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6/7/2018 N O F I N A N C I A L D I S C LO S U R E S PAT I E N T W. N . What testing options should be offered? 37-year-old G5P4004 at 12 57% weeks gestation A. Cell free DNA -3 healthy children -1 son with Wolf-Hirschorn 33% B.

  1. 6/7/2018 N O F I N A N C I A L D I S C LO S U R E S PAT I E N T W. N . What testing options should be offered? • 37-year-old G5P4004 at 12 57% weeks gestation A. Cell free DNA -3 healthy children -1 son with Wolf-Hirschorn 33% B. Invasive testing with CVS syndrome C. Cell free DNA with CVS only if abnormal - Parental testing performed 11% Father is a carrier of a • balanced translocation Cell free DNA Invasive testing with CVS Cell free DNA with CVS only .. ** 25% recurrence risk 1

  2. 6/7/2018 Cell Free Fetal DNA in Maternal Plasma: A b i t o f b a c k g r o u n d … . Characteristics • O RI G I N P R I M A R I LY  cffDNA represents ~10-15% of total DNA in maternal plasma 1 P L A C E N TA • R ES U LT S F RO M A P O P TO S I S  Reliably detected after 7 wks gestation 2 • 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  Higher concentrations late in gestation ( < 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  Short half life (16 min), undetectable by 2 hrs postpartum 3 P L A S M A cf DNA: The Future cfDNA: Common Clinical Applications  Whole genome  Aneuploidy : detect abnormal ratio of a particular testing chromosome  Single gene  Sex determination disorders  Isoimmunization: noninvasively determine fetal Rh type  Microdeletions 2

  3. 6/7/2018 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 The less fetal DNA, the harder to tell normal from 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 abnormal Prenatal Diagnosis 2012; 32(13) p 1233-41 3

  4. 6/7/2018 Cell free DNA screening: Biologic Challenges Cell free DNA screening: Biologic Challenges False Positives  Unrecognized or “vanishing”twin False negatives  Placental Mosaicism  Low level of fetal DNA  Low level maternal mosaicism, esp. sex chromosomes  Placental mosaicism  Maternal genetic variation (copy number variants)  Maternal genetic variation (copy number  Maternal Malignancy variants) Example of report and question Cell free DNA screening: Biologic Challenges Failed Results  Increased BMI  Low level of fetal DNA  Fetal Aneuploidy 4

  5. 6/7/2018 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 What is the next step? A. No further testing needed, referral to GC to discuss diagnosis 55% B. Repeat cell free DNA 26% C. Recommend invasive testing 19% A g . N n . . D t i r s , e e d e e t r e d f e v l l i e e s n c a v g t n The false positives add up…. n a e i t i d p s e n e e t R m e r m h t o r c u e f R o N 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 5

  6. 6/7/2018 S h o u l d a l l w o m e n b e o f f e r e d Microdeletions are rare 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 ? Expanded panels  Trisomies 9, 16 and 22  Rarely seen in viable pregnancies except as mosaics  Microdeletions • Microdeletions can be detected by cfDNA, but…  22q, 1q36, 5p-, 4p-, 15q11-13, 8q-, 11q- • Difficult to validate • Much of the data on in vitro samples  Whole genome • Clinical testing includes small number of syndromes • 22q, 1q36, 5p-, 4p-, 15q11-13 (Prader-Willi/Angelman) 6

  7. 6/7/2018 Prevalence of many microdeletion syndromes is unknown  Many such clinical syndromes are caused by different molecular mechanisms 95 cases were identified as Positive  Only deletions detected 61 positives with follow up dx testing  Smaller deletions more difficult to detect  22q syndrome (~1/4000) 11 true positives 50 false positives  85% have 3Mb deletion, 15% smaller  “97% detection” refers to only 3Mb deletion Ultrasound Obstet Gynecol, 2016 cfDNA for Microdeletions Noninvasive Microarray Sequenced 565 samples: Prevalence of 5 microdeletion syndromes: ~1/2500 31 with CNV and 534 normal Prevalence of CNV by microarray: 1/60 • Detected 83% with CNV >6Mb but only 20% (2/10) that were <6Mb Detection rate of cfDNA for all pathogenic CNV: 4.2% • 2/534 false positives • More sequencing = higher sensitivity 7

  8. 6/7/2018 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 8

  9. 6/7/2018 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  Ehrich et al. (2017) reports on the clinical experience of one lab’s experience with the • RAISES BOTH PRACTICAL AND first 10,000 cases ETHICAL ISSUES  reports on copy number variations >7 Mb • MAY GIVE INFORMATION  25% of samples were sent due to an ultrasound abnormality REGARDING RISK FOR ADULT ONSET CONDITIONS THAT  Screen-positive test results were reported in 554 cases, leading to a screen-positive rate ARE NOT RELEVANT 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 Non-Invasive Single Gene Tests 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  Maternal and fetal cell free DNA cannot be easily distinguished  Current clinically available for limited conditions  Can identify de novo or paternal gene mutation circulating in maternal blood  Achondroplasia  An affected fetus will have an overrepresentation of the mutant allele in  Thanatophoric dysplasia maternal plasma DNA  Apert syndrome  Ideal for autosomal dominant paternally inherited conditions  Cystic fibrosis  Useful if testing for an autosomal recessive condition and parents have different mutations 9

  10. 6/7/2018 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 Special thanks to Mary Norton, MD  Microdeletion/expanded panels for cell free DNA are not recommended 10

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