NTGLH Module ID Module Title Module Format NTGHL001 NHS Genomic Medicine Service Handbook NTGHL002 Ordering from the National Test Directory Handbook NTGHL003 Whole Genome Sequencing consent ID: 1009881
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1. The genomics facilitator’s toolkitNTGLH Module IDModule TitleModule FormatNTGHL_001NHS Genomic Medicine ServiceHandbookNTGHL_002Ordering from the National Test DirectoryHandbookNTGHL_003Whole Genome Sequencing consentHandbookNTGHL_004Whole Genome Sequencing sample requirementsHandbookNTGHL_005Clinical genetic testing methodsPowerpoint with narrationNTGHL_006Clinical testing DNA sequence variant interpretationPowerpoint with narrationNTGHL_007Whole Genome Sequencing resultsPowerpoint with narrationNTGHL_008Introduction to genomicsPowerpoint with narrationNTGHL_009Test cases in cancerHandbook
2. NTGHL005 Clinical genetic testing methodsInformation for healthcare professionals
3. MethodologiesNational Health Service England (NHSE) National Test Directory (TD) ‘genetic’ testing methodologies are grouped broadly into two categories depending on the type of abnormality being measured:Cytogenetic - detect abnormalities in chromosome structureMolecular - detect abnormalities in DNA sequence.NTGLH005 - Clinical genetic testing methods
4. cytogeneticmolecularNTGLH005 - Clinical genetic testing methods
5. TD test methodsGenetic test typeGenetic test methodExampleCytogeneticsDNA repair defect (breakage)Nijmegen breakageCytogeneticsFluorescent in situ hybridization (FISH)EWSR1 rearrangementCytogeneticsKaryotypeDisorders of sexual development/infertilityCytogeneticsChromosomal microarray analysis (CMA)Developmental delay/learning difficultiesCytogeneticsQuantitative fluorescence PCR (QF-PCR) Haematological tumour, RUNX1-RUNX1t1 MolecularComplex targeted variant1Haematological tumour, BCR-ABL1 TKD seqMolecularIdentityIdentityMolecularMethylation2,3X-inactivationMolecularMicrosatellite instability2Solid tumour MSI testingMolecularMultiplex ligation-dependent probe amplification (MLPA)2Prader-Willi syndromeMolecularNon-invasive prenatal genetic diagnosis (NIPD)3NIPD for cystic fibrosisMolecularSingle gene/(simple) targeted variant sequencing1Neutropenia consistent with ELANE mutationsMolecularSmall panel3Sarcoma multi-target, H3F3A, H3F3B, IDH1, IDH2MolecularSmall Tandem Repeat (STR)1Fragile XMolecularuniparental disomy (UPD)2Prader-Willi syndromeMolecularWhole exon sequencing (WES) or large panel3Hypertrophic cardiomyopathyMolecularWhole genome sequencing (WGS)3Eligible rare disease or cancerNTGLH005 - Clinical genetic testing methods
6. Cytogenetic methods detect abnormalities in chromosome structureNTGLH005 - Clinical genetic testing methods
7. KaryotypingGenome-wide image of chromosomes’ banding pattern Used when cultured cells are availableIn ideal conditions aberrations as small as 5 Mb can be detectedProvides a way of screening the whole genome for genetic deletions, duplications and rearrangementsPrimary tool for analysing patients with a variety of congenital anomalies.NTGLH005 - Clinical genetic testing methods
8. Fluorescence in situ hybridization (FISH)Genome-wide image of labelled probes mapped to specific DNA sequences of chromosomesCan detect much smaller chromosomal abnormalities than karyotyping - 100 Kb-1 MbRequires in vitro cultured cells but a much smaller sample than karyotyping - even down to a single cellProvides a way of screening the whole genome for genetic deletions, duplications and rearrangementsPrimary tool for analysing patients with specific microdeletion syndromes and sub-telomere screeningNTGLH005 - Clinical genetic testing methods
9. Fluorescent in situ hybridization (FISH)NTGLH005 - Clinical genetic testing methods
10. Chromosomal microarrays analysis (CMA)Genome-wide chromosome testProvides information on how much genetic material is present, not an image of where it isDoes not require cultured cellsCan detect much smaller chromosomal abnormalities than karyotyping and FISH; can detect abnormalities as low as 10 KbLimitation: will not pick up balanced translocationsFirst line analysis for developmental delay, intellectual disability, congenital defects.ABC
11. Quantitative fluorescence PCR (QF-PCR)A targeted aneuploidy test. Is commonly used for specific-variant testing (i.e. abnormality previously identified in blood relatives)Does not require in vitro cultured cells; uses various sample typesInformation on how much genetic material is present, not an image of where it isLimitation: does not detect structural abnormalitiesQF-PCR commonly assesses a number of locations on chromosomes 13, 15, 16, 18, 21, 22, X and Y; the commonest trisomies found in pregnancy losses.NTGLH005 - Clinical genetic testing methods
12. Molecular methods detect abnormalities in DNA sequenceNTGLH005 - Clinical genetic testing methods
13. Molecular methodsMolecular technologies are broadly grouped into three approaches:Polymerase chain reaction (PCR)Ligation mediated PCR e.g. MLPANext Generation Sequencing (NGS).NTGLH005 - Clinical genetic testing methods
14. Polymerase chain reaction (PCR)DenatureAnnealExtendGenome target of interestAmplified target of interest, two copiesPolymerase chain reaction, or PCR, is a laboratory technique used to amplify/make multiple copies of a segment of DNA. Template DNA is denatured into single strands. Forward (PF) and reverse (PR) primers anneal. An extension step leads to amplified/copies of the original target of interest.PF PRNTGLH005 - Clinical genetic testing methods
15. CTTCTTCTTCTTCTTCTTCTT0 +3 basesExamples of PCR sequence-based testsMicrosatellite-based testingMicrosatellite lengthGenomics England Rare Disease Results GuideHuntington disease is a progressive brain disorder caused by a pathogenic microsatellite CAG-repeat motif. An individual with CAG36-39 may or may not develop symptoms of Huntington disease, whilst people with 40 or more CAG (CAG40+) repeats almost always develop the disorder.NTGLH005 - Clinical genetic testing methods
16. Targeted and single gene testingSequence chromatogramExamples of PCR sequence-based testsNTGLH005 - Clinical genetic testing methods
17. Ligation mediated PCRMultiplex Ligation-dependent Probe Amplification (MLPA)The MLPA reaction can be divided into five steps: DNA denaturation and probe hybridisationLigation reactionPCR amplificationSeparation of amplification products by electrophoresisData analysis.NTGLH005 - Clinical genetic testing methods
18. Genomic imprinting and uniparental disomy disorders tested by MLPAClinical IndicationTarget Regions/GenesAngelman syndromeAS/PWS critical regionPrader-Willi syndromeAS/PWS critical regionHypotonic infant with a likely central causeSNRPN DMRGrowth failure in early childhood11p15 imprinted growth regulatory region and UPD7 growth regulatory critical regionBeckwith-Wiedemann syndrome11p15 imprinted growth regulatory regionIsolated hemihypertrophy or macroglossia11p15 imprinted growth regulatory regionTemple syndrome - maternal uniparental disomy 14UPD14 critical regionKagami-Ogata syndrome - paternal uniparental disomy 14UPD14 critical regionAlbright hereditary osteodystrophy, pseudohypoparathyroidism and pseudopseudohypoparathyroidismGNAS DMRsWilms tumour with features suggestive of predisposition11p15 imprinted growth regulatory regionNTGLH005 - Clinical genetic testing methods
19. NTGLH005 - Clinical genetic testing methodsPMID: 22489151
20. Next generation sequencingGenomeGenome fragmentedPrepare sample - DNA modified and amplified.Cluster generation - results in clonal amplification of all fragments. Sequencing by synthesis - nucleotide added one at a time, using template. Each nucleotide addition is ‘excited’, emitting light specific to the nucleotide (A,C,G.,T) and imaged. Image converted to a sequenceData analysis - all fragments aligned against a reference genome. Variants against reference identified.Reference genomeNTGLH005 - Clinical genetic testing methods
21. Whole genome sequencing (WGS) - sequencing all of the genome.Whole exome sequencing (WES) - targets all known protein genes that represent less than 2% of the genetic code, but contains ~85% of known disease-related variants.Clinical exome - targets genes known to be disease causing. For example, GOSHome targets ~ 6000 genes.Targeted clinical exome - targets specific disease causing genes for the phenotype(s) in question. For example, skeletal panel of GOSHome includes 240 genes where variants can cause skeletal anomalies.Small panel - targets (usually) ten to 100 or more disease causing genes. Types of NGS sequencingNTGLH005 - Clinical genetic testing methods
22. Alignment, depth of coverage, variantReference genomeHeterozygoteErrorHomozygoteAligned sequence readsATTTTTTTTTTTTTAATNTGLH005 - Clinical genetic testing methods
23. Depth of coverageNTGLH005 - Clinical genetic testing methods
24. WES v WGSWESCheaperMajority of disease causing variants will be in the exomeLess analysis time requiredHighly variable coverageWGSCapture info on all genomeFuture proofMore reliable sequence coverageLimited ability to interpretNTGLH005 - Clinical genetic testing methods
25. North Thames Genomic Laboratory Hub Follow us: @NorthThamesGLH Contact us at: gos-tr.norththamesglh@nhs.net Further education (as an example)https://www.genomicseducation.hee.nhs.uk/education/core-concepts/what-is-genomics/https://www.yourgenome.org/topic/in-the-cellhttps://www.futurelearn.com/programs/genomics-in-healthcareAdvice and educational resources
26. Any questions?Francesca Faravelli, Consultant Clinical GeneticistFrancesca.Faravelli@gosh.nhs.ukNorth Thames GLH Education & Training Lead: Corinne Trim corinne.trim@gosh.nhs.uk Includes slides adapted from https://www.genomicseducation.hee.nhs.uk/supporting-the-nhs-genomic-medicine-service/https://www.england.nhs.uk/wp-content/uploads/2018/08/national-genomic-test-directory-faqs.pdfPrimary author - Shazia Mahamdallie, Scientist, GOSH.Narrated by - Dahlia Hopmeier, Genomics Clinical Fellow, GOSH.