Publications by authors named "Richard K Wilson"

318 Publications

Maternal mosaicism for a missense variant in the SMS gene that causes Snyder-Robinson syndrome.

Cold Spring Harb Mol Case Stud 2021 Oct 19. Epub 2021 Oct 19.

Nationwide Children's Hospital.

There is increasing recognition for the contribution of genetic mosaicism to human disease, particularly as high-throughput sequencing has enabled detection of sequence variants at very low allele frequencies. Here, we describe an infant male who presented at 9 months of age with hypotonia, dysmorphic features, congenital heart disease, hyperinsulinemic hypoglycemia, hypothyroidism, and bilateral sensorineural hearing loss. Whole-genome sequencing of the proband and the parents uncovered an apparent mutation in the X-linked gene. SMS encodes spermine synthase, which catalyzes the production of spermine from spermidine. Inactivation of the gene disrupts the spermidine/spermine ratio, resulting in Snyder-Robinson syndrome. The variant in our patient is absent from the gnomAD and ExAC databases and causes a missense change (p.Arg130Cys) predicted to be damaging by most tools. While Sanger sequencing confirmed the status in our proband, PCR and deep targeted resequencing to ~84,000-175,000x depth revealed that the variant is present in blood from the unaffected mother at ~3% variant allele frequency. Our findings thus provided a long-sought diagnosis for the family while highlighting the role of parental mosaicism in severe genetic disorders.
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http://dx.doi.org/10.1101/mcs.a006122DOI Listing
October 2021

Genomic Profiling of Lung Adenocarcinoma in Never-Smokers.

J Clin Oncol 2021 Nov 30;39(33):3747-3758. Epub 2021 Sep 30.

Division of Oncology, Washington University School of Medicine, St Louis, MO.

Purpose: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers.

Methods: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium.

Results: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% 49.5%; < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% 6.9%; = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition.

Conclusion: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.
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http://dx.doi.org/10.1200/JCO.21.01691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601276PMC
November 2021

Hypomorphic alleles pose challenges in rare disease genomic variant interpretation.

Clin Genet 2021 12 3;100(6):775-776. Epub 2021 Sep 3.

The Steve and Cindy Rasmussen Institute for Genomic Medicine, Nationwide Children's Hospital, Columbus, Ohio, USA.

Exon skipping associated with an ATP7B intronic variant in a patient with Wilson's disease. (A) Sashimi plot visualization of aligned RNA sequencing data from proband liver tissue at ATP7B exons 14-13-12. The red track shows traditional RNA-seq data; the blue track shows RNA-seq enriched with exon capture (cDNA-cap) which achieves higher depth of protein-coding transcripts. The histogram indicates overall sequencing depth while arcs tabulate the number of junction-spanning reads supporting exon pairs. (B) The domain structure (top) and exon structure (bottom) of ATP7B. Loss of exon 13 (dashed box) would remove a transmembrane domain and disrupt the first phosphorylation domain.
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http://dx.doi.org/10.1111/cge.14052DOI Listing
December 2021

Genome Assemblies across the Diverse Evolutionary Spectrum of Protozoan Parasites.

Microbiol Resour Announc 2021 Sep 2;10(35):e0054521. Epub 2021 Sep 2.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.

We report the high-quality draft assemblies and gene annotations for 13 species and/or strains of the protozoan parasite genera , , and , which span the phylogenetic diversity of the subfamily Leishmaniinae within the kinetoplastid order of the phylum Euglenazoa. These resources will support studies on the origins of parasitism.
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http://dx.doi.org/10.1128/MRA.00545-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8411921PMC
September 2021

PTEN somatic mutations contribute to spectrum of cerebral overgrowth.

Brain 2021 Nov;144(10):2971-2978

The Steve and Cindy Rasmussen Institute for Genomic Medicine, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.

Phosphatase and tensin homologue (PTEN) regulates cell growth and survival through inhibition of the mammalian target of rapamycin (MTOR) signalling pathway. Germline genetic variation of PTEN is associated with autism, macrocephaly and PTEN hamartoma tumour syndromes. The effect of developmental PTEN somatic mutations on nervous system phenotypes is not well understood, although brain somatic mosaicism of MTOR pathway genes is an emerging cause of cortical dysplasia and epilepsy in the paediatric population. Here we report two somatic variants of PTEN affecting a single patient presenting with intractable epilepsy and hemimegalencephaly that varied in clinical severity throughout the left cerebral hemisphere. High-throughput sequencing analysis of affected brain tissue identified two somatic variants in PTEN. The first variant was present in multiple cell lineages throughout the entire hemisphere and associated with mild cerebral overgrowth. The second variant was restricted to posterior brain regions and affected the opposite PTEN allele, resulting in a segmental region of more severe malformation, and the only neurons in which it was found by single-nuclei RNA-sequencing had a unique disease-related expression profile. This study reveals brain mosaicism of PTEN as a disease mechanism of hemimegalencephaly and furthermore demonstrates the varying effects of single- or bi-allelic disruption of PTEN on cortical phenotypes.
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http://dx.doi.org/10.1093/brain/awab173DOI Listing
November 2021

Gastroblastoma with a novel EWSR1-CTBP1 fusion presenting in adolescence.

Genes Chromosomes Cancer 2021 Sep 7;60(9):640-646. Epub 2021 Jun 7.

Department of Pathology, The Ohio State University, Columbus, Ohio, USA.

Gastroblastomas are rare tumors with a biphasic epithelioid/spindle cell morphology that typically present in early adulthood and have recurrent MALAT1-GLI1 fusions. We describe an adolescent patient with Wiskott-Aldrich syndrome who presented with a large submucosal gastric tumor with biphasic morphology. Despite histologic features consistent with gastroblastoma, a MALAT1-GLI1 fusion was not found in this patient's tumor; instead, comprehensive molecular profiling identified a novel EWSR1-CTBP1 fusion and no other significant genetic alterations. The tumor also overexpressed NOTCH and FGFR by RNA profiling. The novel fusion and expression profile suggest a role for epithelial-mesenchymal transition in this tumor, with potential implications for the pathogenesis of biphasic gastric tumors such as gastroblastoma.
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http://dx.doi.org/10.1002/gcc.22973DOI Listing
September 2021

Novel morphologic findings in PLAG1-rearranged soft tissue tumors.

Genes Chromosomes Cancer 2021 Aug 29;60(8):577-585. Epub 2021 Apr 29.

Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, Columbus, Ohio, USA.

Oncogenesis in PLAG1-rearranged tumors often results from PLAG1 transcription factor overexpression driven by promoter-swapping between constitutively expressed fusion partners. PLAG1-rearranged tumors demonstrate diverse morphologies. This study adds to this morphologic heterogeneity by introducing two tumors with PLAG1 rearrangements that display distinct histologic features. The first arose in the inguinal region of a 3-year-old, appeared well-circumscribed with a multinodular pattern, and harbored two fusions: ZFHX4-PLAG1 and CHCHD7-PLAG1. The second arose in the pelvic cavity of a 15-year-old girl, was extensively infiltrative and vascularized with an adipocytic component, and demonstrated a COL3A1-PLAG1 fusion. Both showed low-grade cytomorphology, scarce mitoses, no necrosis, and expression of CD34 and desmin. The ZFHX4-/CHCHD7-PLAG1-rearranged tumor showed no evidence of recurrence after 5 months. By contrast, the COL3A1-PLAG1-rearranged tumor quickly recurred following primary excision with positive margins; subsequent re-excision with adjuvant chemotherapy resulted in no evidence of recurrence after 2 years. While both tumors show overlap with benign and malignant fibroblastic and fibrovascular neoplasms, they also display divergent features. These cases highlight the importance of appropriate characterization in soft tissue tumors with unusual clinical and histologic characteristics.
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http://dx.doi.org/10.1002/gcc.22953DOI Listing
August 2021

Molecular classification of a complex structural rearrangement of the RB1 locus in an infant with sporadic, isolated, intracranial, sellar region retinoblastoma.

Acta Neuropathol Commun 2021 04 7;9(1):61. Epub 2021 Apr 7.

The Steve and Cindy Rasmussen Institute for Genomic Medicine, Abigail Wexner Research Institute At Nationwide Children's Hospital, 575 Children's Crossroad, Columbus, OH, 43215 , USA.

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.
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http://dx.doi.org/10.1186/s40478-021-01164-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025529PMC
April 2021

The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BMC Biol 2021 03 10;19(1):41. Epub 2021 Mar 10.

Department of Computer Science & Engineering, Department of Biochemistry & Biophysics, Texas A & M University, College Station, TX, USA.

Background: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies.

Results: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways.

Conclusions: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.
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http://dx.doi.org/10.1186/s12915-021-00975-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944917PMC
March 2021

A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis.

PLoS Pathog 2021 01 19;17(1):e1009222. Epub 2021 Jan 19.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor β-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.
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http://dx.doi.org/10.1371/journal.ppat.1009222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846122PMC
January 2021

Sequence analysis in reveals pervasiveness of X-Y arms races in mammalian lineages.

Genome Res 2020 12 18;30(12):1716-1726. Epub 2020 Nov 18.

Whitehead Institute, Cambridge, Massachusetts 02142, USA.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.
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http://dx.doi.org/10.1101/gr.269902.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706723PMC
December 2020

YAP1-FAM118B Fusion Defines a Rare Subset of Childhood and Young Adulthood Meningiomas.

Am J Surg Pathol 2021 03;45(3):329-340

The Steve and Cindy Rasmussen Institute for Genomic Medicine.

Meningiomas are a central nervous system tumor primarily afflicting adults, with <1% of cases diagnosed during childhood or adolescence. Somatic variation in NF2 may be found in ∼50% of meningiomas, with other genetic drivers (eg, SMO, AKT1, TRAF7) contributing to NF2 wild-type tumors. NF2 is an upstream negative regulator of YAP signaling and loss of the NF2 protein product, Merlin, results in YAP overexpression and target gene transcription. This mechanism of dysregulation is described in NF2-driven meningiomas, but further work is necessary to understand the NF2-independent mechanism of tumorigenesis. Amid our institutional patient-centric comprehensive molecular profiling study, we identified an individual with meningioma harboring a YAP1-FAM118B fusion, previously reported only in supratentorial ependymoma. The tumor histopathology was remarkable, characterized by prominent islands of calcifying fibrous nodules within an overall collagen-rich matrix. To gain insight into this finding, we subsequently evaluated the genetic landscape of 11 additional pediatric and adolescent/young adulthood meningioma patients within the Children's Brain Tumor Tissue Consortium. A second individual harboring a YAP1-FAM118B gene fusion was identified within this database. Transcriptomic profiling suggested that YAP1-fusion meningiomas are biologically distinct from NF2-driven meningiomas. Similar to other meningiomas, however, YAP1-fusion meningiomas demonstrated overexpression of EGFR and MET. DNA methylation profiling further distinguished YAP1-fusion meningiomas from those observed in ependymomas. In summary, we expand the genetic spectrum of somatic alteration associated with NF2 wild-type meningioma to include the YAP1-FAM118B fusion and provide support for aberrant signaling pathways potentially targetable by therapeutic intervention.
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http://dx.doi.org/10.1097/PAS.0000000000001597DOI Listing
March 2021

Infantile fibrosarcoma-like tumor driven by novel fusion consolidated with cabozantinib.

Cold Spring Harb Mol Case Stud 2020 10 7;6(5). Epub 2020 Oct 7.

Division of Hematology, Oncology, Blood and Marrow Transplant, Nationwide Children's Hospital, Columbus, Ohio 43205, USA.

Infantile fibrosarcoma (IFS) is nearly universally driven by gene fusions involving the NTRK family. fusions account for ∼85% of alterations; the remainder are attributed to NTRK-variant fusions. Rarely, other genomic aberrations have been described in association with tumors identified as IFS or IFS-like. We describe the utility of genomic characterization of an IFS-like tumor. We also describe the successful treatment combination of VAC (vincristine, actinomycin, cyclophosphamide) with tyrosine kinase inhibitor (TKI) maintenance in this entity. This patient presented at birth with a right facial mass, enlarging at 1 mo to 4.9 × 4.5 × 6.3 cm. Biopsy demonstrated hypercellular fascicles of spindle cells with patchy positivity for smooth muscle actin (SMA) and negativity for S100, desmin, myogenin, and MyoD1. Targeted RNA sequencing identified a novel fusion with confirmed absence of , and the patient was diagnosed with an IFS-like tumor. A positron emission tomography (PET) scan was negative for metastatic disease. VAC was given for a duration of 10 mo. Resection at 13 mo of age demonstrated positive margins. Cabozantinib, a MET-targeting TKI, was initiated. The patient tolerated cabozantinib well and has no evidence of disease at 24 mo of age. We describe a novel driver fusion in association with a locally aggressive IFS-like tumor. MET functions as an oncogene and, when associated with the RNA binding protein RBPMS, forms an in-frame fusion product that retains the MET kinase domain. This fusion is associated with aberrant cell signaling pathway expression and subsequent malignancy. We describe treatment with cabozantinib in a patient with an IFS-like neoplasm.
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http://dx.doi.org/10.1101/mcs.a005645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552925PMC
October 2020

Genetic Characterization of Pediatric Sarcomas by Targeted RNA Sequencing.

J Mol Diagn 2020 10 1;22(10):1238-1245. Epub 2020 Aug 1.

Institute for Genomic Medicine, Nationwide Children's Hospital, Columbus, Ohio; Department of Pathology, Wexner Medical Center, The Ohio State University, Columbus, Ohio. Electronic address:

Somatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR-based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants.
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http://dx.doi.org/10.1016/j.jmoldx.2020.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538815PMC
October 2020

Somatic mosaicism correlates with clinical findings in epilepsy brain tissue.

Neurol Genet 2020 Aug 17;6(4):e460. Epub 2020 Jun 17.

The Steve and Cindy Rasmussen Institute for Genomic Medicine (K.E.M., D.C.K., K.M.S., T.A.B., E.C., K.L., V.M., H.Z., P.B., J.B., J.F., N.B., A.R.M., C.E.C., P.W., R.K.W., E.R.M.), Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH; Division of Genetic and Genomic Medicine (E.C.), Nationwide Children's Hospital, Columbus, OH; Department of Neurosurgery (A.S., J.L., J.A.P.), Nationwide Children's Hospital, Columbus, OH; Department of Pathology and Laboratory Medicine (C.R.P.), Nationwide Children's Hospital, Columbus, OH; Division of Child Neurology (A.P.O.), Nationwide Children's Hospital, Columbus, OH; Department of Radiology (J.A.R., S.H.S), Nationwide Children's Hospital, Columbus, OH; Department of Pediatrics (D.C.K., V.M., C.E.C., J.L., P.W., R.K.W, E.R.M., A.P.O.), The Ohio State University College of Medicine, Columbus, OH; Department of Neurosurgery (J.L., J.A.P., A.P.O.), The Ohio State University College of Medicine, Columbus, OH; Department of Pathology (C.E.C., C.R.P.), The Ohio State University College of Medicine, Columbus, OH; and Department of Biomedical Education & Anatomy (C.R.P.), Division of Anatomy, The Ohio State University College of Medicine, Columbus, OH.

Objective: Many genetic studies of intractable epilepsy in pediatric patients primarily focus on inherited, constitutional genetic deficiencies identified in patient blood. Recently, studies have revealed somatic mosaicism associated with epilepsy in which genetic variants are present only in a subset of brain cells. We hypothesize that tissue-specific, somatic mosaicism represents an important genetic etiology in epilepsy and aim to discover somatic alterations in epilepsy-affected brain tissue.

Methods: We have pursued a research study to identify brain somatic mosaicism, using next-generation sequencing (NGS) technologies, in patients with treatment refractory epilepsy who have undergone surgical resection of affected brain tissue.

Results: We used an integrated combination of NGS techniques and conventional approaches (radiology, histopathology, and electrophysiology) to comprehensively characterize multiple brain regions from a single patient with intractable epilepsy. We present a 3-year-old male patient with West syndrome and intractable tonic seizures in whom we identified a pathogenic frameshift somatic variant in , present at a range of variant allele fractions (4.2%-19.5%) in 12 different brain tissues detected by targeted sequencing. The proportion of the variant correlated with severity and location of neurophysiology and neuroimaging abnormalities for each tissue.

Conclusions: Our findings support the importance of tissue-based sequencing and highlight a correlation in our patient between variant allele fractions and the severity of epileptogenic phenotypes in different brain tissues obtained from a grid-based resection of clinically defined epileptogenic regions.
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http://dx.doi.org/10.1212/NXG.0000000000000460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323482PMC
August 2020

The clonal evolution of metastatic colorectal cancer.

Sci Adv 2020 Jun 10;6(24):eaay9691. Epub 2020 Jun 10.

The Alvin J. Siteman Comprehensive Cancer Center, Washington University in St. Louis, St. Louis, MO, USA.

Tumor heterogeneity and evolution drive treatment resistance in metastatic colorectal cancer (mCRC). Patient-derived xenografts (PDXs) can model mCRC biology; however, their ability to accurately mimic human tumor heterogeneity is unclear. Current genomic studies in mCRC have limited scope and lack matched PDXs. Therefore, the landscape of tumor heterogeneity and its impact on the evolution of metastasis and PDXs remain undefined. We performed whole-genome, deep exome, and targeted validation sequencing of multiple primary regions, matched distant metastases, and PDXs from 11 patients with mCRC. We observed intricate clonal heterogeneity and evolution affecting metastasis dissemination and PDX clonal selection. Metastasis formation followed both monoclonal and polyclonal seeding models. In four cases, metastasis-seeding clones were not identified in any primary region, consistent with a metastasis-seeding-metastasis model. PDXs underrepresented the subclonal heterogeneity of parental tumors. These suggest that single sample tumor sequencing and current PDX models may be insufficient to guide precision medicine.
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http://dx.doi.org/10.1126/sciadv.aay9691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286679PMC
June 2020

Early-onset Wilson disease caused by exon skipping associated with intronic variant.

Cold Spring Harb Mol Case Stud 2020 06 12;6(3). Epub 2020 Jun 12.

Department of Pediatrics, The Ohio State University, Columbus, Ohio 43205, USA.

Wilson disease is a medically actionable rare autosomal recessive disorder of defective copper excretion caused by mutations in , one of two highly evolutionarily conserved copper-transporting ATPases. Hundreds of disease-causing variants in have been reported to public databases; more than half of these are missense changes, and a significant proportion are presumed unequivocal loss-of-function variants (nonsense, frameshift, and canonical splice site). Current molecular genetic testing includes sequencing all coding exons (±10 bp) as well as deletion/duplication testing, with reported sensitivity of >98%. We report a proband from a consanguineous family with a biochemical phenotype consistent with early-onset Wilson disease who tested negative on conventional molecular genetic testing. Using a combination of whole-genome sequencing and transcriptome sequencing, we found that the proband's disease is due to skipping of exons 6-7 of the gene associated with a novel intronic variant (NM_000053.4:c.1947-19T > A) that alters a putative splicing enhancer element. This variant was also homozygous in the proband's younger sister, whose subsequent clinical evaluations revealed biochemical evidence of Wilson disease. Our work adds to emerging evidence that exon skipping from deep intronic variants outside typical splice junctions is an important mechanism of Wilson disease; the variants responsible may elude standard genetic testing.
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http://dx.doi.org/10.1101/mcs.a005306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304350PMC
June 2020

Disease-associated mosaic variation in clinical exome sequencing: a two-year pediatric tertiary care experience.

Cold Spring Harb Mol Case Stud 2020 06 12;6(3). Epub 2020 Jun 12.

The Steve and Cindy Rasmussen Institute for Genomic Medicine, Nationwide Children's Hospital, Columbus, Ohio 43205, USA.

Exome sequencing (ES) has become an important tool in pediatric genomic medicine, improving identification of disease-associated variation due to assay breadth. Depth is also afforded by ES, enabling detection of lower-frequency mosaic variation compared to Sanger sequencing in the studied tissue, thus enhancing diagnostic yield. Within a pediatric tertiary-care hospital, we report two years of clinical ES data from probands evaluated for genetic disease to assess diagnostic yield, characteristics of causal variants, and prevalence of mosaicism among disease-causing variants. Exome-derived, phenotype-driven variant data from 357 probands was analyzed concurrent with parental ES data, when available. Blood was the source of nucleic acid. Sequence read alignments were manually reviewed for all assessed variants. Sanger sequencing was used for suspected de novo or mosaic variation. Clinical provider notes were reviewed to determine concordance between laboratory-reported data and the ordering provider's interpretation of variant-associated disease causality. Laboratory-derived diagnostic yield and provider-substantiated diagnoses had 91.4% concordance. The cohort returned 117 provider-substantiated diagnoses among 115 probands for a diagnostic yield of 32.2%. De novo variants represented 64.9% of disease-associated variation within trio analyses. Among the 115 probands, five harbored disease-associated somatic mosaic variation. Two additional probands were observed to inherit a disease-associated variant from an unaffected mosaic parent. Among inheritance patterns, de novo variation was the most frequent disease etiology. Somatic mosaicism is increasingly recognized as a significant contributor to genetic disease, particularly with increased sequence depth attainable from ES. This report highlights the potential and importance of detecting mosaicism in ES.
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http://dx.doi.org/10.1101/mcs.a005231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304353PMC
June 2020

Long non-coding RNA RAMS11 promotes metastatic colorectal cancer progression.

Nat Commun 2020 05 1;11(1):2156. Epub 2020 May 1.

Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.
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http://dx.doi.org/10.1038/s41467-020-15547-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195452PMC
May 2020

MYCN amplification and ATRX mutations are incompatible in neuroblastoma.

Nat Commun 2020 02 14;11(1):913. Epub 2020 Feb 14.

Basic Science Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41467-020-14682-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021759PMC
February 2020

The Genotypic and Phenotypic Spectrum of BICD2 Variants in Spinal Muscular Atrophy.

Ann Neurol 2020 04;87(4):487-496

Department of Pediatrics, Ohio State University, Columbus, OH.

The bicaudal D cargo adaptor 2 (BICD2) gene encodes a conserved cargo adaptor protein required for dynein-mediated transport. Inherited and de novo variants in BICD2 cause SMALED2 (spinal muscular atrophy lower extremity dominant 2), and a subset have recently been reported to cause severe, often lethal disease. However, a true genotype-phenotype correlation for BICD2 has not been performed, and cases described to date are scattered among at least 14 publications. In this review, we identify the characteristics of disease-causing variants in BICD2 that distinguish them from benign variation and perform genotype-phenotype correlations for 99 BICD2 variant carriers from 35 families. ANN NEUROL 2020;87:487-496.
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http://dx.doi.org/10.1002/ana.25704DOI Listing
April 2020

Novel in-frame deletion causes Larsen syndrome in a three-generation pedigree.

Cold Spring Harb Mol Case Stud 2019 12 13;5(6). Epub 2019 Dec 13.

Department of Pediatrics, The Ohio State University, Columbus, Ohio 43205, USA.

A 4-yr-old female with congenital knee dislocations and joint laxity was noted to have a strong maternal family history comprising multiple individuals with knee problems and clubfeet. As the knee issues were the predominant clinical features, clinical testing included sequencing of , , and , which identified no significant variants. Research genome sequencing was performed in the proband, parents, and maternal grandfather. A heterozygous in-frame deletion in c. 5468_5470delAGG, which predicts p.(Glu1823del), segregated with the disease. The variant is rare in the gnomAD database, removes a residue that is evolutionarily conserved, and is predicted to alter protein length. Larsen syndrome may present with pathology that primarily involves one joint and thus may be difficult to differentiate clinically from other skeletal dysplasias or arthrogryposis syndromes. The p.(Glu1823del) variant maps to a filamin repeat domain where other disease-causing variants are clustered, consistent with a probable gain-of-function mechanism. It has reportedly been observed in two individuals in the gnomAD database, suggesting that mild presentations of Larsen syndrome, like the individual reported here, may be underdiagnosed in the general population.
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http://dx.doi.org/10.1101/mcs.a004176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913154PMC
December 2019

Whole Exome Sequencing of Highly Aggregated Lung Cancer Families Reveals Linked Loci for Increased Cancer Risk on Chromosomes 12q, 7p, and 4q.

Cancer Epidemiol Biomarkers Prev 2020 02 11;29(2):434-442. Epub 2019 Dec 11.

Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Baltimore, Maryland.

Background: Lung cancer kills more people than any other cancer in the United States. In addition to environmental factors, lung cancer has genetic risk factors as well, though the genetic etiology is still not well understood. We have performed whole exome sequencing on 262 individuals from 28 extended families with a family history of lung cancer.

Methods: Parametric genetic linkage analysis was performed on these samples using two distinct analyses-the lung cancer only (LCO) analysis, where only patients with lung cancer were coded as affected, and the all aggregated cancers (AAC) analysis, where other cancers seen in the pedigree were coded as affected.

Results: The AAC analysis yielded a genome-wide significant result at rs61943670 in at 12q23.3. has been implicated somatically in lung cancer, but this germline finding is novel and is a significant expression quantitative trait locus in lung tissue. Interesting genome-wide suggestive haplotypes were also found within individual families, particularly near at 7p36.1 in one family and a large linked haplotype spanning 4q21.3-28.3 in a different family. The 4q haplotype contains potential causal rare variants in at 4q22.1 and at 4q21.3.

Conclusions: Regions on 12q, 7p, and 4q are linked to increased cancer risk in highly aggregated lung cancer families, 12q across families and 7p and 4q within a single family. , and are currently the best candidate genes.

Impact: Functional work on these genes is planned for future studies and if confirmed would lead to potential biomarkers for risk in cancer.
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http://dx.doi.org/10.1158/1055-9965.EPI-19-0887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007362PMC
February 2020

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

Genome Biol 2019 09 2;20(1):187. Epub 2019 Sep 2.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA.

Background: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity.

Results: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges.

Conclusions: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.
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http://dx.doi.org/10.1186/s13059-019-1768-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721284PMC
September 2019

Mutations in PLS1, encoding fimbrin, cause autosomal dominant nonsyndromic hearing loss.

Hum Mutat 2019 12 1;40(12):2286-2295. Epub 2019 Oct 1.

Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.

Nonsyndromic hearing loss (NSHL), a common sensory disorder, is characterized by high clinical and genetic heterogeneity (i.e., approximately 115 genes and 170 loci so far identified). Nevertheless, almost half of patients submitted for genetic testing fail to receive a conclusive molecular diagnosis. We used next-generation sequencing to identify causal variants in PLS1 (c.805G>A, p.[E269K]; c.713G>T, p.[L238R], and c.383T>C, p.[F128S]) in three unrelated families of European ancestry with autosomal dominant NSHL. PLS1 encodes Plastin 1 (also called fimbrin), one of the most abundant actin-bundling proteins of the stereocilia. In silico protein modeling suggests that all variants destabilize the structure of the actin-binding domain 1, likely reducing the protein's ability to bind F actin. The role of PLS1 gene in hearing function is further supported by the recent demonstration that Pls1 mice show a hearing loss phenotype similar to that of our patients. In summary, we report PLS1 as a novel gene for autosomal dominant NSHL, suggesting that this gene is required for normal hearing in humans and mice.
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http://dx.doi.org/10.1002/humu.23891DOI Listing
December 2019
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