implementation of the ddd/clingen ogt (cytosure v3) microarray · west midlands regional genetics...

West Midlands Regional Genetics Laboratory

Implementation of the DDD/ClinGen OGT (CytoSure v3) Microarray

OGT UGM

Birmingham 08/09/2016

Dom McMullan

Birmingham Women's NHS Trust


WM chromosomal microarray (CMA) testing

• Population of ~6 million (10%) • Chromosomal microarray (CMA)

– First-line test for all DD patients – >5,000 per annum plus 15% growth – Prenatal diagnosis (structural anomalies) – Fetal pathology


Typical DD Pathway until 2015

CMA +/- single gene testing

Deciphering Developmental Disorders (DDD)

12,000 trio exomes +/- ultra-high res arrayCGH


Typical DD Pathway from 2016?

CMA Clinical Exome (filtered)

100,000 whole genomes


Balancing factors for platform choice in Clinical Genomics

• Cost • Detection rate • Ease of analysis • Throughput

• Discovery less important ?


CMA platform choices

Lowest cost, highest detection rate Available platforms within UK cost envelope: • OGT / Agilent based 8x60k design (aCGH) • Affymetrix CytoScan 750k (SNP+CN based) • Illumina CytoSNP 8x850k (SNP based)


DDD project • Transformed Clinical Genetics in UK

– DECIPHER – 12,000 exomes back to NHS – Ended diagnostic odyssey – Discovered >12 “new” genes – Expedited interpretation skills – the Genomic MDT – DDG2P evidence based gene list


Evidence Bases for new OGT (v3.0) DDD array

• DDG2P gene list (n=>1,400) – Disorder, Mechanism, Consequence and HI scores

https://decipher.sanger.ac.uk/ddd#ddgenes • ISCA/ClinGen dosage sensitivity map

– http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/ • ISCA/ClinVar submitted clinical cases • DECIPHER recurrent syndromes

• OGT v3.0 60k CNV, 180k CNV and 180k CNV+SNP designs

https://decipher.sanger.ac.uk/ddd#ddgenes

http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/


DDG2P centric gene targeting • ~300 Monoallelic / LoF genes at single exon resolution • ~200 Monoallelic missense /uncertain genes at gene level • Additional coverage in DECIPHER curated syndrome regions • Evidence based “backbone” (ISCA/ClinGen pathogenic + HI score)

Tier 1; ~120kb HI gene rich / reported pathogenic CNVs Tier 2; ~300kb Tier 3; ~500kb agenic / no reported pathogenic CNVs Theoretical resolution based on 3-probe calling


Validation • Bank of 1,000s of known abnormal cases • CNVs at limits of detection previously

– E.g. 2 probe calls • Mosaics • Many samples have data for:

– BlueGnome / Illumina 60K ISCA v2.0 – Affymetrix CytoScan-HD and 750K SNP array – Illumina CytoSNP SNP bead array 850K

• CHALLENGING CASES


DLR spread comparison; 48 sample validation

Mean DLR: OGT v3 DDD = 0.115, Illumina v2 = 0.158


Deletion at 1q41-q42

• 8.7Mb deletion; mean log2 ratio, -0.9 • Highly consistent probe behaviour; all 119 probes < -0.6


PDHA1 deletion (Xp22.2)

• 35 probes on v3 array • Multi-exon 13kb deletion


MEF2C deletion (5q13.4)

• Manually called and validated from ISCA v2.0 array (2 probes only) • 36 probes called on v3 array • Minimum size 68kb; multiple exons


CREBBP (Rubenstein-Taybi) deletion (16p22.2)

• Manually called and validated from ISCA v2.0 array (2 probes only) • 9 probes called on v3 array • Minimum size 2.2kb; 2 exons


MID1 (Opitz-G) intragenic duplication (single exon)

• Previous detection by 2x105k OGT X-array • 4 probes called on DDD array • Minimum size 500bp (single exon)


MID1 (Opitz-G) intragenic duplication (single exon)

• Previous detection by 2x105k OGT X-array • 4 probes called on v3 array • Minimum size 500bp (single exon)


Investigations • 46 XY • 22q- FISH: NAD (brothers) Opitz-G? • MID1 seq ( Naples 2006): NAD • 1Mb BAC array: NAD • X/Y tiling BAC array: NAD • OGT X exon array; intragenic dup • DDD aCGH: NAD • DDD exome: intragenic dup


TBX5 12q24 deletion (Holt-Oram)

• Minimum 450bp • 3 probes, 1 exon


Mosaic trisomy 18?

Low level mosaic on peripheral blood in IUGR neonate Confirmation on buccal swab

17 19 18


Mosaic trisomy 18?

Low level mosaic on peripheral blood in IUGR neonate Confirmation on buccal swab

17 19 18 Aneuploidy summary plot


Validation of CNVs from DDD


Validation of CNVs from DDD

• 12,000 undiagnosed UK trios recruited – Majority had routine aCGH in local centres

• WES and ultra-high resolution aCGH at Wellcome Trust Sanger Institute, Cambridge

• Plausible pathogenic data fed-back to local clinical/laboratory teams for validation

• 1200 trios from WM region – 300 variants so far (SNVs and CNVs)


DDD Research Report


Array-CGH (2x1m Agilent custom) De novo 1.27kb deletion


HDAC8 deletion confirmation

• 1.17kb deletion (minimum) • 2 exons, 7 probes


Predictive comparison in UCSC

Platform

Min Max

OGT /DDD v3.0 ✔ ✔

Illumina 850k ✗ ✗

CytoScan HD ✗ ✗


DDD validation Case 2: History

• Intellectual disability (severe) • Autistic • Self-mutilation • Ptosis


DDD Research Report


De novo 3.8kb

Array-CGH (2x1m Agilent custom) De novo 3.8kb deletion


MED13L deletion confirmation

• 269bp deletion (minimum) • 1 exon (9), 3 probes


MED13L deletion confirmation

• 269bp deletion (minimum) • 1 exon(9), 3 probes


Predictive comparison in UCSC

Platform

Min Max

OGT /DDD v3.0 ✔ ✔

Illumina 850k ✗ ✗

CytoScan HD ✗ ✗


DDD validations

• Genotype-phenotype discussion in local MDT • Both de novo cases fit clinical phenotypes • Clinical reports issued • Rapid, cost-effective validations

• Answers would have been possible for families

years previously via aCGH if DDD array had been available…..


Prospective exonic CNV diagnoses

(OGT/DDD array implemented April 2016)


Single exon deletion (1.2kb); ARSE

Dysmorphic Skeletal anomalies Parental samples requested Chrondrodysplasia Punctata (X-linked)


Single exon deletion (1.2kb); ARSE


Single exon (1kb) PTPN11

• dysmorphic • ?VSD/ASD • puffy hands/feet • short 5th fingers

• Parental samples

requested


Single exon (1kb) PTPN11


PTPN11; DECIPHER


Single exon (1.1kb) GRIN2A

• ILD • Motor delay

• DE NOVO


Balancing factors

• Exonic deletions • SNPs: AOH/UPD


Application of v3 4x180K SNP array • Unusual case of autosomal recessive Bartter syndrome • Clinician; “Homozygous deletion via UPD event? ” • Bartter syndrome type 4B

– Renal salt wasting – Deafness – CLCNKA and CLCNKB

• Bartter syndrome type 3 – Renal salt wasting – CLCNKB


Chr1 (whole chromosome)


Chr1 (whole chromosome)

Regions of UPD (AOH)


Homozygous deletion in region of UPD 1p36.13


Algorithmic deletion call

Deleted probes only mapped to CLCNKB;

NO probes in CLCNKA

Homozygous deletion in region of UPD 1p36.13


OGT/DDD v3 180k SNP Beta test data


OGT/DDD v3 180k SNP Beta test data

CLCNKA not deleted • No deafness • Bartter Type 3


Summary • OGT/DDD 8x60k implemented all referrals • Single exon deletions can be detected if targeted • Will detection rate increase?

– 3/400 (0.75%) WM DDD cases have pathogenic single exon changes – >7/1500 (>0.5%) prospective cases missed previously?

• How much has been missed? • High density does not equate to high resolution • New genes = future versions? BUT have the majority of haploinsufficient genes now been discovered?

– “Prevalence and architecture of dominant developmental disorders” – http://biorxiv.org/content/early/2016/04/20/049056

http://biorxiv.org/content/early/2016/04/20/049056


Summary • Extended life for chromosomal microarray?

– Rapid, cost-effective detection of recurrent CNV syndromes

• Exome/panel CNV calling improvements? • CNV calling in WGS / 100kgp data? • Need for higher resolution data-sets

– ExAC CNV data released August 2016 – Clinical CNV data


Data-sharing

NHS Data-sharing consortium – (semi) auto-upload capability to DECIPHER

• OGT software export plug-in

– Auto-upload via API functionality being explored – SNV and CNV worlds are colliding

• Interpretation requires all variattion

– THIS DATA NEEDS SHARING


NHS Data-sharing Consortium


Data Collection • How common are smaller exonic changes?

– e.g <3 exons – What has been missed ? – Unusual mechanisms/ genotype-phenotype ?

• This UGM as a forum to drive data-collection project

• OGT keen to support collaborative study • DDD team keen to collaborate on joint publication


Acknowledgements

• WMRGL team – Rowena Roberts – Lisa Reali, Sam Martin, Dave Bohanna, Laura Scott,

Gareth Masson, Fiona Togneri

• DDD / DECIPHER team • OGT team

Thank-you for your attention !!

implementation of the ddd/clingen ogt (cytosure v3) microarray · west midlands regional genetics...

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