Translational Diagnostics

Diagnosis is essential for the management and treatment of patients with rare diseases. In a group patients, genetic study identifies variants uncertain significance or inconsistent phenotype; therefore, it urgent to develop novel strategies reach definitive diagnosis. Herein, we in-house Translational Diagnostics Program (TDP) validate as part diagnostic process close collaboration physicians, clinical scientists, research scientists. The first 7 33 consecutive whom exome-based tests were not investigated. TDP pipeline includes four steps: (i) phenotype assessment, (ii) literature review prediction in silico pathogenicity, (iii) experimental functional studies, (iv) decision-making. Re-evaluation re-analysis exome allowed diagnosis one patient. remaining studies included either cDNA cloning PCR-amplified genomic DNA, use patients' fibroblasts. A comparative computational analysis confocal microscopy images related protein function was performed. five these six evidence pathogenicity variant found, which validated by physicians. current demonstrates feasibility support resolve intramural medical problems when patient unknown phenotype. disease (RD) defined condition that affects <5 per 10,000 inhabitants European Union less than total 200,000 affected individuals United States. It estimated number different RDs approximately 7000, many them are ultrarare. usually severe, chronic, disabling, onset pediatric life young adulthood, 80% have origin.1Berman J.J. Rare Disease Orphan Drugs. Academic Press–Elsevier, London, UK2014Google Scholar symptoms signs frequently nonspecific, leading difficulty and/or delay diagnosis, impedes adequate therapeutic planning counseling. remains undiagnosed, deal this category undiagnosed RD (URD) developing actions, including in-depth phenotyping prompt genome analysis, achieve Next-generation sequencing technology has greatly improved ability identify causal particular RD.2Wright C.F. FitzPatrick D.R. Firth H.V. Paediatric genomics: diagnosing children.Nat Rev Genet. 2018; 19: 253-268Crossref PubMed Scopus (179) Google Scholar, 3Tan T.Y. Dillon O.J. Stark Z. Schofield D. Alam K. Shrestha R. Chong B. Phelan Brett G.R. Creed E. Jarmolowicz A. Yap P. Walsh M. Downie L. Amor D.J. Savarirayan McGillivray G. Yeung Peters H. Robertson S.J. Robinson A.J. Macciocca I. Sadedin S. Bell Oshlack Georgeson Thorne N. Gaff C. White S.M. Diagnostic impact cost-effectiveness whole-exome ambulant children suspected monogenic conditions.JAMA Pediatr. 2017; 171: 855Crossref (160) 4Stark Wilson W. Mupfeki Prospective comparison usual care overwhelmingly supports early reimbursement.Genet Med. 867-874Crossref (124) However, massive (VUSs) derived from next-generation and, sometimes, incongruence between candidate gene hindered both downstream interpretations findings translation into practice. There necessity define reliable valid method complex phenotype-genotype correlation VUS classification time help management. To assist relative patient's phenotype, developed. objective delineate potential causality through holistic approach based on triangle phenotyping–clinical genomics–functional genomics. Such program combines stages: comprehensive evaluation, integrates enrichment integrity disease-phenotype annotations Human Phenotype Ontology5Robinson P.N. Köhler Bauer Seelow Horn Mundlos human ontology: tool annotating analyzing hereditary disease.Am J Hum 2008; 83: 610-615Abstract Full Text PDF (0) Scholar; bioinformatic variant/s; validation molecular, cellular, imaging assays; decision-making referring physician recommendations pathogenic potential. presents results applied seven URD patients. effectiveness shown resolving doubts relationship genotype congruent. All procedures complied ethical guidelines Sant Joan de Déu Children's Hospital approved Clinical Research Ethics Committee under references PIC-30-17 PIC-223-19. Informed consent obtained parents legal guardians. January 2017, Pediatric Institute Diseases Department Genetic Medicine started testing URD. consisting solution hybridization (TruSight One Sequencing Panel; Illumina, San Diego, CA) whole (Nextera Flex Enrichment; Illumina) used subsequent performed an Illumina NextSeq500 sequencer. bioinformatics using developed department. quality reads determined (FastQC version 0.11.5; Babraham Institute, Cambridge, UK), low-quality adapters removed (Cutadapt 1.13; National Bioinformatics Infrastructure Sweden, Uppsala, Sweden), subsequently aligned reference (BWA-MEM 0.7.15; Wellcome Trust Sanger UK). Low-mapping duplicates [BEDtools 2.26.0 (University Utah, Salt Lake City, UT) Picard 2.9.0 (Broad MA)] removed. Variants detected programs [SAMtools 1.5 (Wellcome GATK 3.7 MA), FreeBayes 1.1.0 (Boston College, Boston, VarScan 2.4.0 (Washington University, St. Louis, MO)], annotation (SnpEff 4.3k; Wayne State Detroit, MI) databases 1000 Genomes, dbSnp, ExAc, ClinVar. Deletions/duplications (copy variations) R exomeDepth 1.1.10 package (R Inc., MA). date, series been variants. Patients if found met any following criteria: (A) there insufficient despite being genes already (B) partial incomplete reported Online Mendelian Inheritance Man (MIM); (C) showed discrepancies described scientific atypical expression (eg, Man, www.omim.org, last accessed February 24, 2020). addition, parental segregation available parents’ DNA variant. When necessary, paternity PowerPlex 16HS system (Promega, Madison, WI), according manufacturer's protocol. VarSome6Kopanos Tsiolkas V. Kouris Chapple C.E. Albarca Aguilera Meyer Massouras VarSome: search engine.Bioinformatics. 2019; 35: 1978-1980Crossref (429) (https://varsome.com, 2020) American College Medical Genetics Genomics7Richards Aziz Bale Bick Das Gastier-Foster J. Grody W.W. Hegde Lyon Spector Voelkerding Rehm H.L. ACMG Laboratory Quality Assurance Committee: Standards interpretation sequence variants: joint consensus recommendation Genomics Association Molecular Pathology.Genet 2015; 17: 405-423Crossref (10354) standards Genome Aggregation Database (https://gnomad.broadinstitute.org, Centro Investigación Biomédica en Red Enfermedades Raras–Spanish Variant Server8Dopazo Amadoz Bleda Garcia-Alonso Alemán García-García F. Rodriguez J.A. Daub J.T. Muntané Rueda Vela-Boza López-Domingo F.J. Florido J.P. Arce Ruiz-Ferrer Méndez-Vidal Arnold T.E. Spleiss O. Alvarez-Tejado Navarro Bhattacharya S.S. Borrego Santoyo-López Antiñolo 267 Spanish exomes reveal population-specific differences disease-related variation.Mol Biol. 2016; 33: 1205-1218Crossref (45) (http://csvs.babelomics.org, population, respectively. Gene Mutation Database9Stenson P.D. Mort Ball E.V. Evans Hayden Heywood Hussain Phillips A.D. Cooper D.N. mutation database: towards repository inherited data research, studies.Hum 136: 665-677Crossref (652) [http://www.hgmd.cf.ac.uk/ac/index.php (professional 2019.4), 2020] collects published peer-reviewed humans. predict effects function, tools used: Polymorphism Phenotyping 2 (http://genetics.bwh.harvard.edu/pph2, 2020),10Adzhubei I.A. Schmidt Peshkin Ramensky V.E. Gerasimova Bork Kondrashov A.S. Sunyaev S.R. server predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref (8382) Protein Variation Effect Analyzer (http://provean.jcvi.org, 2020),11Choi Y. Chan A.P. PROVEAN web server: effect amino acid substitutions indels.Bioinformatics. 31: 2745-2747Crossref (1109) PMut (http://mmb.irbbarcelona.org/PMut/, 2020),12López-Ferrando Gazzo la Cruz X. Orozco Gelpí J.L. PMut: web-based pathological proteins, 2017 update.Nucleic Acids Res. 45: W222-W228Crossref (75) MutationTaster (http://www.mutatiotaster.org, 2020),13Schwarz J.M. Schuelke MutationTaster2: deep-sequencing age.Nat 2014; 11: 361-362Crossref (2053) Combined Annotation Dependent Depletion (https://cadd.gs.washington.edu, 2020),14Rentzsch Witten G.M. Shendure Kircher CADD: deleteriousness throughout genome.Nucleic 47: D886-D894Crossref (892) probability loss-function intolerance, variation scores (last 2020).15Lek Karczewski K.J. Minikel Samocha K.E. Banks Fennell T. et al.Analysis protein-coding 60,706 humans.Nature. 536: 285-291Crossref (5847) Scholar,16Samocha E.B. Sanders Stevens Sabo McGrath L.M. Kosmicki Rehnström Mallick Kirby Wall D.P. MacArthur D.G. Gabriel S.B. DePristo Purcell Palotie Boerwinkle Buxbaum J.D. Cook Jr., E.H. Gibbs R.A. Schellenberg G.D. Sutcliffe J.S. Devlin Roeder Neale B.M. Daly M.J. framework novo disease.Nat 46: 944-950Crossref (533) Multiple alignment among various species ClustalW. SH-SY5Y neuroblastoma embryonic kidney HEK293T cells ATCC (Manassas, VA). cultured Dulbecco’s modified Eagle’s medium/F-12 HAM (Sigma-Aldrich, MO) supplemented 10% (v/v) fetal bovine serum (Sigma-Aldrich), mmol/L l-glutamine 100 mg/mL penicillin-streptomycin (Sigma-Aldrich). skin fibroblast grown medium high glucose (Sigma-Aldrich) serum, l-glutamine, penicillin-streptomycin. Cell cultures maintained 5% CO2 humidified atmosphere at 37°C until they reached confluence maximum 10 passages. Cells periodically tested Mycoplasma infection. For biopsies after informed representatives. Healthy control fibroblasts Biobank. SWI/SNF matrix-associated actin-dependent regulator chromatin subfamily member 4 (SMARCA4) entry pcDNA6.2/N-EmGFP-DEST mammalian vector gift Kyle Miller (Addgene, MA; Addgene plasmid, 65391),17Gong Chiu L.Y. Cox Aymard Clouaire Leung J.W. Cammarata Perez Agarwal Brodbelt Legube K.M. Screen bromodomain ZMYND8 recognition transcription-associated damage promotes homologous recombination.Genes Dev. 29: 197-211Crossref (136) zinc finger E-box binding homeobox (ZEB2) pRP[Exp]CMV>hZEB2 generated Cyagen (Santa Clara, cloned pEGFP-N1 dynein cytoplasmic 1 heavy chain (DYNC1H1) pDyn1 Andrew Carter (Addgene 64067)18Schlager M.A. Hoang H.T. Urnavicius Bullock S.L. vitro reconstitution highly processive recombinant complex.EMBO 1855-1868Crossref region linker stalk plasmid. SMARCA4 mutant (c.2194T>G; p.Y732D), ZEB2 (c.2801A>G; p.H934R), DYNC1H1 (c.4867C>T; p.R1623W c.4700G>A; p.R1567Q) site-directed mutagenesis QuickChange Lightning Site-Directed kit (Aligent Technologies, La Jolla, CA). Wild-type (WT) constructs verified sequencing. transfected FuGene HD (Promega), protocol, analyzed 48 hours transfection. immunofluorescence staining, 9 × 104 (HEK293T SH-5YHY) seeded onto glass coverslip plated previously treated poly-l-lysine, 40 ?g/mL; Sigma-Aldrich). Two days transfection indicated all samples harvested, washed phosphate-buffered saline (PBS), fixed prewarmed 4% paraformaldehyde 20 minutes room temperature. After PBS washes, coverslips mounted slide glasses presence Fluoromont-G DAPI (Thermo Fisher Scientific, Waltham, Skin (3 104) Fibroblasts permeabilized 0.2% Triton X-100 30 temperature blocked 1% albumin hour Primary antibodies ?-dynamin-related (DRP1; 1:200; BD Transduction Laboratories, Franklin Lakes, NJ), ?–translocase outer mitochondrial membrane (TOM20; 1:100; Laboratories), ?-mediator subunit 13 (MED13; Novus Biological, Littleton, CO) incubated overnight 4°C. visualized Alexa Fluor 488-labeled secondary antibody (1:500; Thermo Scientific). incubation, rinsed DAPI. visualize mitochondria, loaded 200 nmol/L MitoTracker Deep Scientific) 37°C; counting Leica DMI 3000B microscope (Leica Microsystems, Wetzlar, Germany) 63× oil immersion objective. Five independent experiments counted each cell type (at least random experiment). Super resolution acquired TCS SP8 X Light Laser hybrid spectral detectors HyVolution Microsystems) LAS software 3.1.5 100× (HCX Plan APO CS, 1.4 numerical aperture). Negative background setting before image acquisition. original stored 16-bit gray scale spatial 792 1128 pixels. Pixel sizes ranged 0.018 0.031 ?m. punctate pattern, deconvolution Huygens Essential 4.4, supplied Scientific Volume Imaging (Hilversum, Netherlands). Z-stacks 6.5 9.99 ?m z-increments cell. Because variability encountered, two-stage unsupervised segmentation implemented three-dimensional pattern. stage nuclei interest contrast enhancement followed binarization (Otsu method) labeling green channel [green fluorescent (GFP)-SMARCA4/ZEB2]. resulting binary mask improve pattern detection accuracy. second (segmentation) stage, nuclear patterns identified modeling voxel intensity distribution univariate gaussian mixture. model components selected basis Akaike information criterion (Supplemental Figure S1). component highest mean corresponding Feature extraction segmented volume (?m3) analyses MATLAB R_2018a (The MathWorks Natick, (9 24 hours; then, transfection, PBS, washes ice-cold methanol ?20°C minutes. incubation blocking preheated humidity chamber, 4°C specific primary antibodies: rabbit anti-GFP (1:100; Abcam, UK) mouse anti-Dynactin Santa Biotechnology, Dallas, TX). Afterward, proximity ligation assay instructions [Duolink Situ Detection Starter (Mouse/Rabbit) Kit; Sigma-Aldrich], Duolink Mounting Medium antibody, negative experiment performed, where only probes. 12-well plates (2 105 cells/well) hours, then harvested 4°C, 70% ethanol washing stained containing: (final concentration, ?g/mL), 0.1% X-100, RNaseA ?g/mL) 37°C. ACEA NovoCyte Flow Cytometer (ACEA Biosciences, collecting 20,000 events, cycle phase distributions NovoExpress 1.4.1 Biosciences) gating out debris signal. Results experiments. Non-transfected control. (1 trypsinized, resuspended 5 ?mol/L MitoSOX 15 dark 37°C, Cytometer. assay, events collected analyzed. 500 H2O2 positive Final values fluorescence calculated over two healthy controls. Fibroblast 105) 35-mm plate. 2.5 three times PBS. As control, H2O2. Images captured Data color maps (using royal lookup table model) ImageJ 1.52p (NIH, Bethesda, MD; https://imagej.nih.gov/ij, July 2, Exon GATA domain-containing 2B (GATAD2B; NM_020699.2; 316 bp) its flanking introns (48 bp 5? arm 235 3? arm) PCR amplified controls Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA) forward reverse primers carrying restriction sites XhoI NheI fragments pSPL3 exon trapping (Promega) reagent, post-transfection, RNA isolated Trizol reagent manufacturer QIAamp synthesized Maxima first-strand synthesis real-time quantitative RT-PCR Biotools Polymerase (Biotools B&M Labs, Madrid, Spain) SD6 (forward: 5?-TCTGAGTCACCTGGACAACC-3?) SA2 (reverse: 5?-ATCTCAGTGGTATTTGTGAGC-3?). Splice minigene separated electrophoresis sequenced. expressed means ± SD box plots, showing median, edges represent 25th 75th percentiles, whiskers extended minimum values. P asterisks: ?P < 0.05, ??P 0.01, ???P 0.001. normality assessed Kolmogorov-Smirnov test. Mantel-Haenszel ?2 test percentages ZEB2, DYNC1H1, subcellular localization (case 1, 5). U-test evaluate (Patients Nonparametric Kruskal-Wallis test, Dunn post hoc compare dots (Patient 2) 5); case, stacked bar represents percentage within G0/G1 phase, S G2/M phase. parametric one-way variance Tukey oxidative stress levels 6 7). Statistical computing graphs RStudio 1.1.447 (RStudio GraphPad Prism 8.0.1 (GraphPad Software, First, records updated (Table 1). reanalysis yielded 3) helped design Functional strategies: (genetic versus WT) lines, culture (patients control) cellular phenotypes (subcellular location function).Table 1Summary Findings Seven URDPatient no.Recruitment criterion?See selection Materials Methods.Clinical (age sex)Exome findings:gene/variant (reference sequence)†All present heterozygous sequences https://www.ncbi.nlm.nih.gov/nuccore).SegregationMIM no.: phenotypes/inheritanceGenotype-phenotype correlationVariant status HGMD‡Variant class HGMD, professional 2019.4.gnomAD§gnomAD allele frequency./BIER¶BIER frequency. frequencyCADD?Scores ?20 indicate predicted be most deleterious genome. (score)Final diagnosis1AMild ID, speech delay, gross fine motor skills delay. Cerebellar syndrome intention tremor, global progressive cerebellar atrophy, chronic sensory axonal neuropathy, talipes equinovarus, (clumsy gait 18 months, female)ZEB2/c.2801A>G p.H934R (NM_014795.3)M: carrierSegregation studies; bold further studied.F: studied.235730/ADPartialNRNR/NR3.9Impasse diagnosis/undiagnosed diseaseSTIL/c.1455G>C p.L485F (rs139912214)(NM_001048166.1)M: studied.612703/ARNoDCM0.00211/0.00214.0CEP152/c.3313C>G p.L1105V (rs74553953)(NM_001194998.1)M: studied.613823/AR61