rationale application of next generation sequencing (ngs) … · 2020. 12. 7. · 12/7/2020 1...

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©2020 MFMER | slide-1 ©2020 MFMER | slide-1

Rationale Application of Next Generation Sequencing (NGS) Technology in Coagulation Disorders Rajiv K. Pruthi, M.B.B.S. Consultant I Associate Professor of Medicine Director, Mayo Comprehensive Hemophilia Center Co-Director, Special Coagulation and Molecular Hematopathology Laboratories, Chair, Coagulation Diseases Oriented Group (Hematology) Mayo Clinic, Rochester, MN

A Case-based Workshop: Clinical and Laboratory Aspects of Hemophilia and Thrombosis Dec 4th, 2020 [email protected]

©2020 Mayo Foundation for Medical Education and Research | slide-1


Relevant Financial Relationship(s)

• None.

Off Label Usage:

• Not applicable

DISCLOSURES

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LEARNING OBJECTIVE

Primer on PCR & Next Generation Sequencing

Overview of hereditary bleeding disorders and

thrombophilia

Rational application of NGS to hereditary

bleeding disorders

Rational application of NGS to hereditary

thrombophilia


Definitions

Phenotypic diagnosis:

Relies on protein based testing: plasma, platelet aggregation etc

May not be widely available

Platelet aggregation tests cannot be mailed in to reference laboratories

Role of genotypic diagnosis:

Prognosis in established diagnosis

Establishing the diagnosis based on genotype information (when protein based assays are not available/reliable)

Can be mailed into reference laboratories

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©2020 MFMER | slide-5

Schematic Diagram of PCR

Template DNA

dCTP

dATP

dGTP

dTTP

Primer

Primer

Taq DNA

Polymerase

DNA strand separation

DNA at 95oC

Taq DNA polymerase mediated primer extension when reheated

Primers anneal when cooled Repeated cycles lead to exponential increase in number of DNA copies

+


PCR of FVIII gene in multi-exon deleted patient Normal

Control Patient

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Sequencing output: forward and reverse strands


Adapted from: Jill M. Johnsen et al. Blood 2013;122:3268-3275

©2013 by American Society of Hematology

Next Generation Sequencing (NGS)

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NGS Workflow

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Pre-analytic

Library Prep

Sequencing

Template Prep

• Demultiplexing

• Base calling

• Alignment

• Variant calling

• Specialized applications

• Variant Annotation

• Variant significance*

10

20

30

“Wet Bench” Process “Dry Bench” Informatics


NGS sequence output: depth of read varies >500

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Major advantages

•Sequence multiple samples on one run

•Sequence multiple regions on one run

•Whole exome: only coding regions

•Whole genome: coding and non coding regions

•Time consuming analytics


Approach to presentation: Hereditary bleeding disorders Inherited thrombophilia

• Patients evaluated for bleeding symptoms

• Based on phenotype assays

• Diagnosis established

• No diagnosis established

• Is there a role for NGS testing

• Impact on management

• Venous thromboembolism (VTE)

• Asymptomatic individual

• Symptomatic individual

• Phenotype assays

• Diagnosis established

• No diagnosis established

• Is there a role for NGS testing

• Impact on management

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Type and incidence of inherited bleeding disorders

VWD 40%

Hemophilia A 25%

FXI deficieny 10%

Platelet disorder 8%

Hemophilia B 6%

FVII deficiency 4%

Fibrinogen defect 2% Rare/Combined

3% Unspecified 2%

Frequency

Sivapalaratnam, S et al BJH 2017; 179:363


Phenotype diagnosis established: What is the role of genotyping? •Plasmatic bleeding disorders

•Hemophilia A and B

•Von Willebrand disease

•Platelet bleeding disorders

•Syndromic vs Non-Syndromic

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Severe hemophilia A genotypes (MLOF initiative)

34.7

38.3

17.4

7.2

5.9

1.2

1.1 0.6

0.2

0.2

Frequency

Int 22 Inv Type 1

Single nucleotide

Frameshift

Int 22 Inv Type 2

Larger SV

Int 1 inv

Small indel

None

Int 22 Inv complex

Int 1 Inv Complex

Johnsen, JM et al Blood Advances 2017; 1:824


©2011 MFMER |

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Large deletions

Multiple exon

Nonsense

Nonsense light chain

Nonsense non-light chain

Intron 22 inversion

Intron 1 inversion

Small in/dels

In poly A runs Outside poly A runs

Missense

Missense light chain Missense non-light chain

Splice site

Conserved Non-conserved

Unknown

Single exon

3.57 (2.26-5.66)

Pooled OR

9.24 (5.39-15.84)

1.09 (0.54-2.2)

1.37 (1.05-1.79)

1.80 (1.22-2.64)

1.04 (0.73-1.49)

0.92 (0.57-1.50)

0.51 (0.41-0.65)

0.27(0.17-0.43)

0.30 (0.20-0.44)

0.37 (0.21-0.65)

0.23 (0.14-0.36)

Reference

0.65 (0.50-0.86)

0.95 (0.59-1.54)

0.76 (0.33-1.78)

0.31 (0.05-1.92)

0.37 (0.23-0.59)

Genotype to phenotype correlation: Inhibitor risk hemophilia A

Gouw S et al Blood 2012; 119:2922

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Hemophilia B Leyden: Phenotypic evolution

0 10 20 30 40 50 60 70

150

100

50

0

Normal

Leyden

Brandenburg

AR

FIX

:C %

no

rma

l

Age Funnell, APW et al Trends in Genetics 2014; 30: 18-23


Genotyping Platelet Disorders: Prognostic

• Hermansky-Pudlack (HPS 1 and 4)

• Type 1: monitor for pulmonary fibrosis

• MYH9-related disorders

• Nephritis

• RUNX1 variant

• Increased risk for acute myeloid leukemia

• Targeted platelet panels vs while exome or genome sequencing

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Take home message: For patients with established phenotypic diagnosis of bleeding disorders •Genotyping has prognostic value

•Technology used in genotyping depends on cost/efficiency/affordability etc

•PCR/Sequencing: for targeted mutation analysis

•NGS technologies: costs will decline

•Exonic vs intronic mutations


Illustrative case(s): Bleeding disorders

•Mild lifelong bleeding symptoms no diagnosis forthcoming with phenotypic testing

• 41 yr old female:

• Menorrhagia at menarche, excessive spontaneous bruising, prolonged bleeding with minor cuts, frequent epistaxis, excess bleeding with procedures: LEEP, hysterectomy, gum surgery

• ISTH Bleeding assessment tool: Score 13 (normal <6)

• Extensive laboratory testing normal: PT/APTT, thrombin time, VWF assays, platelet aggregation, platelet electron microscopy, PAI-1, alpha 2 antiplasmin, ROTEM, TEG. etc

• Phenotype diagnosis NOT established

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Outcomes of NGS testing in coagulation disorders

• Thrombogenomics HTS group

• 2396 index patients (bleeding with or without phenotypic diagnosis, thrombocytopenia, platelet function defect, thrombosis)

• Referred for NGS assay after diagnosis of

• Bleeding disorder (phenotypic diagnosis established)or

• Undiagnosed bleeding disorder (phenotypic diagnosis NOT established) or

• Thrombophilia

Downes K et al Blood 2019; 134:2082


Diagnostic Yield: Patients with Bleeding disorders

0 20 40 60 80 100

Platelet count (n=335)

Platelet function (n=430)

Coagulation (n=728)

Unexplained bleeding (n=619)

All patients (n=2396)

Proportion of patients (%)

Pathogenic

Likely Pathogenic

Variant of uncertain

significance

No variant


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Whole exome sequencing in bleeding disorders

Thrombocytopenia (n=17) Plt function defect (n=19) Undiagnosed (n=51)

4/17 (23%)

Diagnostic Yield

1/19 (5%)

P2RY12 MYH9 x 2

SLFN14

GP9

7/51 (13%)

F7 & F13A1

F2, F8, VWF

GP1BA

MPL

F2

F5

F11

Mild low VWF

Heterozygous

Saes JL et al Haemophilia 2019; 25: 127


Back to the patient (41 year old female with undiagnosed bleeding): Beighton Score 7/9

Possible hypermobile EHD

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Take home message

•For patients with undiagnosed bleeding disorders: yield of finding a pathogenic variant with NGS is low

•Criteria for pathogenicity is evolving as functional data are generated

•Vascular disorders (e.g. Ehlers Danlos) will not be detected by coagulation assays

•Clinical evaluation is important (Beighton Score)


Illustrative case(s): Thrombophilia Testing

• Who, how and when?

• Asymptomatic patient: genotyping for VTE prophylaxis strategy

• No indication for thrombophilia testing for VTE prophylaxis risk stratification

• Symptomatic patient with VTE

• Provoked

• ASH Choosing Wisely 2018: do not order thrombophilia testing

• Unprovoked Venous Thromboembolism:

• Young individual/Strong family history of VTE

• Recurrent VTE

• Cost effective approach: plasma based assays

• Is there a role for Next generation sequencing?

• Approach: exome vs genome

Blood 2013; 122: 3879 & 2014;124:3524

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Problems associated with phenotypic (plasma/protein) based assays

50

40

30

20

10

0 AT

activity

PC

activity

PS

Tot-Ag

PS

Fr Ag

PS

activity

CV

(%)

Overall error rate 2 to 8%:

false-normal (deficient plasma) OR false-abnormal (normal plasma)

Favaloro et al Sem Thromb Hemost 2005; 31:49


Identification of risk factors for VTE: NGS technology

•Current studies based on candidate gene approach

•Evolving to GWAS studies

•Approach will depend on individual goals:

•Genotype vs Discovery of novel alleles

Cunha MLR et al Thromb Hemost 2015; 114: 920-932

Morange, PE JTH 2011; 9:Supp1: 258-264

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Genetic risk factors for VTE:What is currently known ?

Locus SNP Allele Frequency OR/RR Phenotype

ABO O,A2 vs A1B 0.3 1.5 FVIII/VWF

F2 rs1799963 G/A 0.02 2.5 FII

F5 rs6025 G/A 0.05 3.0 APC-R

FGG rs2066865 C/T 0.25 1.47 Fibrinogen

PROC

Multiple private

10

PC def

PROS1 PS def

SERPINC1 AT def



Outcomes of NGS testing in thrombophilia

•Thrombogenomics HTS group

•2396 index patients (bleeding with or without phenotypic diagnosis, thrombocytopenia, platelet function defect, thrombosis)

•Referred for NGS assay after finding of a thrombophilia, bleeding disorder or undiagnosed.

•Thrombotic patients: abnormality of protein C anticoagulant pathway (PC & PS)

•Diagnostic yield: 48.9%


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Diagnostic Yield

0 20 40 60 80 100

Thrombotic (n=284)

Platelet count (n=335)

Platelet function (n=430)

Coagulation (n=728)

Unexplained bleeding (n=619)

All patients (n=2396)

Proportion of patients (%)

Pathogenic

Likely Pathogenic

Variant of uncertain

significance

No variant



Application of GWAS to determining risk loci

Locus SNP Allele Frequency OR Phenotype

C4BPB/C4BP

A

rs3813948 T/C 0.08 1.18 C4BP

F11 rs2036914

rs2289252

C/T

C/T

0.52

0.41

1.35 FXI

GP6 rs1613662 A/G 0.82 1.15 Plt act.

KNG1 rs710446 T/C 0.45 1.2 aPTT

HIVEP1 rs169713 T/C 0.21 1.2 Unknown

SERPINC1 rs2227589 C/T 0.1 1.29 AT

STXBP5 rs1039084 A/G 0.46 1.11

VWF TC2N rs184841 C/T 0.44 1.27

VWF rs1063856 A/G 0.37 1.15


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Findings from GWAS

• Findings:

• Rare genetic variants influence risk for VTE

• Individually have a low OR/RR for VTE

• Novel risk alleles are still being discovered (inflammation related genes)

• Potential explanations:

• VTE is a multifactorial disease

• Study methodology

• Population studied

• Statistical power

• large numbers approximately 140,000 cases required to identify 10 alleles with an OR of 1.12

Lotta L, et al.BMC Med Genomics 2012; 5: 7.

Lotta L, et al. J Thromb Haemost 2013; 11: 1228–1239.

Reviewed in:Cunha MLR et al Thromb Hemost 2015; 114: 920-932


Conclusions • Next Generation Sequencing is a technology (to be judiciously applied)

• Genotyping patients with established phenotypic diagnoses is relevant (prognostic)

• Currently targeted testing most likely optimal approach

• Single gene vs Limited Panel approach

• Advantages of NGS: develop ‘dynamic’ panels: additional of discovery of new genes

• Option to have whole genome/exome

• (masking of non significant genes)

• Unmasking/analysis of genes as they become relevant

• Indiscriminate genotyping of patients with no phenotypic diagnosis has a low yield

• Efforts to continue research and clinical testing aids in understanding biology of disease

• Important to report unreported variants to databases to advance knowledge

• Current needs and future potential options:

• Current strategy: counsel individual patients based on population studies

• Future strategy: counsel individual patients based on individualized risks

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[email protected]

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