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

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Presentation on theme: "Non-invasive diagnosis of fetal sex using free fetal DNA: our experiences so far Rebecca Woodward, Joanne Dunlop, Stephanie Allen and Fiona Macdonald West."— Presentation transcript:

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: 1.Separation of plasma from cellular components 1.Extraction of ffDNA from maternal plasma 1.Detection of Y sequences from male fetuses 1.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 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 Example PAP results

9 Real-time PCR  Terminology –C T value: The cycle at which the fluorescence passes the threshold  Higher the C T, the lower the amount of PCR product produced –Threshold: the line whose intersection with the amplification plot defines the C T 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  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: C T <40 in ≥5/8 replicates –SRY absent: C T =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 Results: PAP + Real-time PCR combined  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

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


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