The Diagnosis of VWD Interpreting laboratory testing for a complex genetic disorder

Transcription

1 The Diagnosis of VWD Interpreting laboratory testing for a complex genetic disorder David Lillicrap Department of Pathology and Molecular Medicine Queen's University, Kingston, Canada Bangkok, November 2017

2 Disclosures for David Lillicrap 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 Bayer, Bioverativ, CSL, Octapharma 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 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

4 Aland Islands from Space: 6,500 islands

5

6

7 1985 Construction of cdna coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene. Verweij CL, de Vries CJ, Distel B, van Zonneveld AJ, van Kessel AG, van Mourik JA, Pannekoek H. Nucleic Acids Res Jul 11;13(13): Human von Willebrand factor (vwf): isolation of complementary DNA (cdna) clones and chromosomal localization. Ginsburg D, Handin RI, Bonthron DT, Donlon TA, Bruns GA, Latt SA, Orkin SH. Science Jun 21;228(4706): Cloning and characterization of two cdnas coding for human von Willebrand factor. Sadler JE, Shelton-Inloes BB, Sorace JM, Harlan JM, Titani K, Davie EW. Proc Natl Acad Sci U S A Oct;82(19): Molecular cloning of cdna for human von Willebrand factor: authentication by a new method. Lynch DC, Zimmerman TS, Collins CJ, Brown M, Morin MJ, Ling EH, Livingston DM. Cell May;41(1):49-56.

8 VWF Gene: Chromosome 12p 178 kbp

9 D1 D2 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K VWFpp 740 AA VWF mature subunit 2050 AA D Assembly Composition VWD-C8-TIL-E von Willebrand Factor Structure Y-F Zhou et al. Blood 2012

10 FVIII P-selectin VWFpp β2 integrins ADAMTS13 Lenting et al. JTH 2012 αiibβ3 αvβ3 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K GPIbα Collagen VI Collagen IV OPG PSGL-1 β2gpi Collagen I Collagen III TSP1 The Mature VWF Subunit with Associated Ligands

11 Von Willebrand Disease Classification (ISTH 2006) Qualitative Variants 25% Type 1 Type 2 Type3 75% 1 per million Quantitative Variants

12 ISTH 2006 VWD Classification Type 2 - qualitative traits 2A 2B 2M 2N ~25%

13 Diagnosis of von Willebrand disease Personal history of excessive mucocutaneous bleeding - Bleeding assessment tools Coagulation laboratory findings consistent with VWD Family history of VWD

14 Hemostasis Laboratory Diagnosis of VWD VWF:Ag VWF:RCo FVIII:C 1. VWF multimer analysis 2. RIPA 3. VWF:F8 4. VWF:CB 5. VWFpp

15 The Laboratory Diagnosis of von Willebrand Disease Standardized measurement of VWF:Ag VWF platelet binding activity

16 VWF + Risto + Platelets = VWF:RCo VWF + Risto + rgpib = VWF:GPIbR VWF + rmutgpib VWF + A1 domain Ab = VWF:GPIbM = VWF:Ab

17 VWF Platelet Binding Functional Assays New Assays Improved sensitivity < IU/mL Improved reproducibility CV 2 6% Lawrie AS et al. Haemophilia 19:338-42, 2013; Stufano F et al. Haemophilia 20:147-53, 2014

18 Type 2 von Willebrand Disease VWF:RCo:VWF:Ag ratio <0.6 (2A, 2B, 2M) Abnormal VWF multimer pattern (2A, 2B) RIPA +ve (2B) FVIII level 5-30% (2N)

19 Platelet Binding VWF Mutants 1. Loss-of function Type 2A (abnormal multimers) 2. Loss-of function Type 2M (normal multimers) 3. Gain-of-function Type 2B Dominant transmission Complete penetrance Missense substitutions

20 VWF Multimer Analysis N 2A 2B N

21 Genotypic Complementation of Type 2 VWD Diagnosis

22 Type 2A von Willebrand Disease Loss of Platelet-dependent Function (abnormal multimers) >55 missense mutations VWF:RCo VWF:GPIbM / VWF:Ag < 0.6

23 2A (80%) D1 D2 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K 2A (10%) 2A (10%) Location of Type 2A von Willebrand Disease Mutations

24 VWF A1 GPIbα Huizinga et al. Science 2002

25 Type 2M VWD Type 2M (GpIb binding mutants) - 18 A1 domain missense mutations

26 D1 D2 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K 2M (collagen) A3 Domain: Collagen Binding Mutants - Require VWF:CB Assay S1731T W1745C S1783A H1786D Ribba et al Thomb Haemost 2001 Riddell et al Blood 2009 Flood et al JTH 2010 Location of Type 2M Collagen binding von Willebrand Disease Mutations

27 Type IV Type VI D1 D2 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K Type I and III Additional VWF - Collagen Interactions (require VWF:CB Assay)

28 Type 2B VWD Type 2B VWD Mutations - 24 A1 domain missense mutations

29 Type 2N von Willebrand Disease FVIII:C 0.97 VWF:Ag 0.89 VWF:RCo 1.04 FVIII:C 0.96 VWF:Ag 0.89 VWF:RCo 1.11 FVIII:C 0.17 VWF:Ag 0.74 VWF:RCo 0.67 FVIII:C 0.23 VWF:Ag 1.13 VWF:RCo 1.07

30 Genetic Mechanisms in Type 2N VWD Missense 1 + Missense 1 Missense 1 + Missense 2 Missense 1 + Null Homozygosity Compound Heterozygosity Compound Heterozygosity

31 2N D1 D2 D D3 A 1 A 2 A 3 D4 C1 C2 C3 C4 C5 C6 C K Location of Type 2N von Willebrand Disease Mutations

32 Type 2N von Willebrand Disease Recurrent Type 2N VWD Mutations R854Q >> R816W, T791M FVIII:C ~20% FVIII:C ~10% (DDAVP +ve) (DDAVP -ve)

33 Molecular Testing for Inherited Bleeding Disorders Differentiation of Genocopies (identical phenotypes due to mutations in different genes)

34 Molecular Testing for Inherited Bleeding Disorders Differentiation of Genocopies (identical phenotypes due to mutations in different genes) 1. Type 2B VWD vs Platelet-Type VWD Genetic Locus: VWF vs GPIbα Therapy: VWF/FVIII concentrate vs Platelets

35 Molecular Testing for Inherited Bleeding Disorders Differentiation of Genocopies (identical phenotypes due to mutations in different genes) 2. Type 2N VWD vs Mild/Moderate Hemophilia A Genetic Locus: VWF vs F8 Therapy: VWF/FVIII concentrate vs FVIII concentrate DDAVP DDAVP

36 Type 3 von Willebrand Disease Pathogenesis

37 FVIII:C 0.97 VWF:Ag 0.79 VWF:RCo 0.94 FVIII:C 0.63 VWF:Ag 0.52 VWF:RCo 0.61 FVIII:C 0.04 VWF:Ag 0.00 VWF:RCo 0.00 Type 3 VWD - Autosomal Recessive Trait

38 FVIII:C 0.57 VWF:Ag 0.49 VWF:RCo 0.44 FVIII:C 0.44 VWF:Ag 0.37 VWF:RCo 0.34 FVIII:C 0.04 VWF:Ag 0.00 VWF:RCo 0.00 Type 3 VWD - Autosomal Co-Dominant

39

40

41 Type 1 von Willebrand Disease When is this diagnosis helpful? Plasma VWF Levels 50% 30% 0%??? Type 1 VWD Type 1 VWD Low VWF Possible VWD

42 Why Does this Matter? Disease "labelling" Health insurance Access to effective therapy Comparative research studies Correct biological interpretation Growing Consensus: 30-50% VWF. - Low VWF

43 Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease Rydz et al Haemophilia 21: , 2015

44 Diagnosis of Low von Willebrand Factor Patients with an appropriate bleeding history and VWF activity IU/mL should be regarded as having primary haemostatic bleeding with reduced VWF as a risk factor rather than VWD. We suggest referring to this as Low VWF (2C). UKHCDO Guidelines Laffan et al. Br.J.Haematol. 2014

45 Type 1 von Willebrand Disease Classical description. - monogenic, dominant inheritance In 2017 Oligogenic Variable penetrance Variable expressivity

46 Proportion of Type 1 VWD with a Candidate VWF Gene Mutation MCMDM-1VWD 105/150-70% Canada 78/123-63% UKHCDO - 19/32-59% ZPMCB-VWD - 111/179-62% University Clinic Bonn - 19/28-68% 332/512-65%

47 Results from Recent Type 1 VWD Studies 1. Candidate VWF mutations in 65% of index cases 2. >100 different VWF gene mutations 3. Missense mutations predominate 4. ~15% of cases have >1 mutation

48 Storage and release from Weibel-Palade Bodies Endothelial Cell Golgi Apparatus Multimer formation Propeptide cleavage Glycan modification Constitutive secretion Endoplasmic reticulum Dimer formation initial glycan additions VWF mrna Processing Gene expression VWF clearance a) Differential glycan additions b) VWF misfolding Receptor-mediated clearance in liver & spleen a) Macrophages b) Sinusoidal endothelium Sites of Potential Pathogenic Changes Resulting in Quantitative VWF Traits

49 Type 1C von Willebrand Disease VWF:Ag <30% often <15% VWF:pp to VWF:Ag ratio >2 up to >10 Exaggerated DDAVP response >4-fold up to 10-fold Short term benefit from DDAVP (2-4 hrs) Plasma VWF half-lives <3 hrs Subtly abnormal VWF multimer profiles

50 100 R1205H (VWF Vicenza) VWF:Ag% DDAVP Response in Type 1C VWD Hrs post-ddavp

51 Type 1C Comprises the Majority of Severe Type 1 VWD Cases Increased Clearance Reduced Secretion VWFpp/VWF:Ag % 38% 7% % of quadrant consistent with type 1C phenotype Contributions from Synthesis Storage Secretion VWF:Ag (IU/dL) Haberichter et al. 2014

52 Missing Type 1 VWD Pathogenetic Influences VWF Intronic variants VWF Flanking regulatory variants Other modifying loci -CHARGE GWAS meta-analysis (Circulation 2010) Environment - Aging

53 Circulation. 2010;121: ,000 Discovery cohort 7,000 Replication cohort

54 Novel Genetic Loci Associated with Plasma VWF Levels Vesicular trafficking and exocytosis STXBP5 Syntaxin binding protein 5 STX2 Syntaxin 2 Receptor proteins SCARA5 Scavenger receptor class A member 5 STAB2 Stabilin 2 CLEC4M C-type Lectin Type 4 Member M

55 Multi-ethnic Genome-wide Association Study Identifies New Loci Regulating Factor VIII and von Willebrand Factor M. Sabater-Lleal, et al. CHARGE Hemostasis Working Group. ISTH Berlin July ,000 normal cohort GWAS Meta-analysis 9 new loci associated with VWF plasma levels ST3GAL4 - Alpha 2,3-Sialyltransferase IV C2CD4B TAB1/SYNGR1 PDHB FCHO2 -FCH only 2 protein HLA-DGA1 GIMAP7 OR13C5 DAB2IP -Disabled homolog 2-interacting protein 2 new loci associated with both VWF & FVIII: RAB5C & ARSA

56 Contributions from non-vwf Genetic Loci Frequent variants - small effects (<5%) - demonstrated in GWAS? Rare variants - larger effects

57 Complex Genetic Background of Type 1 von Willebrand Disease VWF - 91 polymorphic amino acids ABO At least 16 other genes involved in Biosynthesis, trafficking, storage, secretion Clearance

58 + other genetic loci Major Effect on VWF Level Minor Penetrant VWF Mutations Incompletely Penetrant VWF Mutations ABO Blood Group - regulatory sequences - biosynthetic regulators - clearance receptors VWF SNV Combinations Rare Prevalence of Variant Frequent Model for Pathogenesis of Type 1 von Willebrand Disease

59 Relevance of the Complex Genetics of Type 1 VWD Biologically interesting No obvious clinical relevance except to significantly limit genetic testing for type 1 VWD

60 Queen's University 175 th Anniversary