1. Sue Hamilton West Midlands Regional Genetics Laboratory
PAGE2 – Whole Exome Sequencing (WES) of foetal or early neonatal deaths with prenatally identified structural anomalies.
Sue Hamilton
West Midlands Regional Genetics Laboratory

2. PAGE project Includes Social Science, Ethics and Health Economics
Striving for a better understanding of genetic variants
causing developmental problems during pregnancy.
Aiming to improve prenatal diagnostics and parental counselling in the future
Includes Social Science, Ethics and Health Economics
Two “arms” co-ordinating samples being sent to Wellcome Trust Sanger Centre through Birmingham and Great Ormond Street Centres
Health Innovation Challenge Fund, Department of Health and Wellcome Trust

3. The three primary objectives of the PAGE project are to:
Elucidate the relative contribution of different forms of genetic variation to prenatal structural anomalies
Design cost-effective genome sequencing assays for improved prenatal diagnosis of structural anomalies
Catalyse the adoption by the NHS of prenatal diagnostic sequencing through translation of acquired know-how, rigorous health economic assessment and establishment of an ethical and social science framework for clinical implementation

4. PAGE2
PAGE2 is a separate part of the project run solely through the Birmingham arm of the PAGE project
Referred via the West Midlands Perinatal Pathology Service after PM for structural anomaly in any fetal loss (including TOP) or early NND
from the West Midlands Fetal Medicine Centre at Birmingham Women’s Hospital
from other Fetal Medicine Centres once seen by the West Midlands Regional Genetics Service
Samples had either had Chromosomal Microarray (or sub-telomeric MLPA) testing with no clinically significant CNVs detected
Discussed at local MDT and recruitment suggested

5. PAGE2 recruitment
Requirements - sufficient DNA from stored material, parental DNA
Parents approached by letter to inform of study and offer to recruit
Parental bloods or saliva samples collected at time of recruitment.
Trio DNA forwarded to Sanger Centre for WES includes further DNA quality checks

6. Bioinformatics (Sanger)
BAM
GATK
+ DNG
CONVEX
+ CIFER
UPDio
MrMosaic
INDELible
SNVs/indels
CNV
UPD
Mosaic SV
Complex SV QC QC QC QC QC
Combined VCF
Supplementary File
Annotation
Annotated Combined VCF
Annotation
Clinical Filtering

7. Clinical Filtering – PAGE gene list
DDG2P list
Exclusion list - unrelated to antenatal ultrasound findings
Deafness
Blindness
Behavioural traits
Intellectual disability
Additional relevant genes with prenatal phenotype
All cases re-analysed at each new data freeze

8. Clinical interpretation
Uploaded into Sapientia database along with HPO terms related to phenotype for fetus.
Reviewed using ACMG guidelines
Discussed at a Clinical Review Panel (CRP)
Agree on whether relevant for validation

9. PAGE Clinical Review Panel (CRP)
A UK-wide virtual MDT
1x Clinical Scientist, 1x Clinical Geneticist / centre
WTSI Scientists, Fetal Medicine / Pathology
Agenda produced >2 weeks before review
WebEx/Telecon mediated
Assessing + classifying variants
Recording actions + evidence
Request further information

10. PAGE2 cases to date
55 trios were recruited to the study, 2 were subsequently excluded as a diagnosis was obtained prior to samples being ready to send for WES (both PTPN11 Noonan syndrome – testing after MDT review).
30 have completed the WES process to date
11 diagnoses have been obtained (36.7% detection rate)
10 different genes
4 de novo AD
3 homozygous AR
3 compound heterozygous AR
1 X-linked

11. Results 20 cases reviewed by CRP 11 diagnostic cases
9 cases where variant detected was classified as a VUS with no clear contribution to the fetal phenotype

12. ACMG guidelines & de novo KMT2D variants
EGA ID
VEP consequence
ExAC
Presenting Phenotypes (USS)
EGAN
stop_gained
Multisystem
EGAN
Hydrops
Postnatal features of Kabuki Syndrome
Pulmonary hypoplasia (HP: )
transposition of great arteries, mitral valve atresia (HP: )
Incomplete intestinal rotation, hypoplastic gall bladder (HP: )
Single horseshoe kidney (HP: )
De novo STOP_GAINED KMT2D

13. ACMG guidelines & de novo KMT2D variants
EGA ID
VEP consequence
ExAC
Presenting Phenotypes (USS)
EGAN
stop_gained
Multisystem
EGAN
Hydrops
HP:
Cleft palate
YES
HP:
Abnormality of the thorax
HP:
Abnormal heart morphology
HP:
Transposition of the great arteries
HP:
Hypoplastic left heart
HP:
Abnormality of the gastrointestinal tract
Recurrent Kabuki features

14. ACMG guidelines & de novo KMT2D variants
EGA ID
VEP consequence
ExAC
Presenting Phenotypes (USS)
EGAN
stop_gained
Multisystem
EGAN
Hydrops
PM2
PVS1
PS2
Absent from controls
De novo with fitting phenotype
Null variant
PATHOGENIC

15. Case Study 1
Consanguineous couple with history of stillbirth at 35 weeks, miscarriage at 16 weeks and one live birth
Ultrasound at 26/40 showed:-
minimal fetal movements
severely abnormal posture
mild bilateral hydrothoraces
Fetal Akinesia Deformation Sequence (FADS)
Opted for TOP and had PM, discussed at MDT
Recruited to PAGE2

16. Case Study 1
Homozygous variant in ERCC5, endonuclease involved in excision repair
c.2766dupA, p.Leu932Thrfs*7
Assessed as pathogenic variant, causative of phenotype
Cerebro-oculofacial syndrome
Arthrogryposis
Microcephaly
IUGR
Validated by Sanger sequencing in NHS diagnostic laboratory
Fed back to patient who at appointment revealed she was currently pregnant – CVS showed affected fetus

17. Case study 2
Ultrasound at 20/40 severe fetal hydrops, male fetus, parents opted for TOP
FOXP3 variant (c.1189C>T, p.Arg397Try)
X-linked immunodysregulation, polyendocrinopathy and enteropathy
Assigned pathogenic status, causative of phenotype
Validated and reported back to family
Family studies indicated several carriers in the family and findings explained similar affected male pregnancies

18. Reclassification by system following PM
Classification by prenatal ultrasound alone
Classification following Post Mortem

19. Conclusions Important patient group for exome sequencing
High detection rate (36.7%)
Additional information from post mortem examinations has assisted in assessing phenotype in a prenatal context
All helps in defining prenatal phenotype in developmental disorders where this is currently unclear
Correlation with “partial” phenotypes will be important for use of exomes in a prenatal context

20. Mothers and their partners
Lyn Chitty
Jane Fisher
Matt Hurles
Eamonn Maher
Mike Parker
Mark Kilby
Dom McMullan
Tamas Marton
Phillip Cox
Beata Hargitai
Andre Coetzee
Georgina Carey
Liz Quinlan-Jones
Clive Gould
Denise Williams
Rebecca Keelagher
Lab
Elena Prigmore
Bioinformatics
Ruth Eberhardt
Analysis
Jenny Lord
Project Management
Gabi Rinck
Mothers and their partners
My colleagues in the Prenatal and Reproductive Medicine Team, WMRGL
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