non-invasive molecular prenatal diagnosis

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12/06/2022 Non-invasive molecular prenatal diagnosis Oxford BRC molecular diagnostic laboratory Dr Shirley Henderson Consultant Clinical Scientist

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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 Presentation

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Page 1: Non-invasive molecular prenatal diagnosis

19/04/2023

Non-invasive molecular prenatal diagnosis

Oxford BRC molecular diagnostic laboratory

Dr Shirley HendersonConsultant Clinical Scientist

Page 2: Non-invasive molecular prenatal diagnosis

Conventional approach to Prenatal Diagnosis

Ultrasound

Fetal sampling

Chorionic villi Amniotic fluid

DNA diagnostic test

Page 3: Non-invasive molecular prenatal diagnosis

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

Page 4: Non-invasive molecular prenatal diagnosis

• 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?

Page 5: Non-invasive molecular prenatal diagnosis

Plasma DNA approach

Double spin plasma to remove any remaining white cells and platelets

DNA extraction

Cell free DNA

Page 6: Non-invasive molecular prenatal diagnosis

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

Page 7: Non-invasive molecular prenatal diagnosis

Non-invasive fetal sex determination

Female plasmaMaternal plasma with male fetus

Maternal plasma with female fetus

xx

xx

xx

xx

xx

xx

xx

xx

xx

xx

xx

xxxx

xx

xx

xx

xx

xx

xx

xx

xxxy

xy

xx

xx

Method used to detect the y chromosome, usually RT-PCR

Some success stories – all when allele not present in the mother

Page 8: Non-invasive molecular prenatal diagnosis

• 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:-

Page 9: Non-invasive molecular prenatal diagnosis

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

Page 10: Non-invasive molecular prenatal diagnosis

• 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

Page 11: Non-invasive molecular prenatal diagnosis

Sickle cell disease - first molecular disease

DNA-Sickle mutation ProteinPathophysiology

Inheritance Prevalence Immigration

Page 12: Non-invasive molecular prenatal diagnosis

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

Page 13: Non-invasive molecular prenatal diagnosis

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

Page 14: Non-invasive molecular prenatal diagnosis

?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.

Page 15: Non-invasive molecular prenatal diagnosis

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

Page 16: Non-invasive molecular prenatal diagnosis

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

Page 17: Non-invasive molecular prenatal diagnosis

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

Page 18: Non-invasive molecular prenatal diagnosis

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

Page 19: Non-invasive molecular prenatal diagnosis

• 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

Page 20: Non-invasive molecular prenatal diagnosis

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

Page 21: Non-invasive molecular prenatal diagnosis

Normalised data – amplicons 1, 2, & 3

5% AC 8% AC

Affected (SS) fetus

Page 22: Non-invasive molecular prenatal diagnosis

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

Page 23: Non-invasive molecular prenatal diagnosis

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.

Page 24: Non-invasive molecular prenatal diagnosis

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.

Page 25: Non-invasive molecular prenatal diagnosis

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

Page 26: Non-invasive molecular prenatal diagnosis

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

Page 27: Non-invasive molecular prenatal diagnosis
Page 28: Non-invasive molecular prenatal diagnosis

Normalised data for run 7

5% AC

8% AC

15% AC

25% AC

Page 29: Non-invasive molecular prenatal diagnosis

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

Page 30: Non-invasive molecular prenatal diagnosis

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

Page 31: Non-invasive molecular prenatal diagnosis

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