natalie chandler · ultrasound to confirm gestation, identify multiple pregnancies or empty sacs...

Clinical implementation of Non-invasive prenatal diagnosis (NIPD) for single

gene disorders Natalie Chandler

Senior Clinical Scientist, London North Genomic Laboratory Hub cfDNA 2019 – May 24th 2019

Natalie Chandler

London North Genomic Laboratory Hub

Great Ormond Street NHS Foundation Trust

London, UK

Clinical implementation of Non-invasive prenatal diagnosis (NIPD) for single

gene disorders

Disclosure information: Nothing to declare

Cell-free fetal DNA

Issues

• Relative abundance of maternal cfDNA

• Emanates from the placenta - risk of mosaicism?

• Limitations for use in multiple pregnancies

• When used in early pregnancy, issues associated with ‘vanishing twin’

• Originates from placenta (trophoblast)

o represents whole fetal genome

• Detectable from 4 weeks

o % increases with gestation

o 10-20% of total cfDNA

o Cleared from circulation within 30mins of delivery

Non-invasive prenatal … definitions

NIPD - Diagnosis

Monogenic disorders, fetal sex, RHD

Does not require invasive test for confirmation

NIPT or NIPS

Testing or Screening – aneuploidy

Risk of confined placental mosaicism

‘Screens’ all cfDNA

Requires invasive test for confirmation

NIPD for single gene disorders – where marker/ mutation not present in mother

Test that we offer:

Non-Invasive Fetal sex determination – service started in 2007, report around 17 cases per month – 3 day TAT

FGFR3-related skeletal dysplasia including achondroplasia and thanatophoric dysplasia – service started in 2013, at least 1 run a week – 5 day TAT

FGFR2-related craniosynostosis syndromes e.g. Apert & Crouzon

10 common cystic fibrosis mutations – Paternal exclusion test, low uptake

NIPD bespoke service for paternal and de novo mutations

• 30% of all our molecular prenatal diagnosis since 2015 have been non-invasive

>7 weeks

Ultrasound to confirm gestation, identify multiple pregnancies or empty sacs

qPCR assay

Probes specific to Y chromosome

~17 cases/mnth

Sensitivity 99.5% (95% CI 98.2–99.9%)

Reduces invasive testing rate by ~50%

Economic analysis showed NIPD was no more expensive than IPD (Hill et al, 2011)

NIPD – quantitative PCR for fetal sex determination

Male

Female

CCR5

SRY

SRY CCR5

FRAS1 exon 66

hg19_dna range=chr4:79437011-79437070

ctccctccctggcagAGGCAGGGTTCCTGGATGATGTGGTCTATGATAGCACTGCCCTGGGGCCTGGCTACGATCGCCC

CTTCCAGTTTGACCCCAGCGTGCGAGAGCCGAAGACCATCCAGCTCTACAAACACCTGAACCTGAAGAGCTGCGTGTGG

ACCTTTGATGCTTATTATGACATGACTGAGCTGATTGACGTCTGTGGGGGCTCTGTAACCGCTGACTTCCAGgtaggtg

ccccggggcttgtctgaggactctgc

Amplicon Based Design – panels & bespoke service

C primer

Re familial mutation

G exon

Sample index - 6bp

Illumina sequence adaptor (MiSeq)

60bp

Bioinformatic Analysis

Automated scripting to count wild type and mutant sequences

Monitoring sample indexes for potential contamination

Poor quality data is filtered out ID6 ID7 ID8

cffDNA (1) cffDNA (2) Mat gDNA FRAS1 wt 319077 141751 440538

FRAS1 c.10261C>T 34498 12055 292

ZFX 36743 44017 350940 ZXY 6 0 2

Bioinformatic Analysis

Confirmation of Fetal DNA ZFX/Y, HLA and SNP assays are run in parallel to confirm presence of cffDNA in the same plasma extract undergoing mutation test: • Calculate fetal fraction • Important in mutation negative cases

ID01 ID08 ID02

cfDNA cfDNA gDNA

HLABexon3-VAR01 0 1 2

HLABexon3-VAR02 3509 2734 2805



HLABexon3-VAR05 4108 3582 2876




ZFX 27046 48258 55986

ZFY 764 1456 6

Apert wt 97397 163414 97801

Apert c.755C>G 15 15 13

Apert c.758C>G 16 26 8

Apert c.755_756delCGinsTT 0 2 0

FETAL FRACTION ID01

VARIANT Counts

Maternal allele 1: VAR#2 3509

Maternal allele 2: VAR#5 4108

TOTAL maternal counts: 7617

VARIANT Counts

Fetal HLA variant: VAR#7 336

Fetal Fraction: 8.45

Next generation sequencing – FGFR3 panel

5 amplicons

29 mutations

4 ACH

12 TD1, 1 TD2

1 Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN)

9 Hypochondroplasia

2 Craniosynostosis (p.Pro250Arg/Leu)

Screens multiple mutations in one test

Digital readout

742C>T Arg248Cys TD1

746C>G Ser249Cys TD1

749C>T Pro250Leu CRANIO

749C>G Pro250Arg CRANIO

1108G>T Gly370Cys TD1

1111A>T Ser371Cys TD1

1118A>G Tyr373Cys TD1

1123G>T Gly375Cys ACH

1130T>G Leu377Arg ACH

1138G>A Gly380Arg ACH

1138G>C Gly380Arg ACH

1142T>A Val381Glu HCH

1619A>C Asn540Thr HCH

1619A>G Asn540Ser HCH

1620C>A Asn540Lys HCH

1620C>G Asn540Lys HCH

1948A>C Lys650Gln HCH

1948A>G Lys650Glu TD2

1949A>C Lys650Thr AN

1949A>T Lys650Met SADDAN

1950G>C Lys650Asn HCH

1950G>T Lys650Asn HCH

2419T>A *807Argext*101 TD1

2419T>G *807Glyext*101 TD1

2420G>C *807Serext*101 TD1

2420G>T *807Leuext*101 TD1

2421A>C *807Cysext*101 TD1

2421A>G *807Trpext*101 TD1

2421A>T *807Cysext*101 TD1 Chitty et al. Prenatal Diagnosis 2015, 35:656-62

NIPD for FGFR3 mutations 2013 – 2016 Diagnostic yield by indication

Achondroplasia: Normal limb length <24 wks, shortening >24 wks, relative macrocephaly, frontal bossing, short fingers, polyhydramnios

Thanatophoric dysplasia: Very short limbs from first trimester, short ribs and small chest, polyhydramios, frontal bossing, cloverleaf skull, short fingers

Referral reason Number of FGFR3 NIPD Number positive

Short limbs only 168 28 (17%)

Two ultrasound features 50 28 (56%)

>two features 19 19 (95%)

Affected relative 6 2 (30%)

Previously affected (germ-line mosaic risk)

66 0

Paternal age 1 0

Head circumference

Femur length

achondroplasia

thanatophoric

Phenotyping is key in maximising diagnostic yield

Inclusion criteria 1-3 base pair mutation

gDNA from affected proband or carrier parent

De Novo / Paternal Dominant

For recessive conditions, parents must carry different mutations

Target Population Couples with a pregnancy at risk of a genetic condition where the mutation is known and invasive prenatal diagnosis would otherwise be the only option

Exclusions Pseudogene Polymorphisms

Bespoke NIPD

cffDNA testing in pregnancy

Pre-pregnancy work-up preferred

Minimum 9 weeks gestation

Maternal sample run in parallel

Audit of bespoke service delivered at GOSH Phenotype Gene Mutation

Tuberous sclerosis TSC2, TSC1 c.133_134delCT, c.1525C>T, c.2713C>T c.4351dupC, c.4645T>A

Neurofibromatosis type 1

NF1 c.4823T>C, c.4330A>G c.5170C>T, c.1748A>G

Rhabdoid Tumour Predisposition Syndrome

SMARCB1 c.157C>T c.118C>T

Epileptic encephalopathy, early infantile

SCN8A KCNQ2 SCN1A SCN2A

c.669G>T c.431G>A c.5726C>T c.2627A>C

Skeletal Dysplasia

FGFR3 TCF12

SLC26A2 COL2A1

SOX9

c.779C>G c.1916del

c.296T>C (AR) c.2104G>A c.332C>T

Fraser syndrome (AR) FRAS1 c.10261C>T (x4), c.10261C>T

Battens TPP1 c.509-1G>C

Noonan syndrome PTPN11 c.178G>A, c.923A>G

Porencephaly COL4A1 c.324+1G>A

Marfan syndrome FBN1 c.8268G>A, c.1285C>T, c.5911T>G

Polycystic kidney disease PKHD1 c.1486C>T (AR)

Alpers syndrome POLG c.2542G>A

Gorlin Syndrome PTCH1 c.443del

Carney complex PRKAR1A c.124C>T

Von-Hippel Lindau VHL c.499C>T

Brain malformation disorders EIF2B5 (AR) COL4A1

c.241G>A c.324+1G>A

Phenotype Gene Mutation

Intellectual disability ARID1B KDM5B BCAP31

CASK KAT6A PACS1

c.1488C>A, c.6129del, c.3343C>T c.2359T>C

c.678+1G>A c.37G>T

c.4213G>T c.607C>T

Congenital Nephrotic WT1 c.1301G>A

Cornelia De Lange NIPBL c.4160dupA

Osteogenesis imperfecta COL1A1 COL1A2

c.543+4A>T, c.543+4A>T, c.2299G>A, c.1875+1G>A c.1801G>A, c.1801G>A x3

Renal tubular dysgenesis ACE c.96del (AR)

Epider Palmoplantar Keratoderma

KRT9 c.482A>G, c.482A>G

Hereditary Spastic Paraplegia SPAST c.1361A>G

Familial dilated cardiomyopathy ACTC1 TPM1

c.664G>A c.742A>G

Alexander disease GFAP c.235C>A

Laron syndrome (AR) GHR c.922G>A

MECP2-related disorder MECP2 c.148_152del

Mitochondrial disorders POLG AIFM1

c.2542G>A (AR) c.603_605del, c.1715G>A (AR)

Metabolic disorders OTC ADSL TPP1

GELDC

c.608C>G c.153+1G>T (AR) c.509-1G>C (AR) c.2879G>A (AR)

Eye disorders OTX2 c.534C>A

Immunological disorders STAT3 c.1853G>A

Congenital myopathy DNM2 ACTA1

c.1856C>T c.478C>A

102 bespoke tests offered

23 other conditions worked up

20/102 mutation positive

Challenge: fetal DNA present amongst high background of maternal

Definitive NIPD for autosomal recessive conditions

cffDNA fraction directly affects expected extent of allelic imbalance

Relative mutation dosage-RMD

Definitive NIPD for recessive conditions Linkage analysis and RHDO

High relative concentration of maternal cfDNA complicates the assay requiring linkage analysis when parents carry the same mutation or when the paternal allele has been inherited.

Target SNPs flanking the relevant gene

Genotype maternal, paternal & proband or unaffected sib

Establish linkage of SNPs to the mutation

Use heterozygous SNPs to determine fetal fraction

Maternal

Paternal Proband

Lo et al 2010 Sci Trans Med; New et al 2014 J Clin Endo Metab

RHDO – Cystic Fibrosis Service

Cases (n)

Outcome Gestation (wks)

Fetal Fraction (%)

Comment

10 Both high risk, affected 9 – 10 5 – 14.4%

7 Both low risk, unaffected 9 – 9+4 4.8 – 19%

20 One high risk, unaffected carrier

9 – 14 9 – 19.8%

6 Maternal allele inconclusive Paternal allele low risk

9+2 – 9+6 2 – 6.4% Reported as unaffected carrier

2 Maternal allele inconclusive Paternal allele high risk

9+5 – 10+4 3 – 6.7% Repeated and both maternal allele low risk

1 Maternal allele high risk Paternal allele inconclusive

10+3 10.4% Inconclusive – likely recombination event on paternal allele. Invasive testing required

1 Both maternal & paternal inconclusive

9+3 undetermined No informative SNPs 3’ of gene. ?Consanguineous

Gene dossier approved for clinical use in the NHS in late 2016

47 pregnancies referred for CF NIPD in 2 ½ years compared with ~ 2 per annum invasive tests

We are currently working up RHDO services for CAH, SMA & haemophilia. DMD service is also available in the Birmingham laboratory in the UK.

Bringing RHDO services into Clinical service is costly and requires a large number of samples for validation. Limited to common genetic conditions.

Not able to offer testing to consanguineous families at present

Requires samples from the father and previous child / pregnancy – not always available

- Published proband free RHDO methods are available but at present they are too costly for clinical implementation

Limitations of RHDO

Challenge

fetal DNA present amongst high background of maternal

NIPD for autosomal recessive conditions

cffDNA fraction directly affects expected extent of allelic imbalance

Relative mutation dosage-RMD

Droplet digital PCR (ddPCR)

Relative mutation dosage assays for mutations carried by the mother

X linked example: • Mother carrier of F8 mutation c.6046C>T p.(Arg2016Trp) which causes haemophilia • Fetal fraction 20% obtained by Y chromosome marker • Statistical increase observed in mutant droplets over wild type • Fetus predicted to be affected

Blue = mutant droplets; Green = wild-type droplets

NIPD for mutations carried by the mother

Sickle Cell Disease • 12 ddPCR replicates • Analysis using Poisson correction • Examine ratio of mutant droplets to wild type droplets

3.9%

Heterozygous Homozygous mutant Homozygous wild-type Fetal genotype

Fetal fraction 6.1% 6.8%

• Our assays show that this approach is feasible

• Technically challenging assays to design and optimise

• Currently working on optimising assay design as well as analysis pipelines

• To bring to clinical service – sensitivity & specificity needs to be similar to invasive testing

• May not possible at samples with low fetal fraction (<4%). Sample at a later gestation?

• Fetal fraction determined by Y specific marker in male pregnancies. How can we accurately quantify fetal fraction in female pregnancies?

Relative mutation dosage assays for mutations carried by the mother: The challenges to be addressed

NIPD service delivery in the UK – where are we now?

Condition Tested Fail/ inconclusive

Affected

Achondroplasia 280 5 63

Thanatophoric dysplasia 122 5 37

Apert syndrome 24 0 5

Cystic fibrosis (paternal exclusion) 17 1 7*

Cystic Fibrosis (RHDO) 37 2 8

Crouzon syndrome 15 0 7

Bespoke NIPD 87 **6 not possible 19

Total 582 18 (3%) 146

*one false negative At GOSH >32% of all molecular genetic prenatal diagnosis is NIPD (67% if including fetal sex)

West Midlands regional Genetics Laboratory Spinal Muscular Atrophy, Duchenne and Becker Muscular Dystrophies

NIPD service delivery at GOSH

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Conclusions

Technical challenges

• Delivering an accredited NIPD service for known paternal or de novo mutations is relatively straightforward.

• Expanding the service to make NIPD available for conditions where the mother is a carrier is challenging, time consuming and costly.

• Relative haplotype dosage analysis methods have been successful in clinical service but this approach has its limitations

• Relative mutation dosage assays show potential but more work is required to bring in to clinical service

Acknowledgments

NETRGL Lyn Chitty Fiona McKay Joseph Shaw Sandra Moore Natalie Chandler Helena Ahlfors

Funding NETRGS RAPID NIHR PGfAR GOSH CC Charity GOSH BRC

The work described here was partially funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research Programme (RP-PG-0707-10107 – “RAPID”) and the GOSH BRC. The views expressed are those of the author and not necessarily those of the NHS, the NIHR or the Department of Health.

Research Team Sophie Sheppard Rhiannon Mellis Melissa Hill

natalie chandler · ultrasound to confirm gestation, identify multiple pregnancies or empty sacs...

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