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
 Haematology and Genetics
Blood cancers and inherited haematological diseases

“Sickle Cell Anemia, a Molecular Disease”

Pauling L et al. Science November 25, 1949
 Haematology and Genetics
Blood cancers and inherited haematological diseases

“A minute chromosome in chronic granulocytic leukemia”

Nowell P, Hungerford D. Science November 18, 1960
 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
 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
 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
 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
 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

 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 1.2

0.8

0.6

0.4

0.2

0
HBE1 exon 01

HBBP1 exon 01

HBB promotor
HBG2 exon 03

HBD exon 03
HS4

HS3

HS2

HBB exon 02

HBB exon 03A

11-005.2

11-005.2

11-005.2

c
 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
 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
 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

1.2

0.8

0.6

0.4

0.2

0
HBBP1 exon 01

HBB promotor
HBE1 exon 01

HBD exon 03
HS4

HS3

HS2

HBG2 exon 03

HBB exon 03A

HBB exon 02
11-005.2

11-005.2

11-005.2

c
 Ultimate Resolution in
Mutation Detection
Reading at the single nucleotide level

– Whole gene sequencing

• Sanger sequencing

– Whole genome sequencing

• Next generation sequencing
 Gene Sequencing to Detect
Unknown Mutations

Direct nucleotide sequencing

vs
Mutation scanning followed by targeted sequencing
 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
 Direct or Scanning?
Capacity for sequencing
vs
Setup for mutation scanning
 Principles of Mutation Scanning
Amplify candidate gene in segments (amplicons)

Scan for sequence variation in segments

Sequence the abnormal segment

 Scanning for Sequence Variation in
Amplicons
Detection of a change in physical property
- alteration in gel mobility

- alteration in denaturation
rate (melting)
 Altered Gel Mobility
Single strand secondary structure (folding)

Double strand secondary structure (duplex)

Size (length after chemical cleavage)

 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)
 Altered Melting Property under
Changing Temperature
Binding and release of fluorescent dye from
double-stranded amplicons

- melting curve analysis

Principles of Melting Curve Analysis

Non-specific dsDNA binding dyes that increase in

fluorescence hundreds of folds when bound
Principles of Melting Curve Analysis

Dye binding during amplification –

increase in fluorescence

Dye release during

melting – decrease in
fluorescence
Principles of Melting Curve Analysis

During amplification During melting

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

 Readout for Sequence Variations in an
Amplicon
Change in melting temperature (Tm)
of amplicon
– wild-type vs homozygous variant
 Readout for Sequence Variations in an
Amplicon
Change in shape of melting curve
– Heterozygous variant
 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

Importance of Dye Saturation
Data Presentation

Reference -
Variant

Fluorescence (Y-axis) Temperature (X-axis)

 X-Axis Normalisation –
Temperature Shifting

Minimise well-to-well temperature variation

Disadvantage of Temperature Shifting
 Advantages of HRMA for Mutation
Scanning
Simple
Fast
Low cost
Non-destructive
High analytical sensitivity
 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
 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 112 – 343 bp
 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
 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
HRMA Output
HRMA Results
HRMA Results
 HRMA for Scanning
Beta Globin Gene Mutations - Summary
Mutant/Variant

Heterozygous Homozygous
Normal

Single nucleotide Small Single nucleotide Small

substitution Indel substitution Indel

Normal HRMA 39 1 2 1 1

Abnormal
0 22 5 2 1
HRMA

PolyA tail (A>C), PolyA tail (+A), IVS II splice site (-T)
Hb E homo, 41/42 (-CTTT) homo
 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
Shih HC et al. Clinical Biochemistry
2009;42:1667–1676
 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
 Role of HRMA in Diseases with
Heterogeneous Mutations
Competing techniques
– Direct nucleotide sequencing, multiplex mutation-
specific 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
 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

 Unique Challenge in
Hairy Cell Leukaemia
Pancytopenia > small amount of DNA in blood

Marrow fibrosis > DNA harvest from formalin-

fixed paraffin-embedded trephine biopsy
 Sensitivity of Detection
Established by dilution studies of HT29 cell line
 Sensitivity of Detection
Established by dilution studies of HT29 cell line
Amplification Refractory Mutation System
for BRAF V600E
 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
HRMA Results for HCL and SLVL
from Peripheral Blood
HRMA Results of HCL from
Trephine Biopsy

Poor amplification in trephine samples

HRMA Results of HCL from
Trephine Biopsy

Poor amplification in trephine samples

 HRMA for Scanning
BRAF V600E Mutation - Summary

HCL HCL SLVL

Peripheral Blood* Trephine Peripheral Blood

Normal HRMA 0 0 12

Abnormal HRMA 5 0 0

Poor DNA Quality 1 5 1

* Leukaemia cells range from 1 – 32% by flow cytometry

 Method Comparison for BRAF V600E
Detection in HCL
Cell % by
Diagnosis Sample Type Sample Year HRM ARMS Sequencing
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 negative 1%

HCL Peripheral blood 2009 Cp outlier positive positive 32%

 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

 Method Comparison for BRAF V600E
Detection in HCL
Diagnosis Sample Type Sample Year HRM ARMS Sequencing
SLVL Peripheral blood 2010 negative negative negative
SLVL Peripheral blood 2010 negative negative negative
SLVL Peripheral blood 2006 negative negative negative
SLVL Peripheral blood 2006 negative negative negative
SLVL Peripheral blood 2006 negative negative negative
SLVL Peripheral blood 2005 negative negative negative
SLVL Peripheral blood 2007 negative negative negative
SLVL Peripheral blood 2008 negative negative negative
SLVL Peripheral blood 2008 negative negative negative
SLVL Peripheral blood 2010 negative negative negative
SLVL Peripheral blood 2006 negative negative negative
SLVL Peripheral blood 2006 negative negative negative
SLVL Peripheral blood 2008 Cp outlier negative negative
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 negative 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)
 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
 Formalin-fixed Paraffin-embedded Samples
for HRMA of BRAF Exon 15 Mutations
Reports of success:
– Ney JT et al. Arch Pathol Lab Med. 2012;136:983–992
– Carbonell P et al. J Mol Diagn 2011;13:467–473
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;36:1796–1800
 Role of HRMA in Diseases with a
Unique Mutation
Competing techniques
– Targeted sequencing, ARMS, pyrosequencing

Can replace the more costly and time and labour-

intensive specific mutation detection techniques,
especially when modified for genotyping
– Small amplicon genotyping
– Unlabelled probe genotyping
 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
 Acknowledgements
Ms. Amy Chan
Ms. Mandy Ho
Ms. Pesy Leung

Dr. LP Chung

Children’s Thalassaemia Foundation