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DBA Society Day 2016 Genetic Testing Service

DBA Society Day 2016 Genetic Testing Service. Sandra Hing Principal Clinical Scientist. Introduction. Brief introduction of DBA genetics Laboratory diagnostic testing Update and Progress. Ribosomal Genes. Over 80 ribosomal genes To date16 known to be implicated in DBA

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DBA Society Day 2016 Genetic Testing Service

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  1. DBA Society Day 2016Genetic Testing Service Sandra Hing Principal Clinical Scientist

  2. Introduction • Brief introduction of DBA genetics • Laboratory diagnostic testing • Update and Progress

  3. Ribosomal Genes • Over 80 ribosomal genes • To date16 known to be implicated in DBA • 2 other genes also involved are GATA1 and TSR2

  4. DBA Genetics • Inherited in an autosomal dominant mode in approx. 45% of cases • de novo i.e new mutation in a family in approx. 55-60% of cases • GATA1 and TSR2 are inherited in an X linked manner

  5. DBA Genetics cont’d • Incomplete penetrance is observed • Variable phenotypes

  6. Chromosomes and Location of known DBA genes RPL11 RPS7 RPL5 RPL15 RPS10 RPL35A RPS27 RPL31 RPL26 RPL27 RPS24 RPS29 RPS17 RPS26 GATA1 RPS28 TSR2 RPS19

  7. Molecular Level Approximately 30,000 genes in the human genome

  8. DNA, RNA & Proteins Gene

  9. Imperial DBA Genetic Testing • Next Generation Sequencing (NGS) using custom DBA panel ~ >80 genes • Very high throughput (Millions of reads) • Cost effective per gene/per sample • Multiple patient samples in one NGS run • NGS run performed at regular intervals • Potential to detect all types of variants including allele loss deletions & insertions (CNV) • Variants confirmed by sequencing or MLPA

  10. Imperial DBA Screening Strategy Once identified, if appropriate family members can be screened by conventional sequencing or MLPA Unclassified variants identified are extremely difficult to interpret and therefore challenging

  11. Ion Torrent™ - Ion PGM™ System

  12. Technical Workflow 20ng gDNA Allows screening of 16 samples for >80 genes per run Template & Enrich OneTouch2 & ES PGM Sequence 2 x 8 barcode AmpliSeq Library Prep (1-2 days) qPCR quant & pool KAPA Quant Kit Day 1 Day 2 Day 3 Day 3

  13. Analytical Pipeline Data Analysis Data Analysis IGV Issue Report VCF files MLPA confirmation CNV CNV Ion Reporter v4.4 Yes variant Online Databases Other sources Pathogenic/ suspected pathogenic Sanger confirmation SNV/indels VEP ensembl! Issue Report No variant

  14. Data Analysis workflow Variant Caller TS v4.4.2 VCF files Yes mutation/variant Online Sources /in silico analysis Variant Pathogenic or not No mutation/variant

  15. Example of RPS26 mutation TTC (Phenylalanine) -> TAA (STOP) Phe>* c.Phe157* Described as a nonsense mutation

  16. Current Progress and Update • Have 2 Ion Torrent PGM platforms • Enabled the lab to process many more cases • Dedicated scientists for DBA service and up to date database • Continue to collaborate with research group

  17. Whole Genome Sequencing Genomics England… 100,000 genome project

  18. IMPL Diagnostic Team • Laura Kearney • Ilaria Borghi • Dhara Patel • Faisal Abdillah • Natalie Killeen • Sarmad Toma • VincenzoPacifico • Jamshid Khorashad • Sandra Hing • Hui En Foong • Mary Alikian • Laura Cobbold • Persephone Du Parcq

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