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