Suthat Fucharoen1, Pranee Winichagoon1, Saovaros Svasti1, Orapan Sripichai1, Thongperm Munkongdee1, Kittiphong Paiboonsukwong1, Nantika Panutdaporn2, Kai Tang2

1Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand, 2Nanyang Technological University, School of Biological Sciences, Singapore.

α-Thalassemia !

--THAI

ζ2 α1α2 θ1

0kb 10kb 20kb 30kb

ψζ1ψα2 ψα1inter- ζ HVR 3'HVR

--SEA

-α3.7-α4.2

2 α-globin genes deleted = α–thalassemia 11 α-globin gene deleted = α–thalassemia 2

beta gene cluster

11

β-Thalassemia

point mutation >200 mutations

KNOWN MUTATION: PCR-based methods 1. Electrophoresis:

- Gap-PCR: deletion- Amplification Refractory Mutation System

(ARMS)/Allele specific PCR: point mutation- PCR/RE digestion (RFLPs)

2. Allele-specific oligonucleotide hybridization: - Dot-blot hybridization (population screen) - Reverse dot-blot hybridization (individual screen)

MUTATION DETECTION (1)

UNKNOWN MUTATION1. Southern blot hybridization :

Large deletionRestriction Fragment Length Polymorphism (RFLP) -> Linkage analysis

2. Denaturing gradient gel electrophoresis (DGGE) & Single strand conformation polymorphism SSCP):

Point mutation3. PCR/Sequencing:

Point mutation

MUTATION DETECTION (2)

M -/- +/- +/+ Primer-F Primer-R

Dde I

Exon 1

A/G

300 bp

450 bp

150 bp

* 5’ 3’

A

Restriction Fragment Length Polymorphism (RFLP)

G

300

450

150

Gel electrophoresis

Allele specific PCR (ASPCR)

400

200

Gel electrophoresis

DN

A la

dder

N M

Normal

N M

Heterozygote

N M

Homozygote

Normal

Mutant

Normal

Mutant

Reverse

Reverse

Dot ASO probe on nylon membrane

Hybridization with denatured biotin label PCR product

Wash

Detection

Reverse Dot Blot Hybridization

= Biotinylated PCR

= Streptavin Akaline Phosphatase = BCIP/NBT (Substrate)

-28 CD17 CD26CD19 CD35IVSI-1 IVSI-5

N N N N NNN

N N N N NNN

-28 CD17 CD26CD19 CD35IVSI-1 IVSI-5

normal

Normal Probe

Mutant Probe

-28 CD17 CD26CD19 CD35IVSI-1 IVSI-5

N N M N NNN

N N N N NNN

-28 CD17 CD26CD19 CD35IVSI-1 IVSI-5

CD26 heterozygote or CD26 trait

Normal Probe

Mutant Probe

-28 CD17 CD26 CD19 CD35 IVSI-1 IVSI-5

N N N N N M N

N N N N N M N

-28 CD17 CD26 CD19 CD35 IVSI-1 IVSI-5

Compound heterozygote CD19/IVSI-1

Normal ProbeMutant Probe

Mutations covered by ViennaLab β-, α- thalassemia Globin StripAssays®

•  Mass Spectrometry

•  Melting Curve Analysis

•  High-Resolution Melting Curve Analysis (HRM)

•  Multiplex Ligation-dependent Probe Amplification (MLPA)

•  Microarray assay

More DNA Analysis for β and α-Thalassemia

MassARRAYTM

High throughput SNP detection

Primer extension + Mass spectrometry

TOF v α 1/m

Matrix-Assisted Laser Desorption/ Ionization (MALDI)

•  Biomolecules are mixed with a matrix (usually an organic acid) and dried on sample holder.

•  A laser is focused onto a small area of the sample, and a pulse desorbs a plume of analyte and associated cations that are then accelerated into the analyzer.

Matrix-Assisted Laser-Desorption / Ionization Time-of-Flight Mass Spectrometry

MALDI-TOF MS •  Improved mass resolution in

MALDI-TOF MS has been obtained by the utilization of a reflectron.

•  The reflectron, located at the end of the flight tube, is used to compensate for the difference in flight times of the same m/z ions of slightly different kinetic energies by means of an ion reflector.

1020 1066 1112 1158 1204 1250Mass (m/z)

0

2.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

1087.63

1071.56

1178.65

1088.641072.561149.67

1179.651126.581074.57 1150.68

1192.671127.591089.64 1180.661073.57 1144.70 1193.681098.57 1151.68 1226.63

α

β

G γ

Aγ

β

α

1020 1066 1112 1158 1204 1250Mass (m/z)

0

2.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

1087.63

1071.56

1178.65

1088.641072.561149.67

1179.651126.581074.57 1150.68

1192.671127.591089.64 1180.661073.57 1144.70 1193.681098.57 1151.68 1226.63

1020 1066 1112 1158 1204 1250Mass (m/z)

0

2.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

1087.63

1071.56

1178.65

1088.641072.561149.67

1179.651126.581074.57 1150.68

1192.671127.591089.64 1180.661073.57 1144.70 1193.681098.57 1151.68 1226.63

α

β

G γ

Aγ

β

α

Globin chain separation by mass spectrometry

MILD % Hb F – 39

MILD % Hb F – 36

Evaluate to what extent polymorphism within the β-globin gene region contributes to variation in disease severity

β β αGγAγ β+γXmn I -/-

β β αGγAγ β+γXmn I +/+ 665LP

006M

Association Xmn I genotype with Gamma Expression in β-Thalasemia/HbE

MS Genotyping

HbE 6565

WT at HbE

6894

HbA 6968

HbS 7931

Wt at HbE 6894

β-globin biplex mutation assay

HbS/S & Wt/Wt

HbA/A & Wt/HbE

Several MALDI-TOF mass spectrometry spectra are shown depicting various allele combinations called during the β-globin biplex mutation assay. In this biplex assay, genotypes were called at codons 5,6 & 26 of the β-globin gene.

Cycle #

Log

Targ

et D

NA

Theoretical

Amplification is exponential, but the exponential increase is limited:

Real-Time PCR allows us to ‘see’ the exponential phase so we can calculate how much we started with.

●  A linear increase follows exponential ● Eventually plateaus Real Life

Threshold

CT

Reality vs Theory in PCR

Primer & probe set performance assay for ���tri-plex qPCR of α-, β-, and γ-Globin

Ten-fold serial dilution of standard plasmid cDNA from a tri-plex qPCR

100 ng/µl, first step

Concentration of cDNA dilution series corresponding to total RNA input in first step. 2 µl of cDNA was used in each 25 µl qPCR reaction

10 ng/µl 1 ng/µl 100 pg/µl 1 pg/µl 10 pg/µl

α-, β-, and γ-Globin Standard Graph

qRT-PCR primers sequence

Correctly/aberrantly spliced βE-globin mRNA

α/non α-globin mRNA ratio

Summary

Conventional PCR

DNA extraction

PCR amplification

Agarose gel electrophoresis Post-PCR

Pre-PCR

PCR

M = 100 bp DNA ladder plus Lane 1 = α-thalassemia 1 heterozygote (–SEA type) Lane 2 = Not found α-thalassemi 1 (–SEA type)

1200 1500 2000

M 1 2

Normal

α-thalassemia 1

Gap PCR for α-thalassemia

II: Melting Curve!I: DNA Amplification !

85.5 °C

Melting Curve Analysis

Melting Curve Analysis

α-Thalassemia diagnosis by melting curve analysis

Summary

Point mutation genotyping by High Resolution Melting Curve Analysis (HRM)

Agarose Gel Electrophoresis

High Resolution Melting Curve Analysis

DNA from known genotype, wild type, heterozygote and homozygote mutant

Result of unknown samples

Control 41/42(-CTTT) trait Normal Unknown/ HBA2=4.8%

Beta thalassemia gene scanning: a case of beta thal trait

Normalized and temperature-shifted difference plot

Tm Normal: 75.56 C T rait 3.5 kb: 76.45 / 78.15 C Homoz 3.5 kb: 78.05 C

HRMA beta thalassemia 3.4 kb deletion

normal homozygous beta Thal 3.5 kb deletion

Trait 3.5

ζ ψζ αDψα1 α2 α1 θ

HBZ HBZps HBD HBA1ps HBA2 HBA1 HBQ

150k 160k 170k 140k 180k

Luc7L C16orf35 w

w

w

w 3’HVR interζHVR

- -MC --CAL --Dutch I --THAI --MED II --FIL --KOL --BRIT -(α)20.5 --MA --SA --MED 1 --SEA --PP --CANT --SPAN --GEO -(α)5.2

Example: deletions that cause α0-thalassemia

- -00682 - -00591 - -00653 - -00690 --AW

Novel deletions detected by MLPA

Multiplex Ligation-dependent Probe Amplification (MLPA)

C.L.Harteveld Hemoglobinopathies Laboratory

deletion

Deletion of 1 allele: half the amount of PCR product

MLPA

C.L.Harteveld Hemoglobinopathies Laboratory

ABI 3730 normal control

heterozygote mutant

MLPA

Peak height normal Peak height patient = 1 ratio No deletion:

One allele deleted: ratio = 0.5

Both alleles deleted: ratio = 0

Multiplex Ligation-dependent Probe Amplification

C.L.Harteveld, LUMC !

Probe hybridisation Probe ligation PCR of ligated probes with tag primers

Target A Target A

Target B Target B

3’

3’

3’

3’

5’

5’

5’

5’

5’ tag1 tag2 tag2 tag1

stuffer sequence hybridisation sequence

primer sequence

primer sequence

5’ 3’

3’

Micro-array = large number of DNA sequences, immobilized on a solid surface in an ordered

manner

Target cell !

In-situ synthesized oligonucleotides

micro-array slide

Labeled DNA!

scanning!

Image Generation !

!

Hybridization, wash!

3.7 Kb deletion type by gap PCR!

4.2 Kb deletion type by gap PCR!