Clinical Genomics. Ina E. Amarillo, PhD FACMGG

Transcription

1 Clinical Genomics Ina E. Amarillo, PhD FACMGG Associate Medical Director, Cytogenetics Lab (CaTG), Lab and Genomic Medicine Assistant Professor, Pathology and Immunology

2 Outline Clinical Genomics Testing Conventional/Classical Karyotype Analysis (metaphase chromosomes) FISH (interphase nuclei and/or metaphase chromosomes) Molecular CMA (copy number variations, regions of homozygosity) Genome Sequencing (sequence variations, insertion/deletion) Constitutional - Prenatal, Postnatal Testing; (Cancer) Preimplantation Genomics testing prior to implantation

3 Genomic Testing - Detect genomic aberrations at various resolution Genomic Resolution limit of detection Genome Sequencing: 1 bp to ~50 bp (or more) 2013 ACMG Review Course

4 Prenatal and POC POC

5 Normal Human Karyotype 46 chromosomes (22 pairs of autosomes, 2 sex chromosomes) (23 paternal, 23 maternal) Requires cell culture Humidity for good chromosome spreading Banding/staining Only 20 cells total >20 for mosaicism TAT 7 days Size in Mb pairs Chrom Mb Chrom Mb Chrom Mb 46,XY 46,XX

6 Abnormal Human Karyotype Numerical Trisomy 16 multiple congenital anomalies, incompatible with life Triploidy (3n) incompatible with life Structural Translocation between 9 and 22 (Philadelphia chromosome) in chronic myeloid leukemia Deletion of terminal region of short arm of chromosome 5 Cri-du-chat syndrome

7 Principles of Cytogenetics, 3 rd ed Requires cell culture (metaphase) Humidity for good chromosome spreading Up to 3 target regions per assay TAT ~24 hours to 5 days

8 FISH Prenatal Aneuscreen 13, 18, 21, X, Y Principles of Cytogenetics, 3 rd ed

9 Chromosome Microarray Analysis for studying Copy Number Variations (CNVs) and Regions of Homozygosity (ROH) High Resolution CMA higher probe density (whole genome) shorter distance between probes 2013 ACMG Review Course

10 Protocol and QA/QC steps QA/QC

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12 Clinical CMA Reporting Criteria Prenatal Postnatal Copy Number Loss 1 Mb 200 Kb Copy Number Gain 2 Mb 500 Kb Region of Homozygosity 10 Mb 10 Mb Main Challenge - Variants of Uncertain Clinical Significance Smaller than cutoff but with relevant gene(s) No genes but known to be regulatory Incomplete penetrance or variable expressivity

13 45,X one copy loss of X

14 2.8 Mb Interstitial Copy Number Loss CN=1 22q11.21 Not visible in karyotype

15 Data Analysis CN state and Allele (A) Frequency

16 Summary of CN Loss/Gain A B AA AB BB AAA AAB ABB BBB Does NOT detect: balanced rearrangements Tetraploidy 20% mosaicism

17 ROH (region of homozygosity) CN=2 (copy neutral) Overlaps with: 1. Imprinted Gene? 2. Known Uniparental Disomy? UPD11 (Beckwith- Wiedemann syndrome) UPD 15 (Prader- Willi/Angelman syndrome) 3. Autosomal recessive genes

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19 19-20K proteincoding genes Whole exome Targeted analysis

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21 Non-Invasive Prenatal Testing (screening) Cell-free DNA Positive NIPT/S reflexed to diagnostic AF/CVS tests Future in Pre/Postnatal? WGS, WES Nature Reviews Genetics, Vermeesch et al 2016

22 Preimplantation? Genomics Technologies Prenatal, Postnatal and Beyond Karyotype FISH CMA Sequencing

23 Preimplantation Genetics/Genomics challenge: small start sample few cells single cell Nature Reviews Genetics, Vermeesch et al 2016

24 Nature Reviews Genetics, Vermeesch et al 2016

25 Clustered Regularly Interspaced Short Palindromic Repeats pp17e4e-o8 Anti-viral defense by bacteria Adult cells and animal models

26 Junjiu Huang Sun Yat-sen University in Guangzhou Repair HBB/B-globin (B-thalassemia gene) in non-viable, triponuclear embryos (n=86) ~48 hrs (CRISPR-Cas9 action) Up to first stages of development (~8 cells) 71 survived 54 tested 28 spliced, ONLY A FRACTION replaced Several OFF-TARGET mutations (targeted analysis) Whole genome analysis plethora of mutations Terminated experiments Paper rejected by Nature and Science

27 Reproductive biologist Shoukhrat Mitalipov and his team used genome editing to correct a gene that causes a potentially fatal heart condition in humans (heterotropic cardiomyopathy). Oregon Health and Science University in Portland

28 Fixing Embryos by CRISPR? Targeting MYBPC3 GAGT human zygotes at the S-phase MII oocytes fertilized by sperm from a heterozygous patient with equal numbers of mutant and wild-type (WT) spermatozoa. Embryos at the 4 8-cell stage were collected for genetic analysis. resulted in mosaic embryos consisting of non-targeted mutant, targeted NHEJ-repaired and targeted HDR-repaired blastomeres.

29 Junjiu Huang Sun Yat-sen University in Guangzhou Tweaked individual bases (useful for point mutations) no dsdna cleavage, disable Cas9 enzyme, swap individual base pairs (allows G to A, C to T) First recessive disease edited Patients with A>G mutation in HBB Developed embryonic clones from skin cells (cloning) (up to 14 days) 8 of 20 successful repair of G>A (homozygous or heterozygous repair) Other Applications: Test gene-editing tools Unravel mechanisms behind early embryonic development

30 In the future, Huang says, he plans to ask for oocytes and sperm from donors who have one mutated copy of the gene and so are unaffected by the condition, but are carriers of the disease and use these to produce embryos. Some of those embryos would have two mutated copies, and some one, but Huang wants to edit both types. That raises the contentious idea that gene editing might be used not only to prevent severe disease, but also to eliminate the chance of people becoming carriers of the disorder.