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Samuel S. CHONG, PhD
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of SINGAPORE
Preimplantation Genetic Diagnosis Centre, Khoo Teck Puat National University Childrens Medical Institute, National University Health System, SINGAPORE
Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, National University Health System, SINGAPORE
Applications of Molecular Genetics to the Diagnosis and Prevention
of Thalassemia
Alpha- and Beta-Thalassemia Autosomal recessive disorders of hemoglobin (Hb) synthesis.
Among the most common monogenic diseases globally.
~14,000 births of lethal (Hb Barts hydrops fetalis syndrome ) and severe (Hb H disease) -thalassemia annually.
Within Asia, the -3.7 and -4.2 +-thalassemia single -globin gene deletions, as well as the --SEA, --FIL, --THAI and --SA 0-thalassemia double -globin gene deletions are prevalent.
~23,000 births of severe -thalassemia major (Cooleys anemia) annually.
Predominantly caused by different combinations of + and 0 point mutations within the -globin gene.
No effective cure for - or -thalassemia.
Prevention through prenatal diagnosis, however, involves termination of affected pregnancies.
Genetic Basis of -/- Thalassemia -thalassemia
Due to defects within the chromosome 16p13.3 -globin gene cluster.
Most commonly caused by deletions including HBA2 (2 globin) and HBA1 (1 globin), and sometimes HBZ ( globin) gene.
Point mutations are less common.
-thalassemia Due to defects within the chromosome 11p15.5 -globin gene cluster.
Most commonly caused by point mutations within the HBB ( globin) gene. Deletions are less common.
PCR Sequencing of HBA1, HBA2 and HBB Genes
HBA1 amplicon: 1380 bpHBA2 amplicon: 1303 bp
Exon 1 Exon 2 Exon 3
ATAAACCAAT Cap
HBB amplicon: ~1700 bp
~800 bp ~600 bp
Exon 1 Exon 2 Exon 3
ATAAACCAAT Cap
Common -Globin Point MutationsWildtype HBA2 sequence
Constant Spring mutation: TAACAA
Pakse mutation: TAATAT
IVS II,654 CT Point Mutation
C/T
G/A
sens
ean
tisen
se
-Thalassemia Mutations in Singapore
Dec 2005 KKH
N=2409
Cds41/42 (-TCTT)(27.3%)
IVS2 nt 654 (C/T)(22.3%)
HbE(20.5%)
-28 (A/G)(6.6%)
Cd17 (A/T)(4.9%)
IVS1 nt 5 (G-C)(6.4%)
Cds71/72 (+A)(1.2%)IVS1 nt 1 (G-T)(1.3%)
42 mutations(9.1%)
Unknown(0.6%)
IVS II,654(CT) Minisequencing Assay
Mutant allele (T)
TSNP 65453 AAT TCC A TTA TCG
AC G
Wild-type allele (C)
654-R654-F
35 TTA AGG C/T AAT AGC
AT GSNP 654
53 AAT TCC G TTA TCGC
HeterozygousHomozygous Wildtype Homozygous MutantC TC T
HBB Multiplex Minisequencing
InI 5 Cd41/42 -29 Cd26 -28 Cd17Cd71/72
InII 654
Normal
Cd41/42 / InII 654
CG
CT
-29 /
AG
InI 5-R
Cd41/42-F InII 654-F
-28-RCd17-R Cd26-R
Cd71/72-F-29-F
aat t
gc
AT
GC
CG
GC
cg
HBA2 Multiplex Minisequencing
/
Cd0 CS Cd30 Ps SD QS Cd59
CS / --SEATC
Ps /
TA
Cd30-F
TA
GC
GC
TA
TA
AT
TA
Cd0-F CS-F
Ps-R
SD-F QS-F
Cd59-R
Alpha Globin Gene Deletions
Phenotype Genotype
Normal /
Heterozygous -thalassemia 2silent carrier -/
Heterozygous -thalassemia 1-thal trait --/
Homozygous -thalassemia 2-thal trait -/-
Hb H disease (4) --/-
Barts hydrops fetalis (4) --/--
National Thalassemia Registry, KKH
N=3056
-3.7(19.1%)
-4.2(4.3%)
QS(1.1%)
--SEA(70.6%)
CS(1.5%)
--Unk(0.6%)
--Thai(0.9%)
--Fil(0.4%)
Other(1.2%)
Cd30(0.1%)
Cd59(0.6%)
-Thalassemia Mutations in Singapore
Detecting Deletions by Gap PCR
20kb Deletion
Forward primer Reverse primer
0.5kb 0.5kb
PCR
1.5 kb fragment is amplified;21 kb fragment unamplifiable
1 kb fragment is amplified
5 3
PCR
Reverse primer
1 kb
Multiplex Gap-PCR forCommon -Thalassemia Deletions
112 2 1 1
--FIL
-3.7
--SEA
--THAI
IZ-HVR 3-HVR
-4.2
--MED-()20.5
SEA-F
FIL-F4.2-F
2/3.7-F 2-R4.2-R
3.7/20.5-R
FIL-R
SEA-RTHAI-F THAI-R
--SA
MED-F MED-R20.5-F
52 53
-Thalassemia Multiplex Gap-PCR Results
-()20
.5/--
MED
/
/-
3.7
-3.
7/-
3.7
/--
MED
/-(
)20.5
/-
4.2
-3.
7/-
4.2
/--
SEA
-3.
7/--
SEA
-4.
2/--
SEA
--SEA
/--SE
A
/--
THAI
-3.
7/--
THAI
--MED
/--M
ED
/--
FIL
--SEA
/--FI
L
M3 kb
2.5 kb2 kb
1.5 kb
1 kb
.75 kb
.5 kb
-()20.5
LIS1-3.72-4.2--SEA--THAI
--FIL
--MED
Tan et al, Blood 98:250-251, 2001
Preimplantation Genetic Diagnosis
Genetic testing on blastomere(s)/trophectoderm of morula/blastocyst stage
embryos derived from in vitro fertilization (IVF).
Transfer disease-free (normal/unaffected) embryo(s) for implantation.
Detect inherited monogenic disorders & chromosomal abnormalities.
Unlike prenatal diagnosis, affected pregnancies are avoided altogether - avoids
pregnancy terminations.
Thalassemia PGD
Thalassemia couples may be carriers of different mutations/deletions.
PGD for -thalassemia using gap-PCR entails significant assay customization.
PGD for -thalassemia also requires customization.
Most PGD assays now also include analysis of linked microsatellite markers
To track transmission of normal and mutant alleles.
To detect external DNA contamination, to prevent misdiagnosis.
To monitor allele drop-out (ADO), to prevent misdiagnosis.
Informative markers are required entails significant assay customization.
-thalassemia PGD Simultaneous Deletion and Linkage Analysis
IZ-HVR 3 HVR
11
X2 X1Z2 Z1Y2 Y1
-3.7-4.2 --SEA--FIL
--THAI--SA
--MED-()20.5
1 2 3 4 5 6 7 8 9 10 x105 bp
Tel Cen
2 2 1 1
Hb Barts and Hb H Disease
IZ-HVR 3 HVR
2 11 2 1 1X2 X1Z2 Z1
Y2 Y1
Hb H(-3.7/--SEA)
-3.7
--SEA
Hb H(-4.2/--SEA)
-4.2
--SEA
Hb HCS(CS/--SEA)
--SEA
Constant Spring
--SEA
--SEA
Hb Barts(--SEA/--SEA)
-globincluster
0.65
0.67
0.76
0.73
0.77
0.75
0.81
0.88
0.81
0.80
0.73
0.82
0.77
0.91 0
.94
0.90
0.87
0.59
0.64
0.76
0.86
0.69
0.740
.77
0.63
0.76
0.41
0.88
0.87
0.81 0.
86
0.90
0.89
0.87
0.86
0.75
0.94 0
.95
0.87
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
D16S521 16PTEL03 16PTEL05 16PTEL06 HBA370 HBA572 HBA876 HBA950 D16S525
Obs
erve
d He
tero
zygo
sity
0.70
0.80
0.79
0.90
0.71
0.41
0.66
0.87
0.75
0.85
0.90
CH ML IN CA AA Combined average
Observed heterozygosities of-thalassemia microsatellite markers
PGD Assay for Hb Barts & Hb H Disease
Heat
MultiplexPCR
Lysis Buffer
Blastomere / cell Neutralizing Buffer
Haplotype Analysis
GeneScanAnalysis
DiagnosticReport
155
181
256
380 382
393 429
485
254
179 484169 227
233
281
285
331
16PTEL05
D16S525 HBA876 HBA950 HBA370HBA57216PTEL03 D16S521
16PTEL06 Y1
Mutation Detection
157
184256 374
382
393
429379
180
225
159163
184176 256
429
379
378
488484
484
381384
161
225
282441
282
331
331
Hb Barts embryo: --SEA/--SEA
157 176384
379
441429
488
382
180
163 433
Wife: /--SEA
Husband: /--SEA
260
157378180 256
382
379
441
484
433
331
282225
163
Carrier embryo: /--SEA
184256
379429
484
282
374161
331 Unaffected embryo: /
225
260
433
393
Hb BartsPGD
/ /--SEA
/--SEA /--SEA
--SEA/--SEA
379157
----
488260382441180
381159
----
488262384429176
379157
----
488260382441180
379163282305331225484256378433184
393161282305331225484256374429184
379163282305331225484256378433184
D16S52116PTEL0316PTEL05HBA2Y116PTEL06HBA370HBA572HBA876HBA950D16S525
D16S52116PTEL0316PTEL05HBA2Y116PTEL06HBA370HBA572HBA876HBA950D16S525
381159
----
488262384429176
379163282305331225484256378433184
379157
----
488260382441180
393161282305331225484256374429184
305
488
262305
159
260262
381
305
305
378
184488
IZ-HVR 3 HVR
2 11 2 1 1X2 X1Z2 Z1
Y2 Y1
--SEA
16PTEL05D16S525 HBA876 HBA950