Cutting Edge Genetics Made Easy Karyotype Molecular testing with - PowerPoint PPT Presentation

10/19/2017 The evolution of prenatal genetic testing Cutting Edge Genetics Made Easy • Karyotype • Molecular testing with FISH • Chromosomal microarray Teresa Sparks, MD • Cell free DNA screening Department of Obstetrics, Gynecology, and Reproductive Sciences • Aneuploidy University of California, San Francisco • Microdeletions • Single gene disorders • Whole exome and genome sequencing • CRISPR gene editing The history of prenatal diagnosis is focused largely on Very low resolution: Low resolution: High resolution: Down karyotype microarray Whole genome sequencing syndrome Ccell free DNA screening Single gene test (ie CF) 1

10/19/2017 Why all the focus on Down syndrome? Lejeune, 1959 1979: NICHD Consensus Panel on Amniocentesis 1997 2

10/19/2017 Prenatal diagnosis has largely relied on ultrasound and karyotype with recent introduction of MRI and microarray DOWN SYNDROME!! Normal Female Chromosomes Fluorescence in situ hybridization - FISH William’s Syndrome FISH: uses targeted DNA probe to identify submicroscopic deletions in chromosomes. • Detects syndromes such as: 22q11 deletion William’s syndrome Prader-Willi/Angelman syndrome • Need to know what and where to target ! Two copies present Deletion 7q11.23 (normal) (Williams Syndrome) 3

10/19/2017 Microdeletions are genomic imbalances Chromosomal microarrays detected by microarray but not karyotype • “Lab-on-a-chip” • Detects thousands of microdeletions and duplications at the same time Miller et al, 2010, AJHG Chromosomal Microarray (CMA) for Diagnostic Yield of Chromosomal Microarray in Prenatal Diagnosis Cases with Normal Karyotype Indication for Testing Clinically Relevant (N=96) U/S Anomaly 6.0% N=755 AMA 1.7% N=1,966 Positive Screen 1.7% N=729 Other 1.3% N=372 4

10/19/2017 “Non-invasive Genome” Non-Invasive Single Gene Tests • Maternal and fetal cell free DNA cannot be easily distinguished • However, can identify de novo or paternal gene mutation • This includes blood type if mother is Rh negative and fetus is Rh positive 37 yo G1P0 at 28 wks 5

10/19/2017 • N=47 cases • Correct in 46 (96.2%) • Useful tool in 3 rd trimester to distinguish IUGR from achondroplasia Chitty LS, et al. Prenat Diagn 35:656, 2015 6

10/19/2017 Whole Genome and Whole Exome Sequencing Whole Exome Sequencing • Whole Genome Sequencing • Obtaining the complete sequence of all 6 billion base pairs of DNA in any individual • Whole Exome Sequencing • Obtaining the complete sequence of the ~2% of the genome containing the exons that encode proteins “Trio” 7

10/19/2017 WES results 36 yo with two prior abnormal pregnancies CRB2: p.W759X CRB2: p.N800K • Severe ventriculomegaly • Midface hypoplasia • Bilateral echogenic kidneys • MSAFP: 18 MoM CRB2: p.W759X CRB2: p.W759X • Amnio: AFAFP 45 MoM CRB2: p.N800K • Normal microarray CRB2: p.N800K CRB2: p.W759X The future: Noninvasive whole genome Precision Medicine sequencing? Patient group based Tailored therapy Patient group on genomic variants 8

10/19/2017 What is “CRISPR”? What is “CRISPR”? • Clustered • Regularly • Interspaced • Short • Palindromic • Repeats 9

10/19/2017 What is “CRISPR”? CRISPR 101 • Part of bacterial immune • Clustered system against invading • Regularly viruses • Interspaced • Repeating sequences of genetic code • Short • Used to target and • Palindromic modify “typos” in the • Repeats three-billion-letter sequence of the human genome CRISPR-Cas9 gene editing system Genome editing Cas9 enzyme can cut DNA at specific locations, and allow small segments of DNA to be added or removed. RNA guide can Cas9 is attached to RNA aligns to the The DNA can target any unique the RNA guide target DNA and repair itself, or be DNA sequence Cas9 cuts the DNA amended with an inserted gene CRISPR is a collection of DNA sequences that tells Cas9 where to cut 10

10/19/2017 Clinical applications • Edit pig DNA so organs can be transplanted into humans • Inject CRISPR-edited WBCs into humans to treat cancer • Treat illnesses such as cystic fibrosis, sickle-cell anemia and Huntington's disease • Modify and repair embryos The Brave New World of Fetal Medicine Conclusions Stem cell/gene therapy Fetal Surgery • thalassemia • muscular dystrophy • hemophilia Biology Pregnancy Complications • preterm labor • preeclampsia ‘Omics Fetal Origins of Adult Disease Devices • diabetes • hypertension Rare, Fatal Birth Defects Healthy Beginnings 11

10/19/2017 www.mfprecision.ucsf.edu 10/19/2017 Maternal copy number variants and cfDNA Snyder et al, NEJM, 2015 12

10/19/2017 Meschino et al, Prenatal Diagn, 2016 13