an effective therapeutic strategy against ALS/FTLD-U.
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Proteínas de Unión al ADN/metabolismo , Fosforilación/efectos de los fármacos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Bencimidazoles/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular , Proliferación Celular , Drosophila , Humanos , Immunoblotting , Poliubiquitina/metabolismo , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/metabolismoRESUMEN
BACKGROUND: It is believed that Ras mutations drive the proliferation of leukemic cells. The objective of this study was to investigate the association of Ras mutations with childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) with special reference to the presence or absence of mixed-lineage leukemia gene (MLL) rearrangements. METHODS: Bone marrow samples from 313 children with B-precursor ALL and 130 children with de novo AML were studied at diagnosis. Southern blot analysis was used to detect MLL rearrangements, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to detect common MLL fusion transcripts. Complementary DNA panhandle PCR was used to identify the infrequent or unknown MLL partner genes. DNA PCR or RT-PCR followed by direct sequencing was performed to detect mutations at codons 12, 13, and 61 of the N-Ras and K-Ras genes. RESULTS: Twenty of 313 patients with B-precursor ALL and 17 of 130 patients with de novo AML had MLL rearrangements. N-Ras mutations were detected in 2 of 20 patients with MLL-positive ALL and in 27 of 293 patients with MLL-negative ALL (P = 1.000). N-Ras mutations were detected in 2 of 17 patients with MLL-positive AML and in 14 of 113 patients with MLL-negative AML (P = 1.000). K-Ras mutations were present in 8 of 20 patients with MLL-positive ALL compared with 32 of 293 patients with MLL-negative ALL (P = 0.001). K-Ras mutations were detected in 3 of 17 patients with MLL-positive AML compared with 5 of 113 patients with MLL-negative AML (P = 0.069). CONCLUSIONS: Ras mutations were detected in 20.8% of patients with childhood B-precursor ALL and in 17.7% of patients with childhood AML. MLL-positive B-precursor ALL was associated closely with Ras mutations (50%), especially with K-Ras mutations (40%), whereas MLL-positive AML was not associated with Ras mutations.