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10/19/2017 N O F I N A N C I A L D I S C L O S U R E S P A T I E N T W . N . IN T H E NO T S O D IS T A NT P A S T 37-year-old G5P4004 at 12 weeks gestation History notable for 3 healthy children and one son with Wolf-

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N O F I N A N C I A L D I S C L O S U R E S

  • 37-year-old G5P4004 at 12 weeks

gestation

  • History notable for 3 healthy

children and one son with Wolf- Hirschorn syndrome diagnosed after birth

  • Parental testing was performed
  • father was noted to be a carrier
  • f a balanced translocation
  • Increased recurrence risk
  • What now….

A .Cell free DNA

  • B. Invasive testing with CVS
  • C. Cell free DNA with CVS only if

abnormal

P A T I E N T W . N . IN T H E NO T S O D IS T A NT P A S T …

  • Cell free DNA became

commercially available in 2011

  • Multiple labs- at least 10
  • Significant impact on

number of invasive tests performed

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C E L L F R E E D N A -

A B I T O F B A C K G R O U N D

  • Results from apoptosis
  • Origin thought to be

primarily placenta

  • Circulate in maternal

plasma

  • Made up of short segments
  • f fetal DNA (<200 base

pairs)

Cell Free DNA in Maternal Plasma: Characteristics

  • cfDNA represents ~10-15% of total DNA in maternal plasma (Lo 1998,

Chiu 2011)

  • Reliably detected after 7 wks gestation (Birch 2005)
  • Higher concentrations late in gestation
  • Short half life (16 min), undetectable by 2 hrs postpartum (Lo 1999)

cfDNA: Common Clinical Applications

  • Aneuploidy: detect abnormal ratio of a particular

chromosome

  • Sex determination
  • Isoimmunization: noninvasively determine fetal Rh type
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cf DNA: The Future

  • Microdeletions
  • Whole genome testing
  • Single gene disorders

D I F F E R I N G T E C H N I Q UE S U S E D B Y D I F F E R E N T L A B S

  • Massively parallel shotgun sequencing (MPSS)
  • Comprehensive
  • Easy to add additional analyses
  • Targeted sequencing
  • Less expensive
  • Single nucleotide polymorphisms (SNP) analysis
  • Can distinguish maternal from fetal DNA
  • Can identify triploidy, vanishing twin

U N D E R S T A N D IN G T H E M E T H O D S

  • cfDNA
  • Cell free DNA is isolated from

maternal plasma

  • Total cfDNA is sequenced and

millions of sequence reads are generated

  • Sequence reads are aligned to

the reference genome

  • Aligned reads are counted to

determine chromosome number

Curr Genet Med Rep 2013 Jun; 1: (2):113-121

F E T A L F R A C T I O N

Prenatal Diagnosis 2012; 32(13) p 1233-41

The portion of fetal DNA in maternal circulation

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The less fetal DNA, the harder to tell normal from abnormal

Cell free DNA screening: Biologic Challenges

  • False positives:
  • Unrecognized or “vanishing”

twin

  • Placental mosaicism
  • Low level maternal

mosaicism, esp sex chromosomal

  • Maternal genetic variation

(copy number variants)

  • Maternal malignancy
  • False negatives:
  • Low level of fetal

DNA

  • Placental mosaicism
  • Maternal genetic

variation (copy number variants)

C E L L F R E E D N A S C R E E N I N G : B I O L O G I C C H A L L E N G E S

  • Failed results:
  • Increased BMI (BMI >35)
  • Low level of fetal DNA
  • Fetal aneuploidy

P E R F O R M A N C E O F C F D N A F O R O T H E R A N E U P L O ID I E S

The false positives add up….

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R A T E S O F A N O M A L I E S B Y M A T E R N A L A G E

Microdeletions are More Common Than Down Syndrome for Women Under 40

Adapted from: Snijders, et al. Ultrasound Obstet Gynecol 1999;13:167–170

S H O U L D A L L W O M E N B E O F F E R E D S C R E E NI N G F O R M I C R O D E L E T I O N S ?

M IC R OD E L E T IO N S A RE R A R E

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  • Microdeletions can be detected by cfDNA, but…
  • Difficult to validate
  • Much of the data on in vitro samples
  • Clinical testing includes small number of syndromes
  • 22q, 1q36, 5p-, 4p-, 15q11-13 (Prader-Willi/Angelman)

Ultrasound Obstet Gynecol, 2016

Prevalence of many microdeletion syndromes is unknown

  • Many such clinical syndromes are caused by different molecular

mechanisms

  • Only deletions detected
  • Smaller deletions more difficult to detect
  • 22q syndrome (~1/4000)
  • 85% have 3Mb deletion, 15% smaller
  • “97% detection” refers to only 3Mb deletion
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Sequenced 565 samples: 31 with CNV and 534 normal

  • Detected 83% with CNV >6Mb but only 20% (2/10) that

were <6Mb

  • 2/534 false positives
  • More sequencing = higher sensitivity

cfDNA for microdeletions is NOT a noninvasive microarray

Prevalence of 5 microdeletion syndromes: ~1/2500 Prevalence of CNV by microarray: 1/60 Detection rate of cfDNA for all pathogenic CNV: 4.2%

T H E F A L S E P OS IT I V E S A D D U P …

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  • Screening for these microdeletions has not been validated

in clinical studies.

  • Routine cell-free DNA screening for microdeletion syndromes should not

be performed. T H E F U T U R E : N O N I N V A S I V E W H O L E G E N OM E S E Q U E N C I N G

  • Coding and non coding

portions

  • Single nucleotide variants
  • Deletions and Duplications
  • Copy number variants
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  • Ehrich et al. (2017) reports on the clinical experience of one lab’s experience with

the first 10,000 cases

  • reports on copy number variations >7 Mb
  • 25% of samples were sent due to an ultrasound abnormality
  • Screen-positive test results were reported in 554 cases, leading to a screen-

positive rate of approximately 5.4% (compared with 2.3% in traditional cfDNA screening)

  • However, pregnancy outcomes not available so no data on false positive or false

negatives

I S M OR E A L W A Y S B E T T E R ?

  • Obtain information about the

entire fetal genome

  • Raises both practical and ethical

issues

  • May give information regarding

risk for adult onset conditions that are not relevant

Non-Invasive Single Gene Tests

  • Maternal and fetal cell free DNA cannot be easily distinguished
  • mother and fetus may share the same mutation
  • Can identify de novo or paternal gene mutation circulating in maternal blood
  • An affected fetus will have an overrepresentation of the mutant allele in maternal

plasma DNA

  • Ideal for autosomal dominant paternally inherited conditions
  • Useful if testing for an autosomal recessive condition and parents have different

mutations

  • N=47 cases
  • Correct in 46 (96.2%)
  • Useful tool in 3rd trimester to distinguish IUGR from achondroplasia
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C E L L F R E E D N A T E S T I N G F O R S I N G L E G E N E D I S O R D ER S

  • Current clinically available for limited conditions
  • Achondroplasia
  • Thanatophoric dysplasia
  • Apert syndrome, CF

B A C K T O OU R C A S E …

  • Our patient underwent cell free DNA testing with

microdeletions

  • Her results were normal so she declined any further testing
  • Level II ultrasound was notable for unilateral cleft lip and

palate and abnormal profile

  • Amniocentesis confirmed deletion of 8.9 MB deletion on

4pter consistent with Wolf-Hirshorn

Current guidelines?

A C O G / S M F M 2 0 1 5

  • Conventional screening is most appropriate first line screen for most

patients

  • Ethically any patient may choose cfDNA screening, but should be

counseled regarding limitations and benefits

  • Diagnostic testing is required to confirm abnormal results before

irreversible decisions

  • Testing for microdeletions and in twins should not be performed

Special thanks to Meg Autry, MD & Mary Norton, MD

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