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Genetic Analysis of DBA. Gareth Gerrard Imperial Molecular Pathology / Centre for Haematology Hammersmith Hospital. Molecular Diagnostics Begins With…. DNA, Codons and the Amino Acid Code. Genes: Exons, Introns & Splicing. DNA, RNA & Proteins (& Cake). Amino acids. The Ribosome (80S).

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Genetic Analysis of DBA


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    1. Genetic Analysis of DBA Gareth Gerrard Imperial Molecular Pathology / Centre for Haematology Hammersmith Hospital

    2. Molecular Diagnostics Begins With…

    3. DNA, Codons and the Amino Acid Code

    4. Genes: Exons, Introns & Splicing

    5. DNA, RNA & Proteins (& Cake) Amino acids

    6. The Ribosome (80S) 60S (L) unit:  5S RNA,   28S RNA,  5.8S RNA + ~49 proteins 40S (S) unit:  18S RNA + 33 proteins A cake making machine that uses mRNA as the recipe and amino acids as the ingredients

    7. DBA is a ribosomopathy* Mutations affecting ribosomal protein (RP) genes DBA *probably Mutations affecting: 25% RPS19 25-35% RPL5, RPL11, RPS26, RPS24, RPS17, RPS10, RPL35a,RPS7, RPL26, RPL15 40-50% ?? • ~80 RP genes in total • 10 are known to be affected in DBA • GATA1 may also have a role Heterozygous, autosomal dominant Leading to RP haploinsufficiency

    8. Types of Mutations in DBA – 1) Missense Change in recipe – use salt instead of sugar = cake no good!

    9. Types of Mutations – 2) Nonsense Change in recipe – leave out half of ingredients = cake no good!

    10. Types of Mutations – 3) Frameshift Change in recipe – words become unreadable = cake no good!

    11. Types of Mutations – 4) Splice Site Change in recipe – pages left out or go blank = cake no good!

    12. Types of Mutations – 5) Copy Number Variation (CNV) Change in recipe – pages torn out = cake no good!

    13. Why Screen? • Accurate diagnosis • Donor selection for allogeneichaematopoietic stem cell transplantation • Reproductive choices • Linking genotype to phenotype

    14. 10 Commonly Identified DBA associated RP Genes Mutations are mostly SNVs and indels, but large deletions & insertion are also seen = 7 genes in conventional molecular screen

    15. Mutation Detection Technology – Sanger Sequencing ABI 3130 ABI 3500xl 1 Sample / 1 Gene / day 5 Samples / 1 Gene / day

    16. Standard DBA Screening Pipeline Measure & QC Extract DNA Peripheral Blood RPS19 RPL5 RPL11 RPS24 RPS17 RPL35a PCR target gene exons RPS7 Sanger Sequence

    17. Next Generation Sequencers V2 - Current V1 - Pilot Roche 454 IlluminaMiSeq Ion Torrent PGM Getting on a bit / Expensive Highest throughput Fastest / most flexible

    18. Why Next Generation Seq (NGS)? • Very high throughput (fast) • Can look at all 80+ RP genes at once • Can multiplex many samples at once • Potential to pick up allele-loss deletions & insertions (CNV) • Cost effective per-gene / per-sample • Once identified, family members can be screened by conventional sequencing

    19. RP Gene loci used for V1 Gene Capture Latest Version adds GATA1, but loses RPS17 http:// ribosome.med.miyazaki-u.ac.jp

    20. NGS Workflow – v1 Target Enrichment Hybridise and capture Ribosomal Protein Gene DNA including exons, introns, & regulatory regions 3µg Genomic DNA 20 probands Fragment DNA: Covaris e220 Total Time = 2 weeks High-throughput Sequencing Library quant, pool, clean up and cluster generation Data analysis Sanger seq validation

    21. DBA – NGS v1 – Results from Initial 20 Samples British Journal of Haematology, 2013, 162,530–536 • SG= Stop Gain SNV (Nonsense); FSD= Frame-shift Deletion; FSI= Frame-shift Insertion; • SL= Start Loss SNV (Missense); SSD= Splice Site Defect

    22. DBA – NGS – v2 Workflow: Days 1 - 3 20ng gDNA Allows screening of 16 samples for 80+ Genes per run PGM Sequence 2 x 8 barcode AmpliSeq Library Prep (1-2 days) qPCR quant & pool KAPA Quant Kit Template & Enrich OneTouch2 & ES Day 1 Day 2 Day 3

    23. DBA NGS – Day 4: Analysis... Variant Caller TSv3.6.2 DON’T PANIC! IGV VCF Files VEP Ensembl v72 Virtualbox 4.2 SHOW ME THE KITTEHS NextGene / SeqNext Ion Reporter v1.6 MolDiag team for Sanger validation & reporting CONDEL / Mutation Assessor Human Splicing Finder v2.4.1

    24. DBA – NGS - Analysis

    25. DBA Mutation - IGV PileUp showing RPS26 Nonsense TTC (Phenylalanine) -> TAA (STOP)

    26. DBA-NGS v2 – Initial Results 3 definite hits (1 novel); 2 very likely; 5 interesting Only 1 DBA had no mutation (9); 10-13 non-affected family members

    27. Summary • Screening for mutations in DBA is now an established technology • We now use NGS technology to screen all 80+ Ribosomal protein genes • Family members screened by conventional sequencing (for known mutation) • Will introduce screening for CNV in near future

    28. Thank You! IPML Hammersmith Letizia Foroni Kikkeri Naresh MRD Pierre Foskett ThetMyint Faisal Abdillah MolDiag Mikel Valganon Alex Foong Natalie Killeen SarmadToma R&D Mary Alikian George Nteliopoulos Students Aysha Patel Sakuntala Ale Robin Ferrari Deena Iskander Clinical Team Josu de la Fuente Anastasios Karadimitris Jane Apperley David Marin Dragana Milojkovic JiriPavlu John Goldman ACHS CGL Tim Aitman Michael Müller Dalia Kasperaviciute Laurence Game