Genetic Testing in the Clinic. Anne Goodeve Sheffield Diagnostic Genetics Service Sheffield Children s NHS Foundation Trust

Transcription

1 Genetic Testing in the Clinic Anne Goodeve Sheffield Diagnostic Genetics Service Sheffield Children s NHS Foundation Trust

2 Disclosures for Anne Goodeve In compliance with COI policy, ISTH requires the following disclosures to the session audience: Research Support/P.I. Employee Consultant Major Stockholder Speakers Bureau Honoraria Scientific Advisory Board No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare Octapharma, Baxalta No relevant conflicts of interest to declare Presentation includes discussion of the following off-label use of a drug or medical device: <N/A>

3 Sheffield Diagnostic Genetics Service UK Genetic Testing Network (UKGTN) member laboratories are UK based and provide genetic testing services to NHS patients UKGTN website lists testing available through the NHS Approx 25 regional genetics laboratories serve the UK, along with specialist laboratories that cover specific specialities e.g. haemostasis

4 Sheffield Diagnostic Genetics Service Director n=1 Administration n=7 & 3 apprentices Medical Laboratory Assistants n=17 Genetic Technologists n=31 Clinical Scientists/Health Care Scientists n=30 & 6 trainees Information Technology n=3 Bioinformatics n=1 & 3 trainees Business Manager n=1 Other n=13 Total n=116

5 Aim To describe routine diagnostic service use of next-generation DNA sequencing and of variant classification

6 Introduction Inherited coagulation and some platelet bleeding disorders can be readily diagnosed through phenotypic testing Genetic analysis targets genes associated with specific disorders

7 Genetic analysis Extract DNA from blood Examine for sequence variants Determine pathogenicity of sequence variant(s) Report findings to referring clinician, single page report

8 Haemostatic and platelet gene list ADAMTS13 F5 F8 F9 F13A1 F13B FGA FGB FGG ITGA2B ITGB3 MYH9 VWF Total 277 exons

9 Phenotype data Often minimal; Haemophilia; type & severity in most cases VWD; VWF:Ag, VWF:Ac, FVIII:C levels Other disorders; little information

10 Sequence analysis Illumina Illumina MiSeq HiSeq (Rapid run) 2x 150 bp reads 2x 100 bp reads Gb Gb 24 hours 27 hours 16 samples 96 samples Data analysis 20 min - 4 hr

11 Analysis pipeline Script from fastq to filtered annotated variants QC information Variant calling Alignment to reference Mark PCR Duplicates realignment around discovered indels x2 Downsampling Variant calling Consensus call set Filter to small region annotate Remove GATK low quality calls Filter against polymorphism list x4 Downsampling Variant calling Software used BWA aln Picard GATK Picard GATK (HaplotypeCaller) GATK VCFtools SnpEff, ANNOVAR

12 Variant filtering workflow All variants identified 150 variants Variants within genes of interest 30 variants Frequent variants removed Pathogenicity assessment 4 variants 3 variants Bioinformatics Scientists Candidate mutation(s) 2 variants

13 NGS data output format Gene (with HGVS) F8:NM_000132: exon18: c.5954g>a: p.r1985q Chromo some Start bp End bp Reference base(s) Variant base(s) Zygosity chrx C T hom

14 NGS data output format Gene (with HGVS) F8:NM_000132: exon18: c.5954g>a: p.r1985q FGA:NM_000508: exon5: c.991a>g: p.t331a FGG:NM_000509: exon8: c.901c>t: p.r301c Chromo some Start bp End bp Reference base(s) Variant base(s) Zygosity chrx C T hom chr T C het chr G A het

15 Validation 11 previous Sanger sequenced patients reanalysed using NGS following written informed consent Only previously sequenced gene(s) analysed 30 fold sequence coverage of regions of interest (ROI) (exons +/- 5bp) 18 fold sequence coverage (+/-6 bp to +/-25 bp) using Illumina MiSeq

16 Validation 2-30 variants identified per patient in gene(s) for each disorder all variants documented for validation Complete concordance with variants previously identified in each individual

17 VWF gene and pseudogene kb VWFP1, chromosome 22 2 gene Pseudogene conversions extent detected, c affecting to c end of exon 28 97% sequence similarity to VWF 7 variants identified, no pseudogene detection

18 Confirmation of variants Sanger sequencing used to confirm potentially pathogenic variants Risk assessment recently undertaken Single nucleotide substitutions will not be confirmed by Sanger sequencing in future where coverage is sufficient Other sequence changes will be confirmed using Sanger

19 Unclassified variant guidelines UK Association for Clinical Genetic Science (ACGS) Wallis Y, et al. Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics, Available from American College for Medical Genetics (ACMG) Richards S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):

20 Unclassified variants (ACGS classification) 1. Neutral variant 2. Probably neutral variant 3. Unclassified variant 4. Probably pathogenic variant 5. Pathogenic variant Wallis Y, et al. Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics,

21 Unclassified variant analysis of gene of interest of sequence variant for sequence variant for sequence variant for sequence variant of sequence variant of sequence variant with disease with de novo disease

22 Automated unclassified variant pipeline Designed by Bioinformatics trainee Searches several tools on internet simultaneously Speeds up data gathering process to help assess pathogenicity

23 Is the variant pathogenic? Present on population databases? In one/small number of individuals; Possibly disease-associated Present in many individuals; Unlikely to be disease-associated

24 Exome aggregation consortium (ExAC) F8 allele frequency Rare variants Common variant

25 Locus specific databases; F9

26 Splice site prediction SpliceSiteFinder MaxEntScan NNSplice GeneSplicer Can be accessed through Alamut Human Splicing Finder NetGene2 MIT Splice Predictor Alternative Splice Site Predictor

27 Splice site prediction Aim to achieve a consensus through use of 3 splice prediction tools Some apparent missense variants affect mrna splicing, use splice prediction tools

28 mrna analysis mrna analysis can confirm/exclude pathogenicity of possible splice site variants Fresh blood sample from patient mrna extracted from white cells Sequence region of interest or entire cdna Difference in spliced product from wild-type can indicate effect of variant

29 Functional studies Occasional missense variants expressed in vitro to help determine pathogenicity Ensure that in vitro system mimics natural expression e.g. for VWF, storage in Weibel-Palade like bodies is present Some variants are neutral, e.g. VWF: p.pro2063ser Others affect protein secretion and function

30 De novo variant P12F3I P12F3I New variant with no prior family history of disorder, parents appear unaffected de novo p.y1107c P12F3II1 P12F3II2 P12F3II3 Possibilities; mother is a hemophilia carrier (new variant) parent is a mosaic for variant & wild type alleles P12F3III P12F3III p.y1107c Non linkage family. de novo variant in patient non-paternity

31 Co-segregation Does the variant co-segregate with disease in the family? Analysis can be used where there is no previous literature or database report of the variant Family analysis may provide evidence for segregation of variant and disorder

32 Pathogenic sequence variants identified in 112 individuals referred for genetic testing Gene(s) analysed Disorder No. mutation/analysed ADAMTS13 ADAMTS13 deficiency 1/2 F5 FV deficiency 1st patient 0/0 in progress F8 Haemophilia A 44/58 F8 & F9 Haemophilia A & B 1/1 F8 & VWF Haemophilia A & 2N VWD 3 F8, 2 VWF, 5/94 0 variant F8, F9, F13A1, F13B & VWF Bleeding disorders 0/1 F13A1 & F13B FXIII deficiency 2/3 FGA, FGB & FGG Fibrinogen disorders 6/6 ITGA2B & ITGB3 Glanzmann thrombasthenia 2/3 MYH9 MYH9 related disorders No phenotyping 0/4 done VWF von Willebrand disease 16/25 TOTAL ALL PATIENTS 77/112 (69%)

33 Clinical report Single page document sent to referring clinician Variants in UV classes 3-5 reported Significance for patient; eg inhibitory antibody formation Significance for family members; risk of disorder

34 Advantages over Sanger sequencing Analysis of all genes possibly associated with a disorder analysed simultaneously Single laboratory pipeline for NGS analysis Data can be reanalysed later without further laboratory work if possible diagnosis changes

35 Impact on bleeding disorder testing Complete sequencing of VWF gene coding region Carrier of unknown hemophilia type - F8 and F9 genes Mild haemophilia A/2N VWD - F8 and VWF genes Disorders caused by more than one gene; eg factor XIII & fibrinogen deficiencies, Glanzmann thrombasthenia Analysis of several bleeding disorders simultaneously

36 Impact on genetic testing Cost Significantly reduced for large genes/ multigene disorders Turnaround time 56 calendar days

37 NGS advantages Next generation DNA sequencing provides a single workflow for analysis of multiple gene panels Simultaneous analysis of all genes associated with a disorder speeds up the diagnostic process Data analysis straightforward once common sequence variants removed Analysis costs often reduced

38 Summary Systematic analysis of several evidence types can provide classification of a sequence variant For some variants, insufficient evidence is available to determine possible pathogenicity. Classified as Variant of unknown significance Segregation analysis within affected family may demonstrate possible pathogenicity

39 Haemostatic and platelet gene list ADAMTS13 F5 F8 F9 F13A1 F13B FGA FGB FGG ITGA2B ITGB3 MYH9 VWF Available for routine & research analysis; currently analysing 3WiNTERS-IPS type 3 VWD samples; SDGS@sch.nhs.uk

40 Acknowledgements Kevin Blighe, Lucy Crooks & Matt Parker, NGS data analysis pipeline Nick Beauchamp, Laura Crookes & Nikolas Niksic Sheffield Diagnostic Genetics Service Sheffield Haemostasis Research Group