Non-invasive diagnosis of Non-invasive diagnosis of fetal sex using free fetal fetal sex using free fetal

DNA: our experiences so farDNA: our experiences so far

Rebecca WoodwardRebecca Woodward, Joanne Dunlop, Stephanie , Joanne Dunlop, Stephanie Allen and Fiona MacdonaldAllen and Fiona Macdonald

West Midlands Regional Genetics Laboratory, West Midlands Regional Genetics Laboratory, BirminghamBirmingham

Fetal sexingFetal sexing

Early prenatal determination of sex: fetuses Early prenatal determination of sex: fetuses at risk of X-linked disordersat risk of X-linked disorders– Males hemizygous for XMales hemizygous for X

Useful for management of CAHUseful for management of CAH– Females at risk of virilisationFemales at risk of virilisation

Invasive procedures:Invasive procedures:– CVS (10-13 weeks) CVS (10-13 weeks)

Small but significant risk of miscarriage (~1-3%) Small but significant risk of miscarriage (~1-3%) and limb abnormalities. and limb abnormalities.

Free Fetal DNA (ffDNA)Free Fetal DNA (ffDNA)

Lo Lo et alet al (1997) discovered significant amounts of ffDNA in maternal (1997) discovered significant amounts of ffDNA in maternal plasmaplasma– Source of ffDNA: Placenta (majority) and Haematopoietic cellsSource of ffDNA: Placenta (majority) and Haematopoietic cells– Mechanism of release: apoptosis most likely candidate (characterised Mechanism of release: apoptosis most likely candidate (characterised

by fragmentation of genomic DNA) by fragmentation of genomic DNA)

Direct correlation between amount of ffDNA in plasma and gestationDirect correlation between amount of ffDNA in plasma and gestation– Represents 3.4%-6.2% of total DNA in maternal plasmaRepresents 3.4%-6.2% of total DNA in maternal plasma

Rapid clearance from maternal circulation after delivery (half life = 4 to 30 Rapid clearance from maternal circulation after delivery (half life = 4 to 30 minutes).minutes).– Unlike intact fetal cells – reported to persist for yearsUnlike intact fetal cells – reported to persist for years

Fetal sexing using ffDNA reduces need for invasive testing by 50%Fetal sexing using ffDNA reduces need for invasive testing by 50%

Justification of testingJustification of testing

Three genetic laboratories currently offering fetal sexing using ffDNA:Three genetic laboratories currently offering fetal sexing using ffDNA:– International Blood Reference LaboratoryInternational Blood Reference Laboratory– North East Thames Regional Genetics Laboratory North East Thames Regional Genetics Laboratory – Manchester Regional Genetics ServiceManchester Regional Genetics Service

High number of referrals for X-linked disorders and CAH within the High number of referrals for X-linked disorders and CAH within the West Midlands.West Midlands.

Samples currently sent to North East Thames Regional Genetics Samples currently sent to North East Thames Regional Genetics Laboratory.Laboratory.

Samples need to be processed quickly: sending samples away Samples need to be processed quickly: sending samples away increases turn around times.increases turn around times.

Testing strategyTesting strategy

Testing strategy involves:Testing strategy involves:

1.1. Separation of plasma from cellular componentsSeparation of plasma from cellular components

1.1. Extraction of ffDNA from maternal plasmaExtraction of ffDNA from maternal plasma

1.1. Detection of Y Detection of Y specific sequences from male sequences from male fetusesfetuses Pyrophosphorolysis-activated polymerisation (PAP)Pyrophosphorolysis-activated polymerisation (PAP) Real-Time PCRReal-Time PCR

Isolation of ffDNA from maternal Isolation of ffDNA from maternal plasmaplasma

Plasma separated by centrifugation within 48hrs (3000rpm; Plasma separated by centrifugation within 48hrs (3000rpm; 10mins) 10mins)

Further micro-centrifugation prior to extraction to remove Further micro-centrifugation prior to extraction to remove any remaining intact cells (persist from previous any remaining intact cells (persist from previous pregnancies)pregnancies)

ffDNA extracted using EZ1 Virus minikit v2 (QIAGEN) and ffDNA extracted using EZ1 Virus minikit v2 (QIAGEN) and the EZ1 BioRobot workstation:the EZ1 BioRobot workstation:– Majority ffDNA fragments <300bp: method optimised Majority ffDNA fragments <300bp: method optimised

for viral DNA is ideal for viral DNA is ideal

Once DNA extracted – used within half a dayOnce DNA extracted – used within half a day

Pyrophosphorolysis-activated Pyrophosphorolysis-activated polymerisation (PAP)polymerisation (PAP)

Couples pyrophosphorolysis Couples pyrophosphorolysis and polymerisation by DNA and polymerisation by DNA polymerase using an polymerase using an oligonucleotide (P*) blocked oligonucleotide (P*) blocked by a 3’ddC.by a 3’ddC.

ddC must be removed by ddC must be removed by pyrophosphorolysis for pyrophosphorolysis for extension to occurextension to occur

High specificityHigh specificity

[dNMP]n + PPi [dNMP]n-1 + dNTP

Fetal sexing using PAPFetal sexing using PAP Primer pair specific for the M281 locus on the Y Primer pair specific for the M281 locus on the Y

chromosomechromosome– Y chromosome sequence present if product observed at 93bpY chromosome sequence present if product observed at 93bp– Y chromosome sequence absent if no productY 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

Real-time PCRReal-time PCR

TerminologyTerminology– CCTT value value: The cycle at which the : The cycle at which the

fluorescence passes the thresholdfluorescence passes the threshold Higher the CHigher the CTT, the lower the amount , the lower the amount

of PCR product producedof PCR product produced– ThresholdThreshold: the line whose : the line whose

intersection with the amplification plot intersection with the amplification plot defines the Cdefines the CTT value value

Analysis parameters: Analysis parameters: – SRY present: CT<40 in ≥5/8 or 6/8 replicatesSRY 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 47 samples audited: no result rate decreased from 29.8% to 23.4% using ≥5/8 replicatesreplicates

– SRY absent: CT=45 (no amplification) in 8/8 replicatesSRY absent: CT=45 (no amplification) in 8/8 replicates

Primers and probes specific to:Primers and probes specific to:– SRYSRY: Y chromosome specific probe (8 replicates): Y chromosome specific probe (8 replicates)

– CCR5: CCR5: ‘Housekeeping gene’ located on chromosome 3 (2 replicates)‘Housekeeping gene’ located on chromosome 3 (2 replicates) Confirms success of extraction (maternal and fetal DNA)Confirms success of extraction (maternal and fetal DNA)

Example tracesExample 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

ValidationValidation

Testing strategy validated using 78 samples:Testing strategy validated using 78 samples:– Single frozen plasma aliquots (47) Single frozen plasma aliquots (47)

Manchester Regional Genetics ServiceManchester Regional Genetics Service International Blood Reference Laboratory (Bristol)International Blood Reference Laboratory (Bristol) University College LondonUniversity College London

– Maternal blood samples (31) collected in houseMaternal blood samples (31) collected in house– Mean gestation of samples = 11Mean gestation of samples = 11+6+6 weeks. weeks.

PAP and Real-time PCR performed in parallel using the same plasma PAP and Real-time PCR performed in parallel using the same plasma samplesample– Samples scored using each method separately and in combination to Samples scored using each method separately and in combination to

access the reliability and robustness of each methodaccess the reliability and robustness of each method

Where multiple aliquots of plasma were available, test repeated up to Where multiple aliquots of plasma were available, test repeated up to 3x if calling criteria was not met3x if calling criteria was not met– No result after 3 attemptsNo result after 3 attempts

Results: PAPResults: PAP

Y presentY present: 93bp PCR product: 93bp PCR product Y absentY absent: No PCR product: No PCR product Faint bands were scored as a no resultFaint bands were scored as a no result

Sensitivity (false –ve) = 97.4%Sensitivity (false –ve) = 97.4% Specificity (false +ve) = 96.2%Specificity (false +ve) = 96.2% Failure rate = 0%Failure rate = 0%

Results: Real-time PCRResults: Real-time PCR

Analysis parameters:Analysis parameters:– SRY presentSRY present: C: CTT<40 in<40 in ≥5/8 replicates ≥5/8 replicates

– SRY absentSRY absent: C: CTT=45 in 8/8 replicates=45 in 8/8 replicates

– No result if do not fit criteriaNo result if do not fit criteria

Sensitivity (false –ve) = 98.6%Sensitivity (false –ve) = 98.6% Specificity (false +ve) = 100%Specificity (false +ve) = 100% Failure rate = 17.8%Failure rate = 17.8%

Results: PAP + Real-time PCR Results: PAP + Real-time PCR combinedcombined

Absence of Y sequencesAbsence of Y sequences: No band present + CT=45 in 8/8 : No band present + CT=45 in 8/8 replicatesreplicates

Presence of Y sequencesPresence of Y sequences: Band present + CT<: Band present + CT<40 in ≥5/8 40 in ≥5/8 replicatesreplicates

No resultNo result if do not meet the criteria if do not meet the criteria

Sensitivity (false –ve) = 100% Sensitivity (false –ve) = 100% Specificity (false +ve) = 100% Specificity (false +ve) = 100% Failure rate = 22.5%Failure rate = 22.5%

– 81% did not meet the strict calling criteria for scoring as having Y present81% did not meet the strict calling criteria for scoring as having Y present

Confirming the presence of ffDNAConfirming the presence of ffDNA

If SRY is absent in a sample:If SRY is absent in a sample:– ?Fetal sex is female?Fetal sex is female– ?Absence of ffDNA?Absence of ffDNA

Need a method to confirm the presence of ffDNANeed a method to confirm the presence of ffDNA– Non-Y-associated gene inherited from the father, not present in Non-Y-associated gene inherited from the father, not present in

the maternal genomethe maternal genome 8-10 polymorphic biallelic markers8-10 polymorphic biallelic markers

– Other methods being developed - methylation basedOther methods being developed - methylation based

Biallelic markers NOT validated: reported to be Biallelic markers NOT validated: reported to be informative in only ~40% of patientsinformative in only ~40% of patients

ConclusionsConclusions By using Real-time PCR and PAP assays in parallel, the technique was By using Real-time PCR and PAP assays in parallel, the technique was

found to be:found to be:– Reliable (sensitivity and specificity 100%) Reliable (sensitivity and specificity 100%) – Easy to performEasy to perform– Low in costLow in cost– Capable of providing a diagnosis within 24 hoursCapable of providing a diagnosis within 24 hours

High rate of no results:High rate of no results:– Majority of samples received as plasma aliquots from other laboratoriesMajority of samples received as plasma aliquots from other laboratories

1 aliquot per sample: no possibly of repeating if scored as a no result1 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 Further work is being carried out to determine what gestation to offer testing from.testing from.– Currently validating samples from 7-10 weeks gestationCurrently validating samples from 7-10 weeks gestation

Method to confirm the presence of ffDNA where Y is absentMethod to confirm the presence of ffDNA where Y is absent

AcknowledgementsAcknowledgements

West Midlands Regional Genetics LaboratoryWest Midlands Regional Genetics Laboratory– Joanne DunlopJoanne Dunlop– Stephanie AllenStephanie Allen– Jennie BellJennie Bell– Fiona MacdonaldFiona Macdonald

Manchester Regional Genetics ServiceManchester Regional Genetics Service– Helene SchlechtHelene Schlecht

International Blood Reference Laboratory International Blood Reference Laboratory University College LondonUniversity College London