PAT I E N T W. N . What testing options should be offered? - - PowerPoint PPT Presentation

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

• 37-year-old G5P4004 at 12

weeks gestation

• 3 healthy children
• 1 son with Wolf-Hirschorn

syndrome

• Parental testing performed
• Father is a carrier of a

balanced translocation **25% recurrence risk

PAT I E N T W. N .

What testing options should be offered?

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

Cell free DNA Invasive testing with CVS Cell free DNA with CVS only ..

11% 57% 33%

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A b i t o f b a c k g r o u n d … .

• O RI G I N P R I M A R I LY

P L A C E N TA

• R ES U LT S F RO M A P O P TO S I S
• M A D E UP O F S HO RT

S EG M E N T S O F F E TA L D N A ( < 2 0 0 BA S E PA I RS )

• C I RC U L AT E I N M AT E R N A L

P L A S M A

Cell Free Fetal DNA in Maternal Plasma: Characteristics

cffDNA represents ~10-15% of total DNA in maternal plasma1 Reliably detected after 7 wks gestation2 Higher concentrations late in gestation Short half life (16 min), undetectable by 2 hrs postpartum3

cfDNA: Common Clinical Applications

Aneuploidy: detect abnormal ratio of a particular chromosome Sex determination Isoimmunization: noninvasively determine fetal Rh type

cf DNA: The Future

 Whole genome testing  Single gene disorders  Microdeletions

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Understanding the methods…

D i f f e r i n g t e c h n i q u e 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

F r a c t i o n o f c f D N A t h a t i s fe t a l

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

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

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Cell free DNA screening: Biologic Challenges

False Positives

• Unrecognized or “vanishing”twin
• Placental Mosaicism
• Low level maternal mosaicism, esp. sex chromosomes
• Maternal genetic variation (copy number variants)
• Maternal Malignancy

Cell free DNA screening: Biologic Challenges False negatives

• Low level of fetal DNA
• Placental mosaicism
• Maternal genetic variation (copy number

variants) Cell free DNA screening: Biologic Challenges Failed Results

• Increased BMI
• Low level of fetal DNA
• Fetal Aneuploidy

Example of report and question

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What is the next step?

• A. No further testing needed, referral to GC to

discuss diagnosis

• B. Repeat cell free DNA
• C. Recommend invasive testing

N

• f

u r t h e r t e s t i n g n e e d e d , r . . . R e p e a t c e l l f r e e D N A R e c

• m

m e n d i n v a s i v e t e s t i n g

26% 55% 19%

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 i d i e s

The false positives add up….

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

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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 n i n g f o r m i c r o d e l e t i o n s ?

Microdeletions are rare Expanded panels

Trisomies 9, 16 and 22 Rarely seen in viable pregnancies except as mosaics Microdeletions 22q, 1q36, 5p-, 4p-, 15q11-13, 8q-, 11q- Whole genome

• 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)

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Ultrasound Obstet Gynecol, 2016

95 cases were identified as Positive 61 positives with follow up dx testing 11 true positives 50 false positives

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

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 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%

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The false positives add up…

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 o m 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

• f 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 o r 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 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

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 e r s

 Current clinically available for limited conditions

 Achondroplasia  Thanatophoric dysplasia  Apert syndrome  Cystic fibrosis

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Back to our case…

 Our patient underwent cell free DNA testing with micro deletions  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 4p consistent with Wolf-Hirshorn

Current guidelines ACOG/SMFM guidelines

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 Not recommended in twin pregnancies Microdeletion/expanded panels for cell free DNA are not recommended

Special thanks to Mary Norton, MD

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