Non-invasive diagnosis of fetal sex using free fetal DNA: our experiences so far For my trainee project I was involved in setting up and validating fetal sexing using ffDNA. Today I am going to present our experiences so far… Rebecca Woodward, Joanne Dunlop, Stephanie Allen and Fiona Macdonald West Midlands Regional Genetics Laboratory, Birmingham

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. Couples at risk of transmitting sex-linked diseases, where males are hemizygous for the X chromosome, need early and rapid sex determination. It is also useful in the management of Congenital adrenal hyperplasia; where females exhibit varied degrees of virilisation and develop ambiguous genitalia from as early as 8 weeks gestation. Amongst invasive techniques, CVS is the earliest procedure for fetal sex determination during the first trimester. CVS is usually performed between 10 and 13 weeks gestation. But the procedure carries a small but significant risk of miscarriage and transverse limb abnormalities.

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% In 1997 Lo et al discovered significant amounts of ffDNA in the maternal plasma. The placenta is thought to contribute the majority of ffDNA and apoptosis the most likely mechanism of release. There is a direct correlation between the amount of ffDNA in the plasma and the gestation. Using quantitative real-time PCR, fetal DNA was found to represent 3.4% and 6.2% of the total plasma cell-free DNA in the maternal plasma in early and late pregnancy. Unlike intact fetal cells, which have been reported to persist in the maternal circulation for years, ffDNA has a half life of 4 to 30 minutes and is rapidly cleared from the maternal circulation after delivery, therefore previous pregnancies will not affect analysis. Fetal sexing using ffDNA: reduces invasive testing in up to 50% of cases where fetus no longer at risk of being affected

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.

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 The testing strategy used employed involves………..

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 Prior to extraction, a centrifugation step is essential, as inefficient processing could lead to residual intact cells in the plasma, which could interfere with the accuracy of quantification. Because the majority of fetal DNA fragments are <300bp, an extraction method optimised for viral DNA, which favours small-sized DNA fragments was ideal. Therefore the ffDNA is extracted using the QIAGEN EZ1 Virus minikit. But it is important to note that as ffDNA represents a minor fraction of the DNA present in the plasma, therefore it is important that the analytical methods used must detect the fetal DNA with high sensitivity and differentiate the target in a background of maternal DNA.

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 PAP is a method for nucleic acid amplification, originally developed to enhance the specificity of allele-specific PCR for detection of known mutations in presence if excess WT-DNA. PAP couples pyrophosphorolysis; which is the the reverse reaction of DNA polymerisation and polymerisation, by DNA polymerase using an oligonucleotide (P*) blocked at the 3’ terminus by a dideoxy nucleotide. In the diagram this is represented by C*. The dideoxy nucleotide must be removed from the oligonucleotide by pyrophosphorolysis for extension to occur. In the presence of pyrophosphate, when the blocked oligonucleotide anneals to its complementary strand, the 3’ nucleotide is removed from duplex DNA to generate a triphosphate nucleotide and a 3’ terminal shortened duplex DNA ([dNMP]n + PPi  [dNMP]n-1 + dNTP). Coupling of pyrophosphorolysis and polymerisation gives extreme specificity; non-specific amplification requires mismatch pyrophosphorolysis followed by misincorporation by the DNA polymerase [dNMP]n + PPi  [dNMP]n-1 + dNTP

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 For fetal sexing using PAP, a primer pair specific for the M281 locus on the Y chromosome is used. Products are run on a 3% agarose gel and using this assay, normal male samples show a specific band at 93bp, whilst no band is detected in females. On the gel, L1-5 are samples; L1+4 having the presence of Y indicated by the 93bp band and L2,3+5 having the absence of Y. L6-9 are controls. 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

Real-time PCR Primers and probes specific to: Analysis parameters: SRY: Y chromosome specific probe (8 replicates) CCR5: ‘Housekeeping gene’ located on chromosome 3 (2 replicates) Confirms success of extraction (maternal and fetal DNA) 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

Example traces SRY present SRY absent SRY amplification in 8/8 replicates CCR5:amplified => extraction OK The SRY amplification curves are shown in blue and the CCR5 assay amplification plots are shown in green. The top amplification plot shows a sample where SRY was present in 8 out of 8 replicates. The second amplification plot shows a sample where SRY is absent, CCR5 has amplified in both replicates confirming there is DNA (maternal or fetal) present. SRY absent SRY: no amplification in 8/8 replicates CCR5: amplified => extraction OK

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

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% 78 samples were validated using PAP as a stand-alone technique. Samples were scored as having the presence of Y if a 93bp protein product was produced, faint bands were no resulted. Using PAP, 3 false positives and 2 false negative results were observed giving a sensitivity and specificity of 97.4% and 96.2% respectively.

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% 73 samples were validated using Real-time PCR as a stand alone technique. A sample was scored as having the presence of Y if 5 or more replicates had a CT<40, whereas for a sample to be scored as having the absence of Y, no amplification should be observed in all 8 replicates. The sensitivity and specificity was 98.6% and 100% respectively, however the failure rate was 17.8%.

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 Using both techniques in parallel, 71 samples were validated.

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

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

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