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Motivation: The application of machine learning (ML) techniques in the medical field has demonstrated both successes and challenges in the precision medicine era. The ability to accurately classify a subject as a potential responder versus a nonresponder to a given therapy is still an active area of research pushing the field to create new approaches for applying machine-learning techniques. In this study, we leveraged publicly available data through the BeatAML initiative. Specifically, we used gene count data, generated via RNA-seq, from 451 individuals matched with ex vivo data generated from treatment with RTK-type-III inhibitors. Three feature selection techniques were tested, principal component analysis, Shapley Additive Explanation (SHAP) technique and differential gene expression analysis, with three different classifiers, XGBoost, LightGBM and random forest (RF). Sensitivity versus specificity was analyzed using the area under the curve (AUC)-receiver operating curves (ROCs) for every model developed. Results: Our work demonstrated that feature selection technique, rather than the classifier, had the greatest impact on model performance. The SHAP technique outperformed the other feature selection techniques and was able to with high accuracy predict outcome response, with the highest performing model: Foretinib with 89% AUC using the SHAP technique and RF classifier. Our ML pipelines demonstrate that at the time of diagnosis, a transcriptomics signature exists that can potentially predict response to treatment, demonstrating the potential of using ML applications in precision medicine efforts. Availability and implementation: https://github.com/UD-CRPL/RCDML. Supplementary information: Supplementary data are available at Bioinformatics Advances online.
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INTRODUCTION: The KMT2 family of genes is essential epigenetic regulators promoting gene expression. The gene family contains three subgroups, each with two paralogues: KMT2A and KMT2B; KMT2C and KMT2D; KMT2F and KMT2G. KMT2A-D are among the most frequent somatically altered genes in several different cancer types. Somatic KMT2A rearrangements are well-characterized in infant leukemia (IL), and growing evidence supports the role of additional family members (KMT2B, KMT2C, and KMT2D) in leukemogenesis. Enrichment of rare heterozygous frameshift variants in KMT2A and C has been reported in acute myeloid leukemia (AML), IL, and solid tumors. Currently, the non-synonymous variation, prevalence, and penetrance of these four genes are unknown. METHODS: This study determined the prevalence of pathogenic/likely pathogenic (P/LP) germline KMT2A-D variants in a cancer-free adult population from the Genome Aggregation Database (gnomAD). Two methods of variant interpretation were utilized: a manual genomic variant interpretation and an automated ACMG pipeline. RESULTS: The ACMG pipeline identified considerably fewer P/LP variants (n = 89) compared to the manual method (n = 660) in all 4 genes. Consequently, the total P/LP prevalence and allele frequency (AF) were higher in the manual method (1:112, AF = 4.46E-03) than in ACMG (1:832, AF = 6.01E-04). Multiple ancestry-exclusive P/LP variants were identified along with an increased frequency in males compared to females. Many of these variants identified in this population database are also associated with severe juvenile conditions. CONCLUSION: These data demonstrate that putatively functional germline variation in these developmentally important genes is more common than previously appreciated and identification in cancer-free adults may indicate incomplete penetrance for many of these variants. Future research should examine a genetic predisposing role in IL and other pediatric cancers.
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Leucemia Mieloide Aguda , Masculino , Niño , Lactante , Femenino , Adulto , Humanos , Prevalencia , Virulencia , Frecuencia de los Genes , Leucemia Mieloide Aguda/epidemiología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación de Línea GerminalRESUMEN
Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient-specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC-derived myoblasts (MB) and 1774 CpG sites in differentiating SC-derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo- and hyper-methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.
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OBJECTIVE: The Genomic Medicine Working Group of the National Advisory Council for Human Genome Research virtually hosted its 13th genomic medicine meeting titled "Developing a Clinical Genomic Informatics Research Agenda". The meeting's goal was to articulate a research strategy to develop Genomics-based Clinical Informatics Tools and Resources (GCIT) to improve the detection, treatment, and reporting of genetic disorders in clinical settings. MATERIALS AND METHODS: Experts from government agencies, the private sector, and academia in genomic medicine and clinical informatics were invited to address the meeting's goals. Invitees were also asked to complete a survey to assess important considerations needed to develop a genomic-based clinical informatics research strategy. RESULTS: Outcomes from the meeting included identifying short-term research needs, such as designing and implementing standards-based interfaces between laboratory information systems and electronic health records, as well as long-term projects, such as identifying and addressing barriers related to the establishment and implementation of genomic data exchange systems that, in turn, the research community could help address. DISCUSSION: Discussions centered on identifying gaps and barriers that impede the use of GCIT in genomic medicine. Emergent themes from the meeting included developing an implementation science framework, defining a value proposition for all stakeholders, fostering engagement with patients and partners to develop applications under patient control, promoting the use of relevant clinical workflows in research, and lowering related barriers to regulatory processes. Another key theme was recognizing pervasive biases in data and information systems, algorithms, access, value, and knowledge repositories and identifying ways to resolve them.
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Informática Médica , Registros Electrónicos de Salud , Genoma Humano , Genómica , Humanos , Proyectos de InvestigaciónRESUMEN
Background: Different feeding regimens in infancy alter the gastrointestinal (gut) microbial environment. The fecal microbiota in turn influences gastrointestinal homeostasis including metabolism, immune function, and extra-/intra-intestinal signaling. Advances in next generation sequencing (NGS) have enhanced our ability to study the gut microbiome of breast-fed (BF) and formula-fed (FF) infants with a data-driven hypothesis approach. Methods: Next generation sequencing libraries were constructed from fecal samples of BF (n=24) and FF (n=10) infants and sequenced on an Illumina HiSeq 2500. Taxonomic classification of the NGS data was performed using the Sunbeam/Kraken pipeline and a functional analysis at the gene level was performed using publicly available algorithms, including BLAST, and custom scripts. Differentially represented genera, genes, and NCBI Clusters of Orthologous Genes (COG) were determined between cohorts using count data and R (statistical packages edgeR and DESeq2). Results: Thirty-nine genera were found to be differentially represented between the BF and FF cohorts (FDR ≤ 0.01) including Parabacteroides, Enterococcus, Haemophilus, Gardnerella, and Staphylococcus. A Welch t-test of the Shannon diversity index for BF and FF samples approached significance (p=0.061). Bray-Curtis and Jaccard distance analyses demonstrated clustering and overlap in each analysis. Sixty COGs were significantly overrepresented and those most significantly represented in BF vs. FF samples showed dichotomy of categories representing gene functions. Over 1,700 genes were found to be differentially represented (abundance) between the BF and FF cohorts. Conclusions: Fecal samples analyzed from BF and FF infants demonstrated differences in microbiota genera. The BF cohort includes greater presence of beneficial genus Bifidobacterium. Several genes were identified as present at different abundances between cohorts indicating differences in functional pathways such as cellular defense mechanisms and carbohydrate metabolism influenced by feeding. Confirmation of gene level NGS data via PCR and electrophoresis analysis revealed distinct differences in gene abundances associated with important biologic pathways.
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Microbioma Gastrointestinal , Microbiota , Lactancia Materna , Heces/microbiología , Femenino , Microbioma Gastrointestinal/genética , Humanos , Lactante , Fórmulas Infantiles , MetagenómicaRESUMEN
The use of next generation sequencing is critical for the surveillance of severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, transmission, as single base mutations have been identified with differences in infectivity. A total of 1,459 high quality samples were collected, sequenced, and analyzed in the state of Delaware, a location that offers a unique perspective on transmission given its proximity to large international airports on the east coast. Pangolin and Nextclade were used to classify these sequences into 16 unique clades and 88 lineages. A total of 411 samples belonging to the Alpha 20I/501Y.V1 (B.1.1.7) strain of concern were identified, as well as one sample belonging to Beta 20H/501.V2 (B.1.351), thirteen belonging to Epsilon 20C/S:452R (B.1.427/B.1.429), two belonging to Delta 20A/S:478K (B.1.617.2), and 15 belonging to Gamma 20J/501Y.V3 (p.1). A total of 2217 unique coding mutations were observed with an average of 17.7 coding mutations per genome. These data paired with continued sample collection and sequencing will give a deeper understanding of the spread of SARS-CoV-2 strains within Delaware and its surrounding areas.
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COVID-19/patología , Genoma Viral , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/virología , Delaware/epidemiología , Ligamiento Genético , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogenia , ARN Viral/química , ARN Viral/metabolismo , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificaciónRESUMEN
AIM: To analyze transcriptomes from muscle tissue and cells to improve our understanding of differences in gene expression and molecular function in cerebral palsy (CP) muscle. METHOD: In this case-control study, eight participants with CP (five males, three females; mean [SD] age 14y 2mo [1y 8mo]) and 11 comparison individuals (eight males, three females; mean [SD] age 14y 0mo [2y 6mo]) were enrolled after informed consent/assent and skeletal muscle was obtained during surgery. RNA was extracted from tissue and from primary satellite cells grown to form myotubes in vitro. RNA sequencing data were analyzed using validated informatics pipelines. RESULTS: Analysis identified expression of 6308 genes in the tissue samples and 7459 in the cultured cells. Significant differential expression between CP and control was identified in 87 genes in the tissue and 90 genes in isolated satellite cell-derived myotube cultures. INTERPRETATION: Both tissue and cell analyses identified differential expression of genes associated with muscle development and multiple pathways of interest. What this paper adds Expression differences were found in muscle tissue and in isolated muscle cells. There was low variability in expression among cells isolated from different muscles. Expression differences suggest complex functional alterations in spastic cerebral palsy.
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Parálisis Cerebral/genética , Espasticidad Muscular/genética , Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Adolescente , Estudios de Casos y Controles , Niño , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , RNA-Seq , TranscriptomaRESUMEN
OBJECTIVE: Using sickle cell disease (SCD) as a model, the objective of this study was to create a comprehensive learning healthcare system to support disease management and research. A multidisciplinary team developed a SCD clinical data dictionary to standardize bedside data entry and inform a scalable environment capable of converting complex electronic healthcare records (EHRs) into knowledge accessible in real time. MATERIALS AND METHODS: Clinicians expert in SCD care developed a data dictionary to describe important SCD-associated health maintenance and adverse events. The SCD data dictionary was deployed in the EHR using EPIC SmartForms, an efficient bedside data entry tool. Additional data elements were extracted from the EHR database (Clarity) using Pentaho Data Integration and stored in a data analytics database (SQL). A custom application, the Sickle Cell Knowledgebase, was developed to improve data analysis and visualization. Utilization, accuracy, and completeness of data entry were assessed. RESULTS: The SCD Knowledgebase facilitates generation of patient-level and aggregate data visualization, driving the translation of data into knowledge that can impact care. A single patient can be selected to monitor health maintenance, comorbidities, adverse event frequency and severity, and medication dosing/adherence. CONCLUSIONS: Disease-specific data dictionaries used at the bedside will ultimately increase the meaningful use of EHR datasets to drive consistent clinical data entry, improve data accuracy, and support analytics that will facilitate quality improvement and research.
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PURPOSE: Pediatric germ cell tumors are rare, representing about 3% of childhood malignancies in children less than 15 years of age, presenting in neonates or adolescents with a greater incidence noted in older adolescents. Aberrations in primordial germ cell proliferation/differentiation can lead to a variety of neoplasms, including teratomas, embryonal carcinoma, choriocarcinoma, and yolk sac tumors. PATIENTS AND METHODS: Three Finnish families with varying familial germ cell tumors were identified, and whole-genome sequencing was performed using an Illumina sequencing platform. In total, 22 unique subjects across the three families were sequenced. Family 1 proband (female) was affected by malignant ovarian teratoma, Family 2 proband (female) was affected by sacrococcygeal teratoma with yolk sac tumor in the setting of Cornelia de Lange syndrome, and Family 3 proband (male) was affected by malignant testicular teratoma. Rare variants were identified using an autosomal recessive or de novo model of inheritance. RESULTS: For family 1 proband (female), an autosomal recessive or de novo model of inheritance identified variants of interest in the following genes: CD109, IKBKB, and CTNNA3, SUPT6H, MUC5AC, and FRG1. Family 2 proband (female) analysis identified gene variants of interest in the following genes: LONRF2, ANO7, HS6ST1, PRB2, and DNM2. Family 3 proband (male) analysis identified the following potential genes: CRIPAK, KRTAP5-7, and CACNA1B. CONCLUSION: Leveraging deep pedigrees and next-generation sequencing, rare germline variants were identified that were enriched in three families from Finland with a history of familial germ cell tumors. The data presented support the importance of germline mutations when analyzing complex cancers with a low somatic mutation landscape.
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Acute leukemia represents the most common pediatric malignancy comprising diverse subtypes with varying prognosis and treatment outcomes. New and targeted treatment options are warranted for this disease. Patient-derived xenograft (PDX) models are increasingly being used for preclinical testing of novel treatment modalities. A novel approach involving targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was employed to compare 25 primary pediatric acute leukemia samples and their corresponding PDX samples. A comparison of the primary samples and PDX samples revealed a high concordance between single nucleotide variants and gene fusions whereas other complex structural variants were not as consistent. The presence of gene fusions representing the major driver mutations at similar allelic frequencies in PDX samples compared to primary samples and over multiple passages confirms the utility of PDX models for preclinical drug testing. Characterization and tracking of these novel cryptic fusions and exonal variants in PDX models is critical in assessing response to potential new therapies.
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Advancements in next generation sequencing (NGS) technologies have significantly increased the translational use of genomics data in the medical field as well as the demand for computational infrastructure capable processing that data. To enhance the current understanding of software and hardware used to compute large scale human genomic datasets (NGS), the performance and accuracy of optimized versions of GATK algorithms, including Parabricks and Sentieon, were compared to the results of the original application (GATK V4.1.0, Intel x86 CPUs). Parabricks was able to process a 50× whole-genome sequencing library in under 3 h and Sentieon finished in under 8 h, whereas GATK v4.1.0 needed nearly 24 h. These results were achieved while maintaining greater than 99% accuracy and precision compared to stock GATK. Sentieon's somatic pipeline achieved similar results greater than 99%. Additionally, the IBM POWER9 CPU performed well on bioinformatic workloads when tested with 10 different tools for alignment/mapping.
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BACKGROUND: Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. METHODS: Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4-11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. RESULTS: Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. CONCLUSIONS: Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.
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Secuenciación de Nucleótidos de Alto Rendimiento , Leucemia/diagnóstico , Leucemia/genética , Mutación , Adolescente , Línea Celular Tumoral , Niño , Femenino , Humanos , Leucemia/terapia , Masculino , Neoplasia ResidualRESUMEN
The United States has quickly transitioned into one of the epicenters for the coronavirus pandemic. Limitations for rapid testing for the virus responsible for the pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the single most important barrier for early detection and prevention of future outbreaks. Combining innovative molecular biology techniques, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease systems and next generation sequencing (NGS) may prove to be an effective solution to establish a high-throughput diagnostic and genomic surveillance workflow for COVID-19 in the State of Delaware. Integrating key expertise across the medical institutions in Delaware, including ChristianaCare and Nemours/Alfred I. duPont Hospital for Children, is one potential solution for overcoming current barriers and driving a successful implementation of these techniques.
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Protein citrullination (or deimination), an irreversible post-translational modification, has been implicated in several physiological and pathological processes, including gene expression regulation, apoptosis, rheumatoid arthritis, and Alzheimer's disease. Several research studies have been carried out on citrullination under many conditions. However, until now, challenges in sample preparation and data analysis have made it difficult to confidently identify a citrullinated protein and assign the citrullinated site. To overcome these limitations, we generated a mouse hyper-citrullinated spectral library and set up coordinates to confidently identify and validate citrullinated sites. Using this workflow, we detect a four-fold increase in citrullinated proteome coverage across six mouse organs compared with the current state-of-the art techniques. Our data reveal that the subcellular distribution of citrullinated proteins is tissue-type-dependent and that citrullinated targets are involved in fundamental physiological processes, including the metabolic process. These data represent the first report of a hyper-citrullinated library for the mouse and serve as a central resource for exploring the role of citrullination in this organism.
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Citrulina/metabolismo , Redes y Vías Metabólicas/fisiología , Biblioteca de Péptidos , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Cromatografía Liquida , Biología Computacional/métodos , Riñón/química , Riñón/metabolismo , Hígado/química , Hígado/metabolismo , Pulmón/química , Pulmón/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Muramidasa/química , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miocardio/química , Miocardio/metabolismo , Especificidad de Órganos , Péptidos/química , Desiminasas de la Arginina Proteica/químicaRESUMEN
Genetically isolated populations, such as the Old Order Amish and Old Order Mennonite communities, have an increased incidence of specific autosomal recessive disorders caused by the founder effect. In these populations, robust expanded carrier screening and diagnostic testing have the potential to reduce overall medical costs and improve patient outcomes. A novel next-generation sequencing assay was developed using anchored multiplex PCR technology (ArcherDX) for 162 different genetic syndromes caused by 202 pathogenic variants consisting of 150 single-nucleotide changes, 43 small insertion/deletions, and 9 large deletions (>20 nucleotides). To assess the accuracy of the screening panel results, 48 samples were selected on the basis of prior whole exome sequencing results. An additional 15 samples were chosen specifically to validate SMN1 and SMN2 copy number analyses. Collectively, the screening panel detected 273 pathogenic single-nucleotide or small insertion/deletion variants, 35 copy number variations, and 1 chromosomal abnormality (Klinefelter syndrome). Concordance with prior whole exome sequencing was 100%. By using a novel next-generation sequencing workflow, a successful targeted gene variant panel was developed for the Old Order Amish and Old Order Mennonite populations of Lancaster County, Pennsylvania. Population-wide carrier screening may help decrease the morbidity and mortality of these conditions in the high-risk populations.
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Amish/genética , Etnicidad/genética , Tamización de Portadores Genéticos , Genética de Población , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Factores de Edad , Variaciones en el Número de Copia de ADN , Estudios de Asociación Genética , Tamización de Portadores Genéticos/métodos , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Pennsylvania , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Advancements in sequencing capabilities have enhanced the study of the human microbiome. There are limited studies focused on the gastro-intestinal (gut) microbiome of infants, particularly the impact of diet between breast-fed (BF) versus formula-fed (FF). It is unclear what effect, if any, early feeding has on short-term or long-term composition and function of the gut microbiome. RESULTS: Using a shotgun metagenomics approach, differences in the gut microbiome between BF (n = 10) and FF (n = 5) infants were detected. A Jaccard distance principle coordinate analysis was able to cluster BF versus FF infants based on the presence or absence of species identified in their gut microbiome. Thirty-two genera were identified as statistically different in the gut microbiome sequenced between BF and FF infants. Furthermore, the computational workflow identified 371 bacterial genes that were statistically different between the BF and FF cohorts in abundance. Only seven genes were lower in abundance (or absent) in the FF cohort compared to the BF cohort, including CRISPR/Cas9; whereas, the remaining candidates, including autotransporter adhesins, were higher in abundance in the FF cohort compared to BF cohort. CONCLUSIONS: These studies demonstrated that FF infants have, at an early age, a significantly different gut microbiome with potential implications for function of the fecal microbiota. Interactions between the fecal microbiota and host hinted at here have been linked to numerous diseases. Determining whether these non-abundant or more abundant genes have biological consequence related to infant feeding may aid in understanding the adult gut microbiome, and the pathogenesis of obesity.
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Heterotopic ossification (HO) refers to the abnormal formation of bone in soft tissue. Although some of the underlying processes of HO have been described, there are currently no clinical tests using validated biomarkers for predicting HO formation. As such, the diagnosis is made radiographically after HO has formed. To identify potential and novel biomarkers for HO, we used isobaric tags for relative and absolute quantitation (iTRAQ) and high-throughput antibody arrays to produce a semi-quantitative proteomics survey of serum and tissue from subjects with (HO+) and without (HO-) heterotopic ossification. The resulting data were then analyzed using a systems biology approach. We found that serum samples from subjects experiencing traumatic injuries with resulting HO have a different proteomic expression profile compared to those from the matched controls. Subsequent quantitative ELISA identified five blood serum proteins that were differentially regulated between the HO+ and HO- groups. Compared to HO- samples, the amount of insulin-like growth factor I (IGF1) was up-regulated in HO+ samples, whereas a lower amount of osteopontin (OPN), myeloperoxidase (MPO), runt-related transcription factor 2 (RUNX2), and growth differentiation factor 2 or bone morphogenetic protein 9 (BMP-9) was found in HO+ samples (Welch two sample t-test; Pâ¯<â¯0.05). These proteins, in combination with potential serum biomarkers previously reported, are key candidates for a serum diagnostic panel that may enable early detection of HO prior to radiographic and clinical manifestations.
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Biomarcadores/metabolismo , Osificación Heterotópica/diagnóstico , Proteoma/análisis , Proteómica/métodos , Biología de Sistemas/métodos , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osificación Heterotópica/sangre , Osificación Heterotópica/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Spastic cerebral palsy (CP) is a leading cause of physical disability. Most people with spastic CP are born with it, but early diagnosis is challenging, and no current biomarker platform readily identifies affected individuals. The aim of this study was to evaluate epigenetic profiles as biomarkers for spastic CP. A novel analysis pipeline was employed to assess DNA methylation patterns between peripheral blood cells of adolescent subjects (14.9 ± 0.3 years old) with spastic CP and controls at single CpG site resolution. RESULTS: Significantly hypo- and hyper-methylated CpG sites associated with spastic CP were identified. Nonmetric multidimensional scaling fully discriminated the CP group from the controls. Machine learning based classification modeling indicated a high potential for a diagnostic model, and 252 sets of 40 or fewer CpG sites achieved near-perfect accuracy within our adolescent cohorts. A pilot test on significantly younger subjects (4.0 ± 1.5 years old) identified subjects with 73% accuracy. CONCLUSIONS: Adolescent patients with spastic CP can be distinguished from a non-CP cohort based on DNA methylation patterns in peripheral blood cells. A clinical diagnostic test utilizing a panel of CpG sites may be possible using a simulated classification model. A pilot validation test on patients that were more than 10 years younger than the main adolescent cohorts indicated that distinguishing methylation patterns are present earlier in life. This study is the first to report an epigenetic assay capable of distinguishing a CP cohort.
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Biomarcadores/análisis , Parálisis Cerebral/diagnóstico , Parálisis Cerebral/genética , Metilación de ADN , Epigenómica , Aprendizaje Automático , Reconocimiento de Normas Patrones Automatizadas , Análisis de Secuencia de ADN/métodos , Adolescente , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , MasculinoRESUMEN
STUDY QUESTION: Can subphenotype analysis of genome-wide association study (GWAS) data from subjects with testicular germ cell tumor (TGCT) provide insight into cryptorchidism (undescended testis, UDT) susceptibility? SUMMARY ANSWER: Suggestive intragenic GWAS signals common to UDT, TGCT case-case and TGCT case-control analyses occur in genes encoding RBFOX RNA-binding proteins (RBPs) and their neurodevelopmental targets. WHAT IS KNOWN ALREADY: UDT is a strong risk factor for TGCT, but while genetic risk factors for TGCT are well-known, genetic susceptibility to UDT is poorly understood and appears to be more complex. STUDY DESIGN, SIZE, DURATION: We performed a secondary subphenotype analysis of existing GWAS data from the Testicular Cancer Consortium (TECAC) and compared these results with our previously published UDT GWAS data, and with data previously acquired from studies of the fetal rat gubernaculum. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies from the National Cancer Institute (NCI), United Kingdom (UK) and University of Pennsylvania (Penn) that enrolled white subjects were the source of the TGCT GWAS data. We completed UDT subphenotype case-case (TGCT/UDT vs TGCT/non-UDT) and case-control (TGCT/UDT vs control), collectively referred to as 'TECAC' analyses, followed by a meta-analysis comprising 129 TGCT/UDT cases, 1771 TGCT/non-UDT cases, and 3967 unaffected controls. We reanalyzed our UDT GWAS results comprising 844 cases and 2718 controls by mapping suggestive UDT and TECAC signals (defined as P < 0.001) to genes using Ingenuity Pathway Analysis (IPA®). We compared associated pathways and enriched gene categories common to all analyses after Benjamini-Hochberg multiple testing correction, and analyzed transcript levels and protein expression using qRT-PCR and rat fetal gubernaculum confocal imaging, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: We found suggestive signals within 19 genes common to all three analyses, including RBFOX1 and RBFOX3, neurodevelopmental paralogs that encode RBPs targeting (U)GCATG-containing transcripts. Ten of the 19 genes participate in neurodevelopment and/or contribute to risk of neurodevelopmental disorders. Experimentally predicted RBFOX gene targets were strongly overrepresented among suggestive intragenic signals for the UDT (117 of 628 (19%), P = 3.5 × 10-24), TECAC case-case (129 of 711 (18%), P = 2.5 × 10-27) and TECAC case-control (117 of 679 (17%), P = 2 × 10-21) analyses, and a majority of the genes common to all three analyses (12 of 19 (63%), P = 3 × 10-9) are predicted RBFOX targets. Rbfox1, Rbfox2 and their encoded proteins are expressed in the rat fetal gubernaculum. Predicted RBFOX targets are also enriched among transcripts differentially regulated in the fetal gubernaculum during normal development (P = 3 × 10-31), in response to in vitro hormonal stimulation (P = 5 × 10-45) and in the cryptorchid LE/orl rat (P = 2 × 10-42). LARGE SCALE DATA: GWAS data included in this study are available in the database of Genotypes and Phenotypes (dbGaP accession numbers phs000986.v1.p1 and phs001349.v1p1). LIMITATIONS, REASONS FOR CAUTION: These GWAS data did not reach genome-wide significance for any individual analysis. UDT appears to have a complex etiology that also includes environmental factors, and such complexity may require much larger sample sizes than are currently available. The current methodology may also introduce bias that favors false discovery of larger genes. WIDER IMPLICATIONS OF THE FINDINGS: Common suggestive intragenic GWAS signals suggest that RBFOX paralogs and other neurodevelopmental genes are potential UDT risk candidates, and potential TGCT susceptibility modifiers. Enrichment of predicted RBFOX targets among differentially expressed transcripts in the fetal gubernaculum additionally suggests a role for this RBP family in regulation of testicular descent. As RBFOX proteins regulate alternative splicing of Calca to generate calcitonin gene-related peptide, a protein linked to development and function of the gubernaculum, additional studies that address the role of these proteins in UDT are warranted. STUDY FUNDING/COMPETING INTEREST(S): The Eunice Kennedy Shriver National Institute for Child Health and Human Development (R01HD060769); National Center for Research Resources (P20RR20173), National Institute of General Medical Sciences (P20GM103464), Nemours Biomedical Research, the Testicular Cancer Consortium (U01CA164947), the Intramural Research Program of the NCI, a support services contract HHSN26120130003C with IMS, Inc., the Abramson Cancer Center at Penn, National Cancer Institute (CA114478), the Institute of Cancer Research, UK and the Wellcome Trust Case-Control Consortium (WTCCC) 2. None of the authors reports a conflict of interest.
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Antígenos Nucleares/genética , Criptorquidismo/genética , Proteínas del Tejido Nervioso/genética , Polimorfismo de Nucleótido Simple , Factores de Empalme de ARN/genética , Proteínas Represoras/genética , Neoplasias Testiculares/genética , Alelos , Estudios de Casos y Controles , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , MasculinoRESUMEN
RATIONALE: GSK-3ß (glycogen synthase kinase 3ß) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK-3ß for certain functions. OBJECTIVES: Here, we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK-3ß. SNO-dependent modulation of the localization of GSK-3ß and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3ß regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. METHODS AND RESULTS: We found that GSK-3ß undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes, as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK-3ß significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK-3ß promotes its nuclear translocation and access to novel downstream phosphosubstrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phosphoproteomics pathway analysis reveals that nuclear GSK-3ß plays a central role in cell cycle control, RNA splicing, and DNA damage response. CONCLUSIONS: The results indicate that SNO has a differential effect on the location and activity of GSK-3ß in the cytoplasm versus the nucleus. SNO modification of GSK-3ß occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.