Non-invasive diagnosis of fetal sex using free fetal DNA: our experiences so far

1. Non-invasive diagnosis of fetal sex using free fetal DNA: our experiences so far Rebecca Woodward, Joanne Dunlop, Stephanie Allen and Fiona Macdonald West Midlands Regional Genetics Laboratory, Birmingham

2. Fetal sexing Early prenatal determination of sex: fetuses at risk of X-linked disorders Males hemizygous for X Useful for management of CAH Females at risk of virilisation Invasive procedures: CVS (10-13 weeks) Small but significant risk of miscarriage (~1-3%) and limb abnormalities.

3. Free Fetal DNA (ffDNA) • Lo et al (1997) discovered significant amounts of ffDNA in maternal plasma • Source of ffDNA: Placenta (majority) and Haematopoietic cells • Mechanism of release: apoptosis most likely candidate (characterised by fragmentation of genomic DNA) • Direct correlation between amount of ffDNA in plasma and gestation • Represents 3.4%-6.2% of total DNA in maternal plasma • Rapid clearance from maternal circulation after delivery (half life = 4 to 30 minutes). • Unlike intact fetal cells – reported to persist for years • Fetal sexing using ffDNA reduces need for invasive testing by 50%

4. Justification of testing • Three genetic laboratories currently offering fetal sexing using ffDNA: • International Blood Reference Laboratory • North East Thames Regional Genetics Laboratory • Manchester Regional Genetics Service • High number of referrals for X-linked disorders and CAH within the West Midlands. • Samples currently sent to North East Thames Regional Genetics Laboratory. • Samples need to be processed quickly: sending samples away increases turn around times.

5. Testing strategy • Testing strategy involves: • Separation of plasma from cellular components • Extraction of ffDNA from maternal plasma • Detection of Y specific sequences from male fetuses • Pyrophosphorolysis-activated polymerisation (PAP) • Real-Time PCR

6. Isolation of ffDNA from maternal plasma Plasma separated by centrifugation within 48hrs (3000rpm; 10mins) Further micro-centrifugation prior to extraction to remove any remaining intact cells (persist from previous pregnancies) ffDNA extracted using EZ1 Virus minikit v2 (QIAGEN) and the EZ1 BioRobot workstation: Majority ffDNA fragments <300bp: method optimised for viral DNA is ideal Once DNA extracted – used within half a day

7. Pyrophosphorolysis-activated polymerisation (PAP) • Couples pyrophosphorolysis and polymerisation by DNA polymerase using an oligonucleotide (P*) blocked by a 3’ddC. • ddC must be removed by pyrophosphorolysis for extension to occur • High specificity [dNMP]n + PPi  [dNMP]n-1 + dNTP

8. Fetal sexing using PAP Primer pair specific for the M281 locus on the Y chromosome Y chromosome sequence present if product observed at 93bp Y chromosome sequence absent if no product Example PAP results 3% gel showing Y present in L1 + 4 and Y absent in L2-3 and 5. PAP controls: L6 = 100:1 female to male, L7 = male DNA, L8 = female DNA and L9 = negative control

9. Terminology CT value: The cycle at which the fluorescence passes the threshold Higher the CT, the lower the amount of PCR product produced Threshold: the line whose intersection with the amplification plot defines the CT value Analysis parameters: SRY present: CT<40 in ≥5/8 or 6/8 replicates 47 samples audited: no result rate decreased from 29.8% to 23.4% using ≥5/8 replicates SRY absent: CT=45 (no amplification) in 8/8 replicates Real-time PCR • Primers and probes specific to: • SRY: Y chromosome specific probe (8 replicates) • CCR5: ‘Housekeeping gene’ located on chromosome 3 (2 replicates) • Confirms success of extraction (maternal and fetal DNA)

10. Example traces SRY present SRY amplification in 8/8 replicates CCR5:amplified => extraction OK SRY absent SRY: no amplification in 8/8 replicates CCR5: amplified => extraction OK

11. Validation Testing strategy validated using 78 samples: Single frozen plasma aliquots (47) Manchester Regional Genetics Service International Blood Reference Laboratory (Bristol) University College London Maternal blood samples (31) collected in house Mean gestation of samples = 11+6 weeks. PAP and Real-time PCR performed in parallel using the same plasma sample Samples scored using each method separately and in combination to access the reliability and robustness of each method Where multiple aliquots of plasma were available, test repeated up to 3x if calling criteria was not met No result after 3 attempts

12. Results: PAP • Y present: 93bp PCR product • Y absent: No PCR product • Faint bands were scored as a no result • Sensitivity (false –ve) = 97.4% • Specificity (false +ve) = 96.2% • Failure rate = 0%

13. Results: Real-time PCR • Analysis parameters: • SRY present: CT<40 in ≥5/8 replicates • SRY absent: CT=45 in 8/8 replicates • No result if do not fit criteria • Sensitivity (false –ve) = 98.6% • Specificity (false +ve) = 100% • Failure rate = 17.8%

14. Absence of Y sequences: No band present + CT=45 in 8/8 replicates Presence of Y sequences: Band present + CT<40 in ≥5/8 replicates No result if do not meet the criteria Sensitivity (false –ve) = 100% Specificity (false +ve) = 100% Failure rate = 22.5% 81% did not meet the strict calling criteria for scoring as having Y present Results: PAP + Real-time PCR combined

15. Confirming the presence of ffDNA • If SRY is absent in a sample: • ?Fetal sex is female • ?Absence of ffDNA • Need a method to confirm the presence of ffDNA • Non-Y-associated gene inherited from the father, not present in the maternal genome • 8-10 polymorphic biallelic markers • Other methods being developed - methylation based • Biallelic markers NOT validated: reported to be informative in only ~40% of patients

16. Conclusions • By using Real-time PCR and PAP assays in parallel, the technique was found to be: • Reliable (sensitivity and specificity 100%) • Easy to perform • Low in cost • Capable of providing a diagnosis within 24 hours • High rate of no results: • Majority of samples received as plasma aliquots from other laboratories • 1 aliquot per sample: no possibly of repeating if scored as a no result • Further work is being carried out to determine what gestation to offer testing from. • Currently validating samples from 7-10 weeks gestation • Method to confirm the presence of ffDNA where Y is absent

17. Acknowledgements • West Midlands Regional Genetics Laboratory • Joanne Dunlop • Stephanie Allen • Jennie Bell • Fiona Macdonald • Manchester Regional Genetics Service • Helene Schlecht • International Blood Reference Laboratory • University College London