Implementation and Feasibility of Clinical Genome Sequencing Embedded Into the Outpatient Nephrology Care for Patients With Proteinuric Kidney Disease.

Introduction: The diagnosis and management of proteinuric kidney diseases such as focal segmental glomerulosclerosis (FSGS) are challenging. Genetics holds the promise to improve clinical decision making for these diseases; however, it is often performed too late to enable timely clinical action and it is not implemented within routine outpatient nephrology visits. Methods: We sought to test the implementation and feasibility of clinical rapid genome sequencing (GS) in guiding decision making in patients with proteinuric kidney disease in real-time and embedded in the outpatient nephrology setting. Results: We enrolled 10 children or young adults with biopsy-proven FSGS (9 cases) or minimal change disease (1 case). The mean age at enrollment was 16.2 years (range 2-30). The workflow did not require referral to external genetics clinics but was conducted entirely during the nephrology standard-of-care appointments. The total turn-around-time from enrollment to return-of-results and clinical decision averaged 21.8 days (12.4 for GS), which is well within a time frame that allows clinically relevant treatment decisions. A monogenic or APOL1-related form of kidney disease was diagnosed in 5 of 10 patients. The genetic findings resulted in a rectified diagnosis in 6 patients. Both positive and negative GS findings determined a change in pharmacological treatment. In 3 patients, the results were instrumental for transplant evaluation, donor selection, and the immunosuppressive treatment. All patients and families received genetic counseling. Conclusion: Clinical GS is feasible and can be implemented in real-time in the outpatient care to help guiding clinical management. Additional studies are needed to confirm the cost-effectiveness and broader utility of clinical GS across the phenotypic and demographic spectrum of kidney diseases.

Implementation of Rapid Genome Sequencing for Critically Ill Infants With Complex Congenital Heart Disease.

BACKGROUND: Rapid genome sequencing (rGS) has been shown to improve care of critically ill infants. Congenital heart disease (CHD) is a leading cause of infant mortality and is often caused by genetic disorders, yet the utility of rGS has not been prospectively studied in this population. METHODS: We conducted a prospective evaluation of rGS to improve the care of infants with complex CHD in our cardiac neonatal intensive care unit. RESULTS: In a cohort of 48 infants with complex CHD, rGS diagnosed 14 genetic disorders in 13 (27%) individuals and led to changes in clinical management in 8 (62%) cases with diagnostic results. These included 2 cases in whom genetic diagnoses helped avert intensive, futile interventions before cardiac neonatal intensive care unit discharge, and 3 cases in whom eye disease was diagnosed and treated in early childhood. CONCLUSIONS: Our study provides the first prospective evaluation of rGS for infants with complex CHD to our knowledge. We found that rGS diagnosed genetic disorders in 27% of cases and led to changes in management in 62% of cases with diagnostic results. Our model of care depended on coordination between neonatologists, cardiologists, surgeons, geneticists, and genetic counselors. These findings highlight the important role of rGS in CHD and demonstrate the need for expanded study of how to implement this resource to a broader population of infants with CHD.

Efficacy and Tolerability of Eravacycline in Bacteremic Patients with Complicated Intra-Abdominal Infection: A Pooled Analysis from the IGNITE1 and IGNITE4 Studies.

Background: Eravacycline is a novel, fully synthetic fluorocycline antibiotic that was evaluated for the treatment of complicated intra-abdominal infections (cIAI) in two phase 3 clinical trials. The objective of this analysis was to evaluate the clinical cure and microbiologic response at the test-of-cure (TOC) visit and the safety of eravacycline in patients with cIAI and baseline bacteremia who received eravacycline versus comparators. Patients and Methods: Pooled data of patients with bacteremia from the Investigating Gram-Negative Infections Treated with Eravacycline (IGNITE) 1 and IGNITE4 studies were analyzed. All patients were randomly assigned in a one-to-one ratio to receive eravacycline 1 mg/kg intravenously every 12 hours, ertapenem 1 g intravensouly every 24 hours (IGNITE1), or meropenem 1 g intravenously every eight hours (IGNITE4) for four to 14 days. Blood and intra-abdominal samples were collected from all patients at baseline. Clinical outcome and microbiologic eradiation at the TOC visit (28 days after randomization) and safety in the microbiologic-intent-to-treat population (micro-ITT) were assessed. Results: Of 415 patients treated with eravacycline and 431 treated with carbapenem comparators, concurrent bacteremia was identified in 32 (7.7%) and 31 (7.2%) patients, respectively. Demographic and baseline characteristics were similar among treatment groups. In the micro-ITT population, the pooled clinical response at the TOC visit for eravacycline was 28 of 32 (87.5%) and was 24 of 31 (77.0%) for comparators among the subgroup with baseline bacteremia (treatment difference 5.9; 95% confidence interval [CI], -6.5 to 17.4). At TOC, microbiologic eradication of pathogens isolated from blood specimens occurred for 34 of 35 (97.1%) pathogens with eravacycline and 35 of 36 (97.2%) pathogens with comparators. The incidence of adverse events was comparable between treated groups and similar to that observed in the non-bacteremic population. Conclusion: Eravacycline demonstrated a similar clinical outcome and microbiologic eradication rate as comparator carbapenems in patients with cIAI and associated secondary bacteremia. Future clinical trials of cIAI should report outcomes of this important clinical cohort (cIAI with concurrent bacteremia) given their high risk for adverse outcomes.

Detection of Copy Number Variants by Short Multiply Aggregated Sequence Homologies.

Chromosomal microarray testing is indicated for patients with diagnoses including unexplained developmental delay or intellectual disability, autism spectrum disorders, and multiple congenital anomalies. The short multiply aggregated sequence homologies (SMASH) genomic assay is a novel next-generation sequencing technology that performs copy number analysis at resolution similar to high-coverage whole genome sequencing but requires far less capacity. We benchmarked the performance of SMASH on a panel of genomic DNAs containing known copy number variants (CNVs). SMASH was able to detect pathogenic copy number variants of ≥10 kb in 77 of 77 samples. No pathogenic events were seen in 32 of 32 controls, indicating 100% sensitivity and specificity for detecting pathogenic CNVs >10 kb. Repeatability (interassay precision) and reproducibility (intra-assay precision) were assessed with 13 samples and showed perfect concordance. We also established that SMASH had a limit of detection of 20% for detection of large mosaic CNVs. Finally, we analyzed seven blinded specimens by SMASH analysis and successfully identified all pathogenic events. These results establish the efficacy of the SMASH genomic assay as a clinical test for the detection of pathogenic copy number variants at a resolution comparable to chromosomal microarray analysis.

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

Identification of targetable BRAF ΔN486_P490 variant by whole-genome sequencing leading to dabrafenib-induced remission of a BRAF-mutant pancreatic adenocarcinoma.

The tumor genome of a patient with advanced pancreatic cancer was sequenced to identify potential therapeutic targetable mutations after standard of care failed to produce any significant overall response. Matched tumor-normal whole-genome sequencing revealed somatic mutations in BRAF, TP53, CDKN2A, and a focal deletion of SMAD4 The BRAF variant was an in-frame deletion mutation (ΔN486_P490), which had been previously demonstrated to be a kinase-activating alteration in the BRAF kinase domain. Working with the Novartis patient assistance program allowed us to treat the patient with the BRAF inhibitor, dabrafenib. The patient's overall clinical condition improved dramatically with dabrafenib. Levels of serum tumor marker dropped immediately after treatment, and a subsequent CT scan revealed a significant decrease in the size of both primary and metastatic lesions. The dabrafenib-induced remission lasted for 6 mo. Preclinical studies published concurrently with the patient's treatment showed that the BRAF in-frame mutation (ΔNVTAP) induces oncogenic activation by a mechanism distinct from that induced by V600E, and that this difference dictates the responsiveness to different BRAF inhibitors. This study describes a dramatic instance of how high-level genomic technology and analysis was necessary and sufficient to identify a clinically logical treatment option that was then utilized and shown to be of clinical value for this individual.

Correction to: Sequencing and curation strategies for identifying candidate glioblastoma treatments.

Following publication of the original article [1], it was reported that the given name of the fourteenth author was incorrectly published. The incorrect and the correct names are given below.

Sequencing and curation strategies for identifying candidate glioblastoma treatments.

BACKGROUND: Prompted by the revolution in high-throughput sequencing and its potential impact for treating cancer patients, we initiated a clinical research study to compare the ability of different sequencing assays and analysis methods to analyze glioblastoma tumors and generate real-time potential treatment options for physicians. METHODS: A consortium of seven institutions in New York City enrolled 30 patients with glioblastoma and performed tumor whole genome sequencing (WGS) and RNA sequencing (RNA-seq; collectively WGS/RNA-seq); 20 of these patients were also analyzed with independent targeted panel sequencing. We also compared results of expert manual annotations with those from an automated annotation system, Watson Genomic Analysis (WGA), to assess the reliability and time required to identify potentially relevant pharmacologic interventions. RESULTS: WGS/RNAseq identified more potentially actionable clinical results than targeted panels in 90% of cases, with an average of 16-fold more unique potentially actionable variants identified per individual; 84 clinically actionable calls were made using WGS/RNA-seq that were not identified by panels. Expert annotation and WGA had good agreement on identifying variants [mean sensitivity = 0.71, SD = 0.18 and positive predictive value (PPV) = 0.80, SD = 0.20] and drug targets when the same variants were called (mean sensitivity = 0.74, SD = 0.34 and PPV = 0.79, SD = 0.23) across patients. Clinicians used the information to modify their treatment plan 10% of the time. CONCLUSION: These results present the first comprehensive comparison of technical and machine augmented analysis of targeted panel and WGS/RNA-seq to identify potential cancer treatments.

Prenatal Diagnosis by Whole Exome Sequencing in Fetuses with Ultrasound Abnormalities.

Whole-exome sequencing (WES) has been used as a standard of care for postnatal diagnosis in the clinical setting in the past few years for children and adults with undiagnosed disease. Many rare disorders have been diagnosed through WES, which is less expensive than the traditional serial genetic testing where patients had previously spent years on an uninformative diagnostic odyssey. Seeking a diagnosis often entails enduring time consuming, and sometimes invasive procedures which may be associated with medical risks that are stressful for families and impose a heavy burden on the health-care system. However, the use of WES is considered impractical in the prenatal and neonatal testing period because of the technical and computational challenges of performing genomic sequencing from small amounts of genetic material, and the need for faster turnaround time (TAT) than the current 6-8 weeks TAT provided by most clinical labs offering postnatal testing. With the rapidly evolving methods of sequence analysis, there are clinical challenges such as the constantly increasing number of genes being identified which are not yet fully phenotypically characterized, especially when ascertained prenatally or neonatally before all the clinical features may be evident. Despite these challenges, there are many clinical benefits to acquiring genomic information in the prenatal and neonatal period. These include superior prognostic information which allows for prenatal planning of mode of delivery and hospital for delivery and optimized neonatal management. We have developed a clinical WES assay using small amounts of DNA with a TAT of 10 days for use in the prenatal or neonatal setting. This test is used to detect small nucleotide variants and indels in fetuses and neonates with structural abnormalities.

A recurrent novel MGA-NUTM1 fusion identifies a new subtype of high-grade spindle cell sarcoma.

NUTM1-rearranged tumors are defined by the presence of a gene fusion between NUTM1 and various gene partners and typically follow a clinically aggressive disease course with poor outcomes despite conventional multimodality therapy. NUTM1-rearranged tumors display histologic features of a poorly differentiated carcinoma with areas of focal squamous differentiation and typically express the BRD4-NUTM1 fusion gene defining a distinct clinicopathologic entity-NUT carcinoma (NC). NCs with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high-grade spindle cell sarcoma harboring a novel MGA-NUTM1 fusion. Whole-genome sequencing identified the presence of complex rearrangements resulting in a MGA-NUTM1 fusion gene in the absence of other significant somatic mutations. Genetic rearrangement was confirmed by fluorescence in situ hybridization, and expression of the fusion gene product was confirmed by transcriptomic analysis. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1-22) and NUTM1 (exons 3-8). Histopathologically, both cases were high-grade spindle cell sarcomas without specific differentiation markers. In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA-NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high-grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer.

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Whole Genome Sequencing-Based Discovery of Structural Variants in Glioblastoma.

Next-generation DNA sequencing (NGS) technologies are currently being applied in both research and clinical settings for the understanding and management of disease. The goal is to use high-throughput sequencing to identify specific variants that drive tumorigenesis within each individual's tumor genomic profile. The significance of copy number and structural variants in glioblastoma makes it essential to broaden the search beyond oncogenic single nucleotide variants toward whole genome profiles of genetic aberrations that may contribute to disease progression. The heterogeneity of glioblastoma and its variability of cancer driver mutations necessitate a more robust examination of a patient's tumor genome. Here, we present patient whole genome sequencing (WGS) information to identify oncogenic structural variants that may contribute to glioblastoma pathogenesis. We provide WGS protocols and bioinformatics approaches to identify copy number and structural variations in 41 glioblastoma patient samples. We present how WGS can identify structural diversity within glioblastoma samples. We specifically show how to apply current bioinformatics tools to detect EGFR variants and other structural aberrations from DNA whole genome sequencing and how to validate those variants within the laboratory. These comprehensive WGS protocols can provide additional information directing more precise therapeutic options in the treatment of glioblastoma.

Clinical whole exome sequencing from dried blood spot identifies novel genetic defect underlying asparagine synthetase deficiency.

We add two novel variants to the existing mutation spectrum of ASNS gene. Loss of ASNS function should be suspected in newborns presenting with congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. Acquisition and sequencing of stored newborn blood spot can be a valuable option when no biological samples are available from a deceased child.

Mechanisms of pigmentation loss in subterranean fishes

Troglobitic (exclusively subterranean) organisms usually present, among their apomorphies related to the subterranean life (troglomorphisms), the regression of eyes and melanic pigmentation. The degree of regression varies among species, from a slight reduction to the complete loss of eyes and dark pigmentation, without a taxonomic correlation. While mechanisms of eye reduction have been intensively investigated in some troglobites such as the Mexican blind tetra characins, genus Astyanax, and the European salamander, Proteus anguinus, few studies have focused on pigmentation. The Brazilian subterranean ichthyofauna distinguishes not only by the species richness (23 troglobitic fishes so far known) but also by the variation in the degree of reduction of eyes and pigmentation. This study focused on Brazilian fishes completely devoid of melanic pigmentation: the characiform Stygichthys typhlops (Characidae) and the siluriforms Ancistrus formoso (Loricariidae), Rhamdiopsis sp.1 (Heptapteridae; from caves in the Chapada Diamantina, Bahia) and Rhamdiopsis sp. 2 (cave in Campo Formoso, Bahia). In order to investigate if such depigmentation is the result of blockage in some step in the melanogenesis, in vitro tests of administration of L-DOPA were done, using caudal-fin fragments extracted from living fish. Except for Rhamdiopsis sp. 2, all the studied species were DOPA(+), i.e., melanin was synthesized after L-DOPA administration. This indicates these fish do have melanophores but they are unable to convert L-tyrosine to L-DOPA. On the other hand, Rhamdiopsis sp. 2, like the albino specimens of Trichomycterus itacarambiensis previously studied (which correspond to one third of the population), are DOPA(-), either because the block of melanin synthesis occurs downstream in melanogenesis, which is probably the case with T. itacarambiensis (monogenic system in view of the phenotypic discontinuity), or because the so-called albinos do no possess...

Organismos exclusivamente subterrâneos (troglóbios) usualmente exibem, entre as apomorfias relacionadas à evolução em isolamento nesse ambiente (troglomorfismos), a redução, até perda total, das estruturas visuais e da pigmentação melânica. Os mecanismos de regressão ocular em troglóbios têm sido intensivamente estudados, sobretudo em peixes como os lambaris cegos mexicanos do gênero Astyanax, e salamandras como Proteus anguinus. Por outro lado, poucos são os trabalhos abordando a perda da pigmentação nesses organismos. A ictiofauna subterrânea brasileira destaca-se não só pela riqueza de espécies (23 conhecidas até o momento) como também pelas diferenças no seu grau de troglomorfismo, sem uma correlação taxonômica. O presente estudo abordou as espécies brasileiras totalmente desprovidas de qualquer traço de pigmentação melânica: o caraciforme Stygichthys typhlops (Characidae) e os siluriformes Ancistrus formoso (Loricariidae), Rhamdiopsis sp.1 (Heptapteridae; cavernas da Chapada Diamantina, Bahia) e Rhamdiopsis sp. 2 (caverna de Campo Formoso, Bahia). Com a finalidade de investigar se essa despigmentação é resultado de bloqueio em algum passo da cadeia de síntese de melanina, foram feitos testes in vitro, utilizando-se fragmentos da nadadeira caudal extraídos de exemplares mantidos vivos, de reação à administração de L-DOPA. Com exceção dos de Rhamdiopsis sp. 2, os exemplares estudados revelaram-se DOPA(+), i.e., houve produção de melanina após a administração de L-DOPA, o que indica que sua despigmentação é devida a uma disfunção da tirosinase, enzima responsável pela transformação de tirosina em L-DOPA nos melanóforos, os quais, portanto, ainda existem nesses peixes. Já os exemplares Rhamdiopsis sp. 2, assim como o terço conspicuamente despigmentado da população de Trichomycterus itacarambiensis, espécie estudada anteriormente sob esse aspecto, são DOPA(-), seja porque o bloqueio na síntese de melanina ocorre em um passo a jusante...