Cutting Edge Genetics Research funding from Natera Made Easy Mary - - PDF document

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Cutting Edge Genetics Made Easy

Mary E Norton, MD Department of Obstetrics, Gynecology, and Reproductive Sciences University of California, San Francisco

Disclosures

• Research funding from Natera

The history of prenatal diagnosis is focused largely on Down syndrome Why all the focus on Down syndrome?

12/8/2017 2 Lejeune, 1959 Tijo and Levan, 1956 1979: NICHD Consensus Panel on Amniocentesis

1979 Cost Benefit Analysis

Amniocentesis

Care for child with Down syndrome Everyone would want it Everyone would terminate

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“Remote shopping, while entirely feasible, will flop—because women like to get out of the house, like to handle merchandise, like to be able to change their minds.”

• --Time magazine, 1966

1997

Cell free DNA from apoptosis

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Noninvasive Prenatal Testing (NIPT) for aneuploidy using cell free DNA (cfDNA)

• Detection requires accurate quantification of DNA

from a specific chromosome

• Somewhat different methods are utilized by

different laboratories

Analysis of cell free DNA

Massively Parallel Shotgun Sequencing (MPSS):

Palomaki et al, 2011

• Random sampling of cfDNA fragments from all chromosomes
• A z-score value is used as a cut-off for trisomy (z-score of 3)

Palomaki GE et al. (2011), Genet. Med

N=1696

DR: 99.2% (98.5 - 99.6)

cfDNA screening for T21: meta-analysis

(Gil et al, Ultrasound Obstet Gynecol, 2015

FPR: 0.09% (0.05 - 0.14) DR: 99.7% (99.1- 99.9) FPR: 0.04% (0.02 - 0.07)

cfDNA screening for T21: meta-analysis

(Gil et al, Ultrasound Obstet Gynecol, 2017)

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20 40 60 80 100 120

Detection rate of prenatal screening for Down syndrome has improved over time

Detection Rate (%)

Cell free DNA and cancer

Estimate the Risk of Metastatic Relapse Estimate the Risk of Progression Monitor the efficacy of treatment Identifying Molecular Targets

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Meschino et al, Prenatal Diagn, 2016

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It’s a boy!

Prenatal diagnosis has largely relied on ultrasound and karyotype with recent introduction of MRI and microarray

DOWN SYNDROME!!

William’s Syndrome

Normal Female Chromosomes Deletion 7q11.23 (Williams Syndrome) Two copies present (normal)

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Chromosomal microarrays

• “Lab-on-a-chip”
• Detects thousands of

microdeletions and duplications at the same time

Microdeletions are genomic imbalances detected by microarray but not karyotype

Miller et al, 2010, AJHG

Chromosomal Microarray (CMA) for Prenatal Diagnosis

“Non-invasive Genome”

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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

• N=47 cases
• Correct in 46 (96.2%)
• Useful tool in 3rd trimester to distinguish IUGR from

achondroplasia

Chitty LS, et al. Prenat Diagn 35:656, 2015

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• De novo mutations
• Many associated with

advanced paternal age

Whole Genome and Whole Exome Sequencing

• Whole Genome Sequencing
• Obtaining the complete sequence of all 6 billion base pairs
• f DNA in any individual
• Whole Exome Sequencing
• Obtaining the complete sequence of the ~2% of the

genome containing the exons that encode proteins

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Whole Exome Sequencing

“Trio”

36 yo with two prior abnormal pregnancies

• Severe ventriculomegaly
• Midface hypoplasia
• Bilateral echogenic kidneys
• MSAFP: 18 MoM
• Amnio: AFAFP 45 MoM
• Normal microarray

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WES results

CRB2: p.W759X CRB2: p.W759X CRB2: p.W759X CRB2: p.N800K CRB2: p.W759X CRB2: p.N800K CRB2: p.N800K

Next pregnancy:

• In vitro fertilization with preimplantation genetic

testing

• Embryos testing for chromosome abnormalities

and for gene variants in CRB2

Next pregnancy:

• In vitro fertilization with preimplantation genetic

testing

• Embryos testing for chromosome abnormalities

and for gene variants in CRB2

• Embryo transferred that carried the maternal

variant

• Noninvasive prenatal test developed for the

paternal variant as confirmatory reassurance

The future: Noninvasive whole genome sequencing?

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Designer babies?

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What is “CRISPR”? What is “CRISPR”?

• Clustered
• Regularly
• Interspaced
• Short
• Palindromic
• Repeats

What is “CRISPR”?

• Clustered
• Regularly
• Interspaced
• Short
• Palindromic
• Repeats
• Part of bacterial immune

system against invading viruses

• Repeating sequences of

genetic code

• Used to target and

modify “typos” in the three-billion-letter sequence of the human genome

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CRISPR 101

Cas9 enzyme can cut DNA at specific locations, and allow small segments of DNA to be added or removed. CRISPR is a collection of DNA sequences that tells Cas9 where to cut

CRISPR-Cas9 gene editing system 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

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Gene correction in human embryos

H Ma et al. Nature 1–7 (2017) doi:10.1038/nature23305

Gene correction in human embryos

H Ma et al. Nature 1–7 (2017) doi:10.1038/nature23305

• Technical details aside,

Crispr-Cas9 makes it easy, cheap, and fast to move genes around— any genes, in any living thing, from bacteria to people. –David Baltimore

Conclusions