Application of High Resolution Melting Analysis in Haematological Diagnosis
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1 Application of High Resolution Melting Analysis in Haematological Diagnosis Dr Jason C C So Clinical Associate Professor Department of Pathology The University of Hong Kong
2 Haematology and Genetics Blood cancers and inherited haematological diseases Sickle Cell Anemia, a Molecular Disease Pauling L et al. Science November 25, 1949
3 Haematology and Genetics Blood cancers and inherited haematological diseases A minute chromosome in chronic granulocytic leukemia Nowell P, Hungerford D. Science November 18, 1960
4 Haematology and Genetics Blood cancers and inherited haematological diseases Antenatal diagnosis of sickle-cell anaemia by D.N.A. analysis of amniotic-fluid cells. Kan YW, Dozy AM. Lancet. Oct 28, 1978
5 Haematology and Genetics Blood cancers and inherited haematological diseases Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. Druker BJ et al. N Engl J Med. Apr 5, 2001
6 Mutations in Haematological Diseases are Diverse Involving one gene 1) Gross rearrangement HBA1/2 deletion in alpha thalassaemia F8 inversion in haemophilia A 2) Small deletion and insertion of nucleotides NPM1 mutation in acute myeloid leukaemia 3) Single nucleotide substitution JAK2 V617F in myeloproliferative neoplasms
7 Mutations in Haematological Diseases are Diverse Involving two genes 1) Gene fusion BCR-ABL1 in chronic myelogenous leukaemia 2) Gene transposition IGH-BCL2 in follicular lymphoma
8 Mutation Detection in Diagnostic Haematology Strategy depends on the types of mutation to be detected in a particular blood disease Gene fusion due to chromosomal translocation Single nucleotide substitution
9 Mutation Detection in Diagnostic Haematology Strategy depends on the types of mutation to be detected in a particular blood disease Single nucleotide substitution, small indel Gross gene deletion HS4 HS3 HS2 on 01 on 03 on on 03 motor on 02 n 03A c c c c c c c
10 Mutation Detection in Diagnostic Haematology Strategy depends on whether the identity of mutation is known in a particular blood disease Known single nucleotide substitution, small indel Targeted sequencing Restriction fragment length polymorphism Mutation-specific PCR priming or probe hybridisation
11 Mutation Detection in Diagnostic Haematology Strategy depends on whether the identity of mutation is known in a particular blood disease Known large deletion Gap-PCR Known gene fusion, transposition Reverse transcription-pcr Fluorescence in-situ hybridisation
12 Mutation Detection in Diagnostic Haematology Strategy depends on whether the identity of mutation is known in a particular blood disease Unknown Direct nucleotide sequencing??? Multiplex ligation-dependent probe amplification HS4 HS3 HS2 on 01 on 03 on on 03 c c c c c c c motor on 02 n 03A 005.2
13 Ultimate Resolution in Mutation Detection Reading at the single nucleotide level Whole gene sequencing Sanger sequencing Whole genome sequencing Next generation sequencing
14 Gene Sequencing to Detect Unknown Mutations Direct nucleotide sequencing vs Mutation scanning followed by targeted sequencing
15 Direct or Scanning? Size of target gene HBB 3 kb vs VWF 175 kb Number of target genes 1 candidate gene for G6PD deficiency vs 15 in Fanconi anaemia Heterogeneity and concentration of mutations Single mutation BRAF V600E in hairy cell leukaemia vs 1500 spreadout mutations in F8 in haemophilia A
16 Direct or Scanning? Capacity for sequencing vs Setup for mutation scanning
17 Principles of Mutation Scanning Amplify candidate gene in segments (amplicons) Scan for sequence variation in segments Sequence the abnormal segment
18 Scanning for Sequence Variation in Amplicons Detection of a change in physical property - alteration in gel mobility - alteration in denaturation rate (melting)
19 Altered Gel Mobility Single strand secondary structure (folding) Double strand secondary structure (duplex) Size (length after chemical cleavage)
20 Altered Melting Property under Changing Ionic Strength of Medium Binding and elution of amplicons from a solid cartridge support - denaturing high performance liquid chromatography (dhplc)
21 Altered Melting Property under Changing Temperature Binding and release of fluorescent dye from double-stranded amplicons - melting curve analysis
22 Principles of Melting Curve Analysis Non-specific dsdna binding dyes that increase in fluorescence hundreds of folds when bound
23 Principles of Melting Curve Analysis Dye binding during amplification increase in fluorescence Dye release during melting decrease in fluorescence
24 Principles of Melting Curve Analysis During amplification During melting
25 Factors Affecting Melting Properties of an Amplicon Length of amplicon GC content Type and position of base change Complementarity of two strands Ionic strength of buffer
26 Readout for Sequence Variations in an Amplicon Change in melting temperature (Tm) of amplicon wild-type vs homozygous variant
27 Readout for Sequence Variations in an Amplicon Change in shape of melting curve Heterozygous variant
28 How to Detect Subtle Changes in Amplicon Sequence? Must detect subtle changes in melting curve DNA quality and PCR efficiency Amplicon size the smaller the better DNA binding dye non-inhibitory, saturating Instrumentation high temperature precision, increased measurements per time unit and drop of temperature, high sensitivity optics, data analysis software > High Resolution Melting Analysis
29 Importance of Dye Saturation
30 Data Presentation Reference - Variant Fluorescence (Y-axis) Temperature (X-axis)
31 X-Axis Normalisation Temperature Shifting Minimise well-to-well temperature variation
32 Disadvantage of Temperature Shifting
33 Advantages of HRMA for Mutation Scanning Simple Fast Low cost Non-destructive High analytical sensitivity
34 Application of HRMA in Haematological Diagnosis Scanning small sequence variations, especially heterozygous Considerations: Known or unknown mutations Single or heterogeneous/widespread mutations Size and number of target gene Sensitivity required Instrumentation
35 HRMA for Scanning Beta Globin Gene Mutations HBB is 3 kb, heterogeneous mutations spreading over all 3 exons and introns 6 amplicons, covering from the promoter to 3 polya tail, size bp
36 HRMA for Scanning Beta Globin Gene Mutations 39 sequencing confirmed wide-type samples and 35 mutants/variants Mutants/variants include small indels and single nucleotide substitutions All 4 SNP classes included (Tm shift from <0.2 to >0.5 C) Mostly heterozygous, some homozygous and compound heterozygous samples
37 HRMA for Scanning Beta Globin Gene Mutations Common thalassaemia mutations in Hong Kong CD41-42 (-CTTT), CD17 (A>T), CD43 (G>T), CD71-72 (+A), IVSII nt 654 (C>T) and Hb E (CD26 G>A) A variety of Hb variants Hb D-Iran, Hb G-Taipei, Hb G-Coushatta, Hb Rothschild, Hb Pokfulam, Hb New York, Hb Hope, Hb S/C, Hb J-Bangkok, Hb D-Los Angeles, Hb Tak
38 HRMA Output
39 HRMA Results
40 HRMA Results
41 HRMA for Scanning Beta Globin Gene Mutations - Summary Mutant/Variant Normal Heterozygous Homozygous Single nucleotide substitution Small Indel Single nucleotide substitution Small Indel Normal HRMA Abnormal HRMA PolyA tail (A>C), PolyA tail (+A), IVS II splice site (-T) Hb E homo, 41/42 (-CTTT) homo
42 Performance of HRMA for Scanning Beta Globin Gene Mutations Heterozygous variations Sensitivity = 90% (a few rare mutations not detected,? need re-design of primers) Specificity = 100% Good pre-sequencing scanning test in clinical samples when pre-test probability is high Not sufficient for genotype calling
43 Shih HC et al. Clinical Biochemistry 2009;42:
44 Performance of HRMA for Scanning Beta Globin Gene Mutations Homozygous variations Sensitivity = 60% Specificity = 100% Not suitable for pre-sequencing scanning in clinical samples Mixing with normal amplicon to generate heteroduplex when pre-test probability is high or use smaller amplicon
45 Role of HRMA in Diseases with Heterogeneous Mutations Competing techniques Direct nucleotide sequencing, multiplex mutationspecific PCR, reverse dot-blot array Best for scanning diseases encoded by large gene(s) with no mutation hotspots e.g. haemophilia A, von Willebrand disease
46 HRMA for Scanning of Mutation in Hairy Cell Leukaemia A single BRAF V600E (GTG>GAG) mutation, highly sensitive and specific Class 4 SNP with <0.2 C difference One amplicon of 136 bp in exon 15
47 Unique Challenge in Hairy Cell Leukaemia Pancytopenia > small amount of DNA in blood Marrow fibrosis > DNA harvest from formalinfixed paraffin-embedded trephine biopsy
48 Sensitivity of Detection Established by dilution studies of HT29 cell line
49 Sensitivity of Detection Established by dilution studies of HT29 cell line
50 Amplification Refractory Mutation System for BRAF V600E
51 HRMA for Scanning of Mutation in Hairy Cell Leukaemia 6 archive peripheral blood samples of hairy cell leukaemia 5 formic acid-decalcified archive trephine biopsy samples of hairy cell leukaemia 13 archive peripheral blood samples of splenic lymphoma with circulating villous lymphocytes
52 HRMA Results for HCL and SLVL from Peripheral Blood
53 HRMA Results of HCL from Trephine Biopsy Poor amplification in trephine samples
54 HRMA Results of HCL from Trephine Biopsy Poor amplification in trephine samples
55 HRMA for Scanning BRAF V600E Mutation - Summary HCL Peripheral Blood* HCL Trephine SLVL Peripheral Blood Normal HRMA Abnormal HRMA Poor DNA Quality * Leukaemia cells range from 1 32% by flow cytometry
56 Method Comparison for BRAF V600E Detection in HCL Diagnosis Sample Type Sample Year HRM ARMS Sequencing Cell % by Flow HCL Peripheral blood 2006 positive positive positive 32% HCL Peripheral blood 2009 positive positive positive 27% HCL Peripheral blood 2012 positive positive positive 24% HCL Peripheral blood 2012 positive positive positive 4% HCL Peripheral blood 2010 positive positive 1% HCL Peripheral blood 2009 Cp outlier positive positive 32%
57 Method Comparison for BRAF V600E Detection in HCL Diagnosis Sample Type Sample Year HRM ARMS Morphology HCL Trephine 2009 Cp outlier positive Extensive HCL Trephine 2010 Cp outlier positive Moderate HCL Trephine 2005 Cp outlier poor DNA quality Extensive HCL Trephine 2005 Cp outlier poor DNA quality Extensive HCL Trephine 2008 Cp outlier poor DNA quality Extensive
58 Method Comparison for BRAF V600E Detection in HCL Diagnosis Sample Type Sample Year HRM ARMS Sequencing SLVL Peripheral blood 2010 SLVL Peripheral blood 2010 SLVL Peripheral blood 2006 SLVL Peripheral blood 2006 SLVL Peripheral blood 2006 SLVL Peripheral blood 2005 SLVL Peripheral blood 2007 SLVL Peripheral blood 2008 SLVL Peripheral blood 2008 SLVL Peripheral blood 2010 SLVL Peripheral blood 2006 SLVL Peripheral blood 2006 SLVL Peripheral blood 2008 Cp outlier
59 Performance of HRMA for Scanning BRAF V600E in HCL Sensitivity for peripheral blood specimens = 83% (limitation by DNA quality, not false negativity) Specificity for peripheral blood specimens = 100% A result excludes presence of the mutation A positive result indicates presence of a mutation and supports the diagnosis in the correct setting (BRAF V600E very rarely found in chronic lymphocytic leukaemia and B-acute lymphoblastic leukaemia)
60 Sufficient for Genotype Calling? Need for Sequencing Confirmation? Variant mutations in BRAF exon 15 reported BRAF V600E + D594D in a HCL patient BRAF K601E in a SLVL patient BRAF D564N in a plasma cell myeloma patient BRAF V600V in a plasma cell myeloma patient Sequencing confirmation advisable when phenotype or HRM curve shape is atypical
61 Formalin-fixed Paraffin-embedded Samples for HRMA of BRAF Exon 15 Mutations Reports of success: Ney JT et al. Arch Pathol Lab Med. 2012;136: Carbonell P et al. J Mol Diagn 2011;13: None on decalcified samples Application of a BRAF V600E Mutation-specific Antibody for the Diagnosis of Hairy Cell Leukemia Andrulis M et al. Am J Surg Pathol 2012;36:
62 Role of HRMA in Diseases with a Unique Mutation Competing techniques Targeted sequencing, ARMS, pyrosequencing Can replace the more costly and time and labourintensive specific mutation detection techniques, especially when modified for genotyping Small amplicon genotyping Unlabelled probe genotyping
63 Conclusions HRMA is a promising mutation scanning tool Good quality specimens and PCR setup are essential Suitable for investigation of a variety of inherited and acquired haematological diseases
64 Acknowledgements Ms. Amy Chan Ms. Mandy Ho Ms. Pesy Leung Dr. LP Chung Children s Thalassaemia Foundation
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