This document provides an overview of non-invasive prenatal diagnosis (NIPD) for single gene disorders using cell-free fetal DNA. It summarizes the development and validation of two NIPD approaches through the NIPSIGEN study: 1) Bespoke assays for specific mutations and 2) Relative haplotype dosage analysis using SNPs to track inheritance of mutated genes. It reports on the clinical implementation of these approaches in the West Midlands Regional Genetics Lab for conditions like DMD/BMD, SMA, CF, and CAH. Results from over 50 NIPD tests conducted to date show 100% accuracy compared to invasive testing. The document concludes by outlining the clinical service and pathways that are now available

1. Stephanie Allen
Consultant Clinical Scientist
West Midlands Regional Genetics Lab,
Birmingham UK
Stephanie.allen@bwnft.nhs.uk

2. • Clinical Teams:
- Germline – Developmental Disorders,
Rare diseases, Prenatal diagnosis &
Reproductive Medicine
- Cancer – Haemato-oncology, Familial
Cancer, Molecular Pathology
• Translational research
• Over 55,000 samples/pa
• Over 250 staff
• Regional service (and associated
Clinical Genetics service)
• UKGTN (UK Genetic Testing
Network)
West Midlands Regional Genetics
Service
Population: ~5.6 million (9% of UK)
West Midlands Regional
Genetics Lab

3. Overview
• Cell free DNA and use for Prenatal
Diagnosis
• Prenatal diagnosis for SGDs in UK
• Aims, Methods, Results NIPSIGEN study
- 1. Bespoke, 2. RHDO
• Clinical Service
• Future work

4. ccfDNA is released from cells undergoing programmed cell
death
Quantity: 1- 5 ng/ml of
plasma
Half-life: 16-17 minutes
Fragment size: 166 bp
Released from: all
tissues
Circulating cell free DNA

5. • Fetal portion of cfDNA during pregnancy: 2 – 25%
• Detectable from 6-7 weeks gestation
• Technical difficulties – small fragments, low
concentration, predominantly maternal
Cell free fetal DNA

6. Determination of fetal sex
Detection of SRY sequences in maternal plasma
Sex linked and sex limited disorders.
RhesusD genotyping for haemolytic diseases of the newborn
Detection of RhD+ sequences in RhD-/- pregnant women
NICE guidelines 2016
Aneuploidy testing for chromosomes 21, 18, 13, X and Y
Detection of excess chromosome copies; driven by private sector
Incorporated into screening pathway 2018
Diagnosis of single gene disorders
Where do we stand?
Cell free fetal DNA for
prenatal diagnosis

7. • Relatively rare, collectively common – WHO “global prevalence of all
single gene diseases at birth is approximately 10/1000”
• Alpha and beta thalassaemia
• Haemoglobinopathies 1-2 per 10,000 in England
• Sickle cell anaemia
• CF 1 in 2,500
• HD 5-7 / 100,000
• DMD
• SMA
Definition rare disease - one that affects fewer than five in every 10,000.
More than 6,000 rare diseases
Mendelian SGDs

8. 0
100
200
300
400
500
600
700
800
900
Number PND 2014-15
Data from 27 UK labs
Majority of these will be due to family history
Several hundred different disorders
Availability of NIPD limited
Disorder PND per
annum
(UK)
Per
week
DMD/BM
D
29 <1
SMA 71 1-2
CF 152 3
CAH 17 <1
Total 269 5-6
ACGS Audit Prenatal
Testing 2014-15

9. NIPSIGEN Study - objectives
• Increase repertoire NIPD tests available
• Secured funding from Health Innovation Challenge
Fund ~£400k 2013-16
• Translation of NIPD for single gene disorders into a
clinical setting
- Generic approach similar to linkage-based prenatal
diagnosis
- DMD/BMD, SMA, CAH, CF
- Bespoke assay approach – individual assays
- Very rare disorders, private mutations

10. Group 2: healthy patients
• Pregnant
• Offered invasive testing
• Some recruited
nationally (UK)
 maternal and paternal blood sample
(20 ml) taken at 7-9 weeks gestation
 DNA sample from proband
(affected sibling)
• Pregnant
• High risk for aneuploidy
• Offered invasive testing
• Recruited locally
 maternal and paternal blood sample
(20 ml) taken at CVS/amnio
appointment
 DNA sample from CVS/amnio
Samples needed for 1
patient
cfDNA
(4 ml plasma)
maternal
gDNA
paternal
gDNA
proband
gDNA
Group 1: patient carriers of
DMD/BMD, SMA, CAH, CF
and others
Patient Recruitment

11. Detection of paternal and/or de novo mutations in cffDNA
Develop assay prior to pregnancy
Partner
(mut B)
Patient
(mutA)
P
Autosomal
recessive
Paternal detection
PartnerPatient
P
Autosomal dominant
Paternal detection
PartnerPatient
P
Autosomal dominant
de novo detection
1. Bespoke NIPD for
SGD

12. 1) PCR across region of interest
1) Next generation Sequencing
- High depth sequencing
3) Separate assay Fetal Marker
(Fetal fraction) – panel of SNPs
Mutation
50-90
bp
Bespoke NIPD workflow

13. delF508 /
c.3889dupT
Disorder: Cystic fibrosis (CFTR gene)
Fetal fraction: 11.96%
Sequence
Sequencing counts
Maternal
plasma
Maternal
genomic
Paternal
genomic
Normal 124052 128642 73497
delF508 mutation 5077 79 66443
% of mutation 3.93% 0.06% 47.47%
Result: mutation detected
Outcome: fetus 50% chance
of being affected
Further action warranted:
invasive testing
delF508c.3889dupT
delF508
baseline
PND: unaffected (carrier)
P

14. c.586+5G>A
Disorder: Neurofibromatosis type I (NF1 gene)
Fetal fraction: 3.27%
Sequence
Sequencing counts
Maternal
plasma
Maternal
genomic
Paternal
genomic
Normal 150480 143961 98065
c.586+5G>A mutation 76 76 67320
% of mutation 0.05% 0.05% 40.70%
Result: mutation undetected
Outcome: fetus unaffected
Further action warranted:
nonebaseline
PND result: unaffected
P

15. * All assays developed prior to a pregnancy – final column indicates
where testing in pregnancy has occurred.
Bespoke assays
Disorder Gene Variant ID Mutation NIPD offered
Congenital myopathy RYR1 - c.14582G>T (p.Arg4861Leu) √
Cystic fibrosis CFTR rs113993960 c.1521_1523delCTT (p.Phe508del) √
- c.1117-1G>A splice site mutation
rs80055610 c.1679G>C (p.Arg560Thr)
rs78756941 c.489+1G>T splice site mutation √
Gaucher's disease GBA rs78973108 c.887G>A p.(Arg296Gln)
Goltz Gorlin syndrome PORCN c.1093C>T p.(Arg365Trp)
Hypophosphatasia ALPL rs763159520 c.550C>T p.(Arg184Trp) √
Infantile epilepsy CDKL5 - c.104C>T p.(Thr35Ile)
Loyes Dietz syndrome TGFBR2 rs104893810 c.1582C>T (p.Arg528Cys)
MICPCH CASK - c.845dupA (p.Y282*) √
Micro syndrome RAB3GAP1 - c.691 C>T (p.Arg231ter)
Neurofibromatosis-I NF1 - c.586+5 G>A splice site mutation √
- c.6364+2 T>G splice site mutation
c.4076delC p.(Pro1359Leufs*26)
Osteogenesis imperfecta COL1A1 - c.3443G>A p.(Gly1148Asp)
c.1706G>A p.(Gly569Asp)
Retinoblastoma RB1 rs121913305 c.1735 C>T (p.Arg579X)
c.1498+1G>C √
c.1072C>T p.(Arg358X) √
Tuberous sclerosis TSC2 rs137854113 c.4279delA (p.Ser1427Val fs*49)
rs45517222 c.2251C>T (p.Arg751Ter)
- c.3110delA (p.Asn1037Thr fs*16)
Wilson's disease ATP7B rs137853280 c.1708-1G>C splice site mutation

16. P
Autosomal
recessive
P
X-linked recessive
P
Autosomal dominant
Maternal
inheritance
Same mutations
Technically more difficult!

17. Detecting exons in the
dystrophin gene

18. Massively Parallel
Sequencing
Compared to ddPCR:
• Better sensitivity
(30 million reads)
• Higher multiplexing
(up to 96 patients)
• Qualitative +
quantitative data
• Complex analysis
(bioinformatics
needed)
• Increased TAT
(lengthy protocols)
• Increased cost
• Streamlining of
NIPD

19. 2. Relative Haplotype
Dosage
• Generic approach
• Linkage based, using SNPs
• Capture regions of interest using probe libraries
• Tracks inheritance of the mutated genes
• NIPSIGEN study – adapted this for routine use
• Method quite complex and relatively expensive, 2 week
TAT
• Multiplexing of different disorders / genomic regions

20. SNP
rsXXX
rsXXX
rsXXX
rsXXX
rsXXX
rsXXX
AvHet>0.4
C
G
A
A
T
G
Hap A
(mut)
100%
Hap A
(mut)
50%
Hap B
(nor)
50%
Only heterozygous SNPs
in the mother are
informative
Allele
ratios
C > T
A > C
G > C
Hap A
(mut)
Mother
(45%) +
fetus (10%)
Hap B
(nor)
Mother (45%)
Proband
(genomic
DNA)
Maternal
(genomic
DNA)
Maternal
(cell free
DNA)
C
G
A
A
T
G
C
G
A
A
T
G
T
G
C
A
T
C
T
G
C
G
T
C
If > 1 =
Mut
If < 1 =
Nor
RHDO analysis for X-
linked disorders

21. Informative
SNPs
Paternal
inheritance (KS
test):
• Hom in mother
• Het in father
NIPD for SGD by relative
haplotype dosage (autosomal
recessive conditions)

22. Informative
SNPs
Maternal
inheritance (RHDO
analysis):
• Het in mother
• Hom in father
Fetal fraction:
• Hom in mother (A)
• Hom in father (B)
NIPD for SGD by relative
haplotype dosage (autosomal
recessive conditions)

23. Cell free DNA
SNPs Chr Pos
Counts
Hap A
Counts
Hap B
rs5927145 33136379 21 22
rs6631741 33136164 105 94
rs5927144 33135440 58 63
rs73190205 33133951 100 112
rs12556796 33132186 50 50
rs2179815 33129716 91 74
rs2143844 33129493 85 78
rs17283533 33128962 70 82
rs7887022 33127097 14 19
rs2182289 33125527 68 68
rs4829279 33124424 98 90
rs5972753 33124037 51 77
rs5972752 33123266 82 74
rs6631737 33120348 55 62
rs5927142 33119092 138 106
rs6527261 33117564 106 110
rs6631735 33116211 77 81
rs5927140 33115938 21 39
Reference haplotype:
Over-represented
Under-represented

24. X-linked
Duchenne and Becker muscular
dystrophies (DMD/BMD)
• 1 in 3500 (DMD)
• Dystrophin gene: 2.2 Mb long; 79
exons
• Mutational profile: 70% exon
deletion/duplication
• Recombination rate: 10% across
the whole gene
Autosomal recessive
Spinal muscular atrophy (SMA)
• 1 in 10,000 live births
• Mutations in the SMN1 gene
Congenital adrenal hyperplasia
(CAH)
• 1 in 15,000 live births
• Mutations in the CYP21A2 gene
Cystic fibrosis (CF)
• 1 in 2,500 live births
• Mutations in the CFTR gene

25. SMA SNPs targeted: 3078
Probe coverage: 67%
CF
SNPs targeted: 2951
Probe coverage: 79%
Custom built probe libraries
SNPs targeted: 1802
Probe coverage: 69%DMD/BMD
CAH SNPs targeted: 4174
Probe coverage: 64%

26. Proband
del ex 45-52
Gestation: 8 w + 6 d
Fetal fraction: 7.26%
SNPs sequencing depth: 169
Informative SNPs used: 284
SPRT classifications: 11 for / 11 rev
Outcome: NORMAL
?Normal
P

27. Proband
c.3889dupT
P
Gestation: 11 w + 2 d
Fetal fraction: 20.99%
SNPs sequencing depth: 205
Informative SNPs used: 728
SPRT classifications: 17 mat / 8 pat
Outcome: CARRIER
?Carrier
c.1521-1523delCTT

28. Overall performance of NIPD assay
Patients
DMD / BMD SMA CAH CF
Num % Num % Num % Num %
True results 20 95% 19 95% 4 100% 7 100%
False results 0 0% 0 0% 0 0% 0 0%
Inconclusive 1 5% 0 0% 0 0% 0 0%
Failed 5* 1 5% 0 0% 0 0%
TOTAL 26 (13 controls) 20 (12 controls) 4 (3 controls) 7 (4 controls)
DMD/BMD SMA CAH CF
Sensitivity 100% 100% 100% 100%
Specificity 100% 100% 100% 100%
Accuracy of haplotype calling 96.35% 99.16% 99.29% 97.54%
*Isolated issue early in validation – not included as a failure rate

29. Prenat Diagn. 2016 Apr;36(4):312-20. doi: 10.1002/pd.4781. Epub 2016 Feb
23.
Non-invasive prenatal diagnosis of Duchenne and Becker
muscular dystrophies by relative haplotype dosage.
Parks M1, Court S1, Cleary S1, Clokie S1, Hewitt J1, Williams D1, Cole
T1, MacDonald F1, Griffiths M1, Allen S1.
Eur J Hum Genet. 2017 Apr;25(4):416-422. doi:
10.1038/ejhg.2016.195.
Non-invasive prenatal diagnosis of spinal muscular atrophy by
relative haplotype dosage Parks M1, Court S1, Bowns B Cleary
S1, Clokie S1, Hewitt J1, Williams D1, Cole T1, MacDonald F1, Griffiths
M1, Allen S1.
Publications

30. Clinical Service
• RHDO
- Accepting samples
DMD/BMD and SMA since
Sept 2016
- CF and CAH to follow
- DMD/BMD and SMA gene
dossiers accepted
- Cost £1200 (UK NHS)
- Reporting time 14 days
• Bespoke
- West Midlands since July
2015
- Will now accept external
referrals
- Cost £773 test
development, £900 PND
(UK NHS)
- Up to 8 weeks test
development, reporting
time PND <14 days.

31. Care pathway DMD
blood sample 20mls,
post 8 weeks gestation
Refer to flow charts
For further details of
DNA samples required
Non-invasive fetal
sexing first

32. Testing from Sept 2016-
Disorder Number
prenatals
Unaffected Affected Second
sample
requested
Fail / partial
result
Comments
SMA 13 10 3 2 1* * High risk paternal
haplotype; invasive
testing confirmed
affected
DMD 4 3 1 0 1* * Likely to be
unaffected; CVS
confirmed result
BMD 3 2 1* 0 1* * Likely to be
affected
(recombination),
recommend CVS;
TOP no follow up
Reporting time – mean 10.4 days (range 7-17)
Fetal fraction – mean 7.5% (range 2.4-16.9%)

33. 0.95
0.97
0.99
1.01
1.03
1.05
66,900 67,900 68,900 69,900 70,900 71,900 72,900
Maternal inheritance (RHDO analysis)
Chr 5 SMN1 SMN2 Exons
alpha SNPs Beta SNPs M-ma blocks N-ma blocks
0.99
1
1.01
1.02
1.03
1.04
1.05
66,900 67,900 68,900 69,900 70,900 71,900 72,900
Paternal inheritance (KS test)
Chr 5 SMN1 SMN2 Exons Paternal SNPs M-pa blocks N-pa blocks
First diagnostic sample -
SMA

34. 0
2
4
6
8
10
12
14
16
0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 16.00% 18.00%
Fetal fraction and results
Gestation
(weeks)
Fetal fraction
Sample 1:
High risk
paternal haplotype
Sample 2:
Unaffected
Both samples:
High risk
paternal
haplotype
• No absolute cut off for fetal fraction, depends on a number of factors

35. Service considerations
and reporting
• Diagnostic
• SMA / DMD / BMD
- “There is a small risk of misdiagnosis due to recombination, (de
novo deletion / gene conversion), or the presence of a vanishing
twin which would complicate the analysis. This is likely to be very
low (approx. 1:500-1,000).”
• All NIPD
- Whatever the outcome of this pregnancy it is important that we
receive appropriate follow-up samples, such as a cord blood sample
(EDTA) and/or placenta (1.5cm²), and that evidence is provided that
parental consent for follow-up studies has been obtained.
• Funding complex – gene dossiers accepted UKGTN
• Referrals through Clinical Genetics (Control samples)

36. P? P
DMD SMA
??
?
• Mum not a carrier
• Residual risk germline mosaicism
• Can test for low risk or increased risk
maternal X
• One parent not a confirmed carrier
• Can test whether fetus has inherited
same haplotype as affected sib
1 copy SMN1 2 copies SMN1
“Linkage-based” NIPD

37. *
*
*
*
*
* *
***
*
******

38. Summary
• NIPD by RHDO is feasible in a clinical
setting
• Centralisation helps to reduce costs
• Increased accessibility to many more
couples with a pregnancy at risk of a SGD
• Future
- addition of probes for other disorders in
“grouped” capture libraries – CF, CAH…
- Validation for other disorders as tests are run
and follow up data obtained

39. Lab team:
Elizabeth Young
Amy Gerrish
Benjamin Bowns
Michael Parks
Samantha Court
Jess Lander
Chipo Mashayamombe
Siobhan Cleary
Samuel Clokie
Fiona MacDonald
Mike Griffiths
Patients, donors and
national collaborators
involved in recruitment to
NIPSIGEN study
Health Innovation Challenge Fund
Contact details:
Stephanie Allen: Stephanie.Allen@bwnft.nhs.uk
Elizabeth Young: Elizabeth.Young@bwnft.nhs.uk
Clinical team:
Trevor Cole
Denise Williams
Julie Hewitt