non-invasive molecular prenatal diagnosis
DESCRIPTION
Non-invasive molecular prenatal diagnosis. Oxford BRC molecular diagnostic laboratory. Dr Shirley Henderson Consultant Clinical Scientist. Conventional approach to Prenatal Diagnosis. Fetal sampling. Ultrasound. Chorionic villi. Amniotic fluid. DNA diagnostic test. Prenatal Diagnosis. - PowerPoint PPT PresentationTRANSCRIPT
19/04/2023
Non-invasive molecular prenatal diagnosis
Oxford BRC molecular diagnostic laboratory
Dr Shirley HendersonConsultant Clinical Scientist
Conventional approach to Prenatal Diagnosis
Ultrasound
Fetal sampling
Chorionic villi Amniotic fluid
DNA diagnostic test
Prenatal Diagnosis
• 1982:- The first ever prenatal diagnoses by fetal DNA analysis in the UK (β-thalassaemia and sickle cell disease). John Old et.al
• Carried out in the National Haemoglobinopathy Reference Laboratory (NHRL), John Radcliffe Hospital
• This was performed on DNA extracted from an amniotic fluid (AF) sample.
• Constituted a major breakthrough for prenatal diagnosis.
• Now the standard approach – 30 years
• 1997 :- Discovered in John Radcliffe Hospital, Oxford by Jim Wainscoat & Dennis Lo et. al.
• Most cell free DNA in plasma is maternal (from haematopoietic system)
• Free fetal DNA is from placenta (trophoblasts)
• 5-15% of plasma DNA is from the fetus
Another safer source of fetal DNA for diagnosis?
Plasma DNA approach
Double spin plasma to remove any remaining white cells and platelets
DNA extraction
Cell free DNA
Main problems with cff-DNA
• Concentration of cff-DNA is low and it is degraded
• Once a blood sample is taken the fetal DNA deteriorates very rapidly, background maternal DNA will increase as white blood cells in the sample breakdown. Plasma needs to be separated within 6 hours of the sample being taken.
• Cell free fetal DNA molecules substantially out-numbered by cell free maternal molecules
• Currently no way to separate fetal DNA from the maternal plasma DNA. Fetus inherits 50% of it’s genetic sequence maternally- making much of the cffDNA indistinguishable from the mother
Non-invasive fetal sex determination
Female plasmaMaternal plasma with male fetus
Maternal plasma with female fetus
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xxxy
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Method used to detect the y chromosome, usually RT-PCR
Some success stories – all when allele not present in the mother
• Fetal Rh status• Achondroplasia
In development for other conditions but not straightforward:-– Need sensitive technique to assay low levels
– DNA is degraded and can be difficult to work with - assays can need a lot of optimisation
– Negative depends on “no result”
Other examples:-
Down’s syndrome (Trisomy 21)
• Main focus for NIPND research so far has been Down’s syndrome
• Next generation sequencing (NGS), whole genome approach :-involves the random sequencing of millions of DNA molecules in maternal plasma.
• Individual sequence tags are aligned to the human genome to determine the chromosome of origin of a particular sequence tag.
• An increase in representation of sequence tags aligned to a particular chromosome indicating a potential trisomy.
• Effective but very expensive and requires access to high-throughput NGS platforms.
• World-wide there are currently only limited clinical services offering this service, none in the UK
• What about the classic single gene disorders where the mother is a carrier for the condition?
- Haemophilia- Cystic fibrosis- Sickle cell disease- Thalassaemia
Autosomal recessive inheritance X-linked inheritance
Sickle cell disease - first molecular disease
DNA-Sickle mutation ProteinPathophysiology
Inheritance Prevalence Immigration
Sickle Cell Disease in the UK
• Birth Incidence:- Nationally 1:2000 (higher than cystic fibrosis)
S.E. London 3:1000
• Most common indication for invasive PND in the UK
• Approximately 440 PNDs carried out per year in UK
NHS National Antenatal screening programme
• Main purpose to identify couples at risk of having a child with a major haemoglobinopathy (sickle cell and thalassaemia) so prenatal diagnosis can be offered.
• UK divided into 2 types of area:-
• High prevalence areas - non-selective screening
• Low prevalence areas - selective screening based on family origins
?NIPND for sickle cell disease
• Recent development has seen the launch of bench top sequencers which are scaled down cost-effective NGS platforms, driven by the need for faster and more cost-effective sequencing both in research and for the determination of genetic variants in patients:-– Life Technologies Ion torrent, Roche Junior, Illumina MiSeq
• Targeted approach cheaper than whole genome approach.
Principle of allelic imbalance in maternal plasma
Sickle copies
Normal copies
8
8
12 8
8 12 10
10
Sickle carrier plasma
Sickle carrier plasma with
disease (SS) fetus
Sickle carrier plasma with normal
(AA) fetus
Sickle carrier plasma with carrier
(AS)fetus
Plan
Amplify the sickle cell gene in maternal plasma – will result in millions of copies of the sickle cell gene
Sequence all these reads using NGS technology
Count how many of the reads have the sickle mutation and how many have the normal sequence.
>50% sickle cell reads <50% sickle cell reads50% sickle cell reads
Sickle Disease Fetus Sickle Carrier Fetus Normal Fetus
Challenges
• Obtaining fresh enough plasma
• Measuring the proportion of fetal DNA in the sample
• Efficient amplification of low copy number poor quality DNA– Need all fetal molecules to be amplified
• Preservation of the starting ratio of sickle and normal molecules– PCR amplification can change ratios (cold PCR effects)
• Development of NGS technology for this application
Plasma separation
DNA extraction
Plasma DNA concentration and fetal fraction determination
Library preparation
Nested PCR
Purification
Quantification and Normalisation
Paired-end sequencing (84|6|84)
QIAamp Circulating Nucleic Acid Kit
RASSF1 assay / Qubit
MinElute PCR Purification kit
Bioanalyser
Miseq
Process
x3 amplicons
amplicon based
• Design 3 overlapping small amplicons
• Amplicon1 • chr11:5248185-5248276• TCACTAGCAACCTCAAACAGACACCATggtgcatctgactcctgaggagaagtctgccgttactgccCTGTGGGGCAAGGTGAACGTGGATG
• Amplicon 2• chr11:5248192-5248274• ACTAGCAACCTCAAACAGACACCATGgtgcatctgactcctgaggagaagtctgccgtTACTGCCCTGTGGGGCAAGGTGAAC
• Amplicon 3• chr11:5248202-5248270
• GCAACCTCAAACAGACACCATggtgcatctgactcctgaggagaagtctgcCGTTACTGCCCTGTGGGG
• bp targets are in red: chr11:5,248,243; chr11:5,248,233; chr11:5,248,232
Targets
Reads data
Amplicon 1 Amplicon 2 Amplicon 3Sample ID Sample description Run
AS (50:50) control (%
mutant reads)
Variation from control of %
mutant
AS (50:50) control (%
mutant reads)2
Variation from control of %
mutant3
AS (50:50) control (%
mutant reads)4
Variation from control of %
mutant5
S-01 5% AC sample 1 48.2 -1.19 49.2 -0.77 50.2 -1.71S-02 8% AC sample 2 48.2 -3.30 49.2 -4.23 50.2 -4.13S-11 Affected (SS) fetus 3 48.2 4.40 49.2 4.74 50.2 3.02
Normalised data for runs 1, 2 and 3
5% AC 8% AC Affected (SS) fetus
S-01 S-02 S-11AMPLICON 1Mutation WT MT percent WT MT percent WT MT percent chr11:5,248,232 267771 237390 47.0 291018 236903 44.87 47392 52557 52.6chr11:5,248,233 493982 11179 2.2 503983 23938 4.53 99941 8 0.0chr11:5,248,243 401 504760 99.9 1039 526882 99.80 47323 52626 52.7AMPLICON 2Mutation WT MT percent WT MT percent WT MT percent chr11:5,248,232 284777 267010 48.4 210305 171594 44.93 33602 39284 53.90chr11:5,248,233 538543 13244 2.4 364673 17226 4.51 72876 10 0.01chr11:5,248,243 520 551267 99.9 968 380931 99.75 33577 39309 53.93AMPLICON 3Mutation WT MT percent WT MT percent WT MT percent chr11:5,248,232 718 676 48.49 265723 227082 46.08 43767 49798 53.22chr11:5,248,233 1367 27 1.94 472809 19996 4.06 93554 11 0.01chr11:5,248,243 4 1390 99.71 90 492715 99.98 43687 49878 53.31
Normalised data – amplicons 1, 2, & 3
5% AC 8% AC
Affected (SS) fetus
Normalised data - Amplicon 3, S mutation
run 8 6% C run 8 S-13 run 8 S-14 run 8 S-15 run 9 S-16 run 9 S-17 run 9 S-18 run 9 S-19 run 10 S-20
run 10 S-21
run 10 S-23
S -3.10982618899741
-0.328583749901505
2.2088615375608
-0.193537285569704
0.979175505854898
4.105698126193
-1.1207197
508907
5.5319957511713
-0.954112492053398
-3.64372093820231
-3.4661413
524661
-5.00
-3.00
-1.00
1.00
3.00
5.00
7.00
Vari
ation
from
con
trol
of %
mut
ant
AS
SS
AA
AA
AS
SS
AS AS
SS
AS
SS
AS AA AA
Conclusions
Successful proof of principle, spiking experiments and real samples – it works!
Cost effective and rapid:-New bench top NGS analyserTargeted sequencing approach rather than whole genome.Simple amplicon based library prep – low cost and quick
Next stage – proper clinical evaluation, validation etc.
Summary
• cff-DNA is a potential “safe” source of fetal DNA for PND• NIVPND in routine use for a small number of conditions
where allele in fetus is not present in the mother• NIVPND currently possible for aneuploidy – but
expensive and not widely available• In development for single gene disorders – new lower
cost NGS platforms hold great promise, NIVPND could become a cost effective reality in routine molecular diagnostic laboratories.
Acknowledgements Oxford BRC molecular diagnostic lab Illumina
Pauline Robbe David McBride
Alice Gallienne Mark Ross
Adele Timbs
Helene Dreau
Jenny EglintonMichelle Rugless
John Old
Anna Schuh
Acknowledgements
Oxford BRC Genomics
Jenny Taylor
Sam Knight
Chris Yau
Chris Holmes
Oxford BRC/NHS SIHMDS
Anna Schuh
John Old
Adam Burns
Ruth Clifford
Adele Timbs
Oxford Hematology/OncologyChris HattonTim LittlewoodParesh VyasBass HassanMark Middleton
ORB Tissue BankingMaite Cabes
Research NurseChristopher Levett
UK NCRN CLL SubgroupPeter HillmenAndy Pettitt
Illumina
David Bentley
Mark Ross
Jennifer Becq
Sean Humphray
David McBride
Our Patients
Oxford PathologyRunjan ChettyLiz Soilleux
Clinical (Bio-)InformaticsJim DaviesJoe WoodMinji DingJean Baptiste CazierMichalis Titsias
EUROSARC
Normalised data for run 7
5% AC
8% AC
15% AC
25% AC
Spiking experiments
5% AC8% AC15% AC25% ACAS (50:50) controlAS-08 S-06 S-07 S-08 S-09
AMPLICON 1Mutation WT MT percent WT MT percent WT MT percent WT MT percent WT MT percent S (chr11:5,248,232) 138103 124960 47.50 160738 65332 28.90 190247 100634 34.60 157339 104587 39.93 130690 97033 42.61C (chr11:5,248,233) 262803 260 0.10 183017 43053 19.04 253039 37842 13.01 242327 19599 7.48 219177 8546 3.75Polymorphism (chr11:5,248,243) 1554 261509 99.41 46527 179543 79.42 42535 248346 85.38 20612 241314 92.13 10796 216927 95.26AMPLICON 3
Mutation WT MT percent WT MT percent WT MT percent WT MT percent WT MT percent
S (chr11:5,248,232) 103820 104772 50.23 110555 53372 32.56 151079 89028 37.08 153617 113229 42.43 129273 109119 45.77C (chr11:5,248,233) 208250 342 0.16 134813 29114 17.76 210634 29473 12.27 249010 17836 6.68 229872 8520 3.57Polymorphism (chr11:5,248,243) 786 207806 99.62 29622 134305 81.93 31030 209077 87.08 20229 246617 92.42 10215 228177 95.72
A1 oxford run A1 chesterford run A3 oxford run A3 chesterford run
S-06 18.5435006989362 18.9185459339253 17.5489133509599 18.1042524334683
S-07 12.7455905042139 13.0776035788019 12.0961897763203 12.2489157930951
S-08 7.29080250965705 7.42541211021849 6.58635681179484 6.69885630004798
S-09 3.59210255467939 3.74513224970838 3.40165970438794 3.43959389030448
1.00
3.00
5.00
7.00
9.00
11.00
13.00
15.00
17.00
19.00
% m
utan
t rea
ds o
n ch
r11:
5,24
8,23
3
Reproducibility comparison histograms of run 7
5% AC
8% AC
15% AC
25% AC
6% C 2005 2091 3258 3324 2091 2005 G126696 4747 2600 1973 1971
Am-plicon 3
46.836396043220
2
49.665734099545
6
52.247059055882
49.803115902561
2
51.000243262376
7
54.194035556978
48.855167053160
3
55.651021134127
3
49.004209713063
9
46.197113077559
3
4.9352411459715
2
46.382449741848
8
41.00
43.00
45.00
47.00
49.00
51.00
53.00
55.00
57.00
59.00
Amplicon 3
Axis Title
AA AS SS AS AS SS AS SS AS AA AA?
AS
SS
AA