Suthat Fucharoen M.D.

Thalassemia Research CenterInstitute of Molecular Biosciences

Mahidol University, Thailand

(suthat.fuc@mahidol.ac.th)

Precision Medicine and Thalassemia

OUTLINE

• Definition: Precision/Personalized medicine

• Global problem of thalassemia

• Precison medicine and thalassemia

– Molecular defects in thalassemia

• Prenatal diagnosis and PGD

• Genotype-phenotype interaction

• Gene therapy and targeted therapy

• NGS and mass screening for thalassemia

“Use of new methods of molecular analysis to better manage a patient’s disease or predisposition towards a disease.

It aims to achieve optimal medical outcomes by helping physicians and patients choose the disease management approaches likely to work best in the context of that patient’s genetic and environmental profile”

(from the Personalized Medicine Coalition)

Precision/Personalized Medicine

From personalized medicine to precisionmedicine

• Precision medicine implies that diseases are defined by underlying molecular mechanisms rather than traditionalsigns and symptoms.

• Some are skeptical that medicine will really be personalized, while others say that physicians have always adaptedtreatment to their individual patients.

Lancet 378 : 1678, 2011

Toward precision medicine

US National Research Council, Nov 2011

Distribution of thalassemia disorders worldwide

thalassemia: China, SE Asia, Africa, Middle East, Mediteranian

thalassemia: Mediteranian , Middle East, India, Pakistan, Africa, SE Asia

Thal

HbS

HbC

HbE

Thalassemia is now common worldwide due to migration

spreading to much of Europe, the Americas, Asia and Australia

Anemia

Massive erythropoiesis

Blood transfusion

-globin genes

Excess unbound α-globin chains

Inclusion bodies

Increased RBC destruction

Extramedullary hematopoiesis

Inadequate nutrition

Defective development

Iron overloadCardiac failure, Cirrhosis, DM

Jaundice

Hyperuricemia

RE hyperplasia

Hepatosplenomegaly

Hypersplenism

Increased infection

Bone changes

Increased bilirubin production

Gallstones

Membrane damage

Increased iron absorption

Pathophysiology of -Thalassemia/Hb E Disease

NUCLEUS

Transcription

mRNA precursor

Processing

mRNA

GT AG GT AG

AATAAA

IVSI IVSII

Exon 1 Exon 2 Exon 3

CAP AAAAA….

NH2

TAAATGTATA

CCAAT

CACCC

5’ 3’

CYTOPLASM Translation

CAP AAAAA….

NH2

NH2

GlobinGrowing chain

(modified from Higgs DR, Disorder of Hemoglobin, 2009)

Interstitial deletional -thal 1 and -thal 2

--/

-/

-thal 1 (--SEA, --THAI & --FIL)

and -thal 2 (-3.7 & -4.2)

Hb Constant Spring

Hb Paksé

Chromosome 16p 13.3

Total 58 non-deletional mutations affecting either a 2, a 1 or –a alleles

Molecular spectrum of non-deletional -thalassemias

T / or T/ or - T/

69 Point mutations Initiation Codon

Splicing Defects

Termination Codon

Polyadenylation

Missense

Non-deletional thalassemias

CS = Hb Constant Spring

PS = Hb Paksé

131-132,+T = Hb Pak Num Po

125Leu->Pro = Hb Quong Sze

109Leu->Arg = Hb Suan Dok

59Gly->Asp = Hb Adana

intA-G = Initiation codon mutation

Cd 30,del = Codon 30 deletion

AATA-- = Poly A mutation (Indian type)

UAA

Constant Spring

CAA, Gln

Icaria

AAA, LysSeal Rock

GAA, Glu

Koya Dora

UCA, Ser

Pakse

UAU, Tyr

UGA

(Stop)UAG

(Stop)

UUA

(Leu)

UAC

(Tyr)

Stop codon mutations -globin gene

Not compatible with two a-chain loci,

or there is always an a-thal gene adjacent to that of Hb J-Tongariki.

(74Asp->His)

(115Ala->Asp)

A?? -thal 1

? -thal 2,Hb A

?? no A, but not hydrops

Duplication of the a- chain locus

-Globin Genotypes

Hb Bart’s hydropsfetalis

Hb H disease

Normal -Thalassemia 1-Thalassemia 2

Homozygous-Thalassemia 2

-Globin genotype in common alpha thalassemic diseases

Normal -Thalassemia 2 trait -Thalassemia 1 trait

Hb Bart’s hydrops fetalis Hb H diesease

Hb Constant Spring Hb H-CS disease Homozygous Hb CS

CS CSCSCS

1. /

2. /Int

3. /CD30del

4. /PS

5. /QS

6. /CS

Multiplex ARMS for common non-deletional

thalassaemia mutations in Thailand

Worrawut Chinchang & Vip Viprakasit; TSH Meeting 2006

Codon 30

(∆GAG)

Exon 1 Exon 2 Exon 3

Initiation codon

(ATGA-G) Codon 125 (Hb Quang Sze)

(CTGCCG)

Codon 142 (Hb CS)

(TAACAA)

R1

F1 Codon 142 (Hb Pakse)

(TAATAT)

M 1 2 3 4 5 6

869 bp772 bp

430 bp

253 bp234 bp184 bp

Olivieri NF. New England J. Med. 1999; 341(12):99-109.

Types of Mutations Resulting in β-Thalassemia

β-globin gene cluster on chromosome 11 (11p15.5)

β-Thalassemia: The β- globin chain synthesis is impaired

-globin cluster -globin cluster

Excess free -globin chain

-chain -chain

Promoter mutations

CACCC CCAAT ATAAAA ACA

-86 -28 +1

G,C

G

A,C

C,G

G,A

G,T,A

A,T

C

AATAAA

G

G

G

C

Poly A addition site mutations

STOP

745 C->G

pre-mRNA

mRNA

protein

579 654 C->T705 T->G

Cryptic splice site in intron 2 of -globin pre-mRNA

3’ 5’

AA

STOP

pre-mRNA

mRNA

proteinE

Splicing of E-globin pre-mRNA

CD26 G->A

AAG GTG AAC…GGT G GT GAG GCC CTG GGC AG GTTGGTATCAAGGTTACA

17 18 19 24 25 26 27 28 29 30 IVS-1

bE

A

… Arg Phe Phe Glu Ser …..

Normal … AGG TTC TTT GAG TCC…..

4 base deletion

… Arg Leu Met ….. STOP

Normal … AGG TTG AGT ….. TGA

Frameshift Mutation

Codons 41/42 (-TTCT)

Codons 71/72 (+A)

41 59

4140 42

β-Thalassemia Mutations

β+

Thalassemia: -86, C->G

ATA -28, A->G

ATA -29, A->G

Codon 19, A->G (Hb Malay)

Codon 126, T->G (Hb Dhonburi)

β0

Thalassemia: Codon 17, A->T

IVS I-1, G->T

IVS I-5, G->C

Codon 35, C->A

Codon 41, -C

Codons 41/42, -TCTT

Codons 71/72, +A

IVS II-654, C->T

3.4 kb deletion

Deletional -thalassemia

Detection of -Thalassemia Mutations by

Reverse Dot Blot Analysis

- Cividalli G, Nathan DG, Kan YW, Santamarina B, Frigoletto F.

Relationship of beta to gamma synthesis during the first trimester.

An approach to prenatal diagnosis of thalassemia. Pediatric Research,

8:553, 1974.

- Chang H, Modell CB, Alter BP, Dickinson MJ, Frigoletto FD,

Huehns ER, Nathan DG. Expression of the beta-thalassemia gene in the

first trimester fetus. PNAS, 72:3633, 1975

- Kan YW, Dozy AM, Alter BP, Frigoletto FD, Nathan DG.

Detection of the sickle cell gene in the human fetus: potential for

intrauterine diagnosis of sickle cell anemia. N Engl J Med 287:1,1972.

- CAO Antonio, Furbetta M, Galanello R, Melis MA,Angius A, Ximene A,

Rosatellli C,Ruggieri R,Addis M, Tuveri T,Falchi AM, Paglietti E, Scalas MT.

Prevention of homozygous beta-thalassemia by carrier screening

and prenatal diagnosis in Sardinia. J Hum Genetics, 33:592, 1981

Demonstration that β-thalassemia major can be diagnosed in utero

Case Registration (year)

35

31

2624

22 22

15

119

3

25 2421

11 10 9

5 4 300

5

10

15

20

25

30

35

40

93 94 95 96 97 98 99 00 01 02

Year

Nu

mb

er

Hb E/beta-thalbeta-Thal major

CMU MOPH

Preconception Prenatal

Preimplantation

Fertilization Implantation

Preimplantation Genetic Diagnosis (PGD/NIPD)

1. Better understanding of gene-gene interaction,

natural history, prognosis

2. Decision of therapeutic intervention

eg. Stem cell transplantation

3. Prenatal diagnosis and selective abortion

4. New therapeutic intervention

Thalassemia: Genotype-phenotype Interaction

Modifying factors of disease severity in -thal/HbE

E

E

g

g

E

Normal Asymtomatic +Thal

(Mild)

Severity Percentage HbF Absolute HbF (g/dL)

Mild (n = 233) 41.0 ± 11.2 3.1 ± 1.2

Moderate (n = 310) 35.3 ± 11.1 2.0 ± 0.9

Severe (n = 407) 31.2 ± 11.2 1.4 ± 0.7

Distribution of fetal hemoglobin (HbF; 2g2) in Thai 0-thalassemia/HbE cohort (n = 950)

Sripichai O, 2009

Severe 0/E0/E::-thal

(Mild)

E

g

0/E::High Hb F

(Mild)

/::/ /::0/ /::+/E /-::0/E /::0/E /::0/E

Results of genome-wide association

- Blood transfusion

- Iron chelators

- Antioxidants

- BM and Stem cell transplantation

- Hb F enhancer

- Gene therapy + siRNA/antisense oligonucleotide)

Treatment

CD34+ cells

-thal/HbE patient

HSC

Cavazzana et.al, Nature, 467: 318-322 (2010)

9 g/dL

βA(T87Q)-globin lentiviral vector

However:

- Risk of insertional mutagenesis

- Control gene expression

Gene therapy/transplantation 5 years ago

Complete transfusion independence since 4 years ago

The first conversion of a patient with severe beta-thalassemia/HbE to

transfusion independence by gene therapy

3838

Thai Patients Treated with Gene Therapy

Genotype Age at Tx

(yr)

CD34+

(cell/kg)

VCN Stop

Transfusion

(mo after Tx)

Last Hb

(gm/dL)

F/U time

(mo)

1 β thal/HbE 21 5.3 1.4 1 9.2 56

2 β thal/HbE 20 8.0 0.8 6 10.3 42

3 β thal/HbE 21 13.0 0.4 4 8.7 38

4 β thal/HbE 24 10.8 4.0 1 13.3 22

VCN = vector copy number

Zynteglo: Gene Therapy for Beta thalassemia

Brad ZakesFounder of Ethan Zakes

Foundation/CEO of CerevastMedical

Nick LeschlyCEO of bluebird bio

• Zynteglo was approved in Europe forBeta thalassemia patients in 2019

• Criteria for Beta thalassemia patients- Age 12 and older- require regular blood transfusions- have no matching donor for a stem cell

transplant

• Prices 1.58 million euros over 5 years

• An installment plan, with 315,000euros paid up front and four additionalannual payments due only if thetreatment continues to be effective

41

Non homologous end joining (NHEJ)

CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats

42

CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats

CRISPR Therapeutics and Vertex Pharmaceuticals listed

the first clinical trial for a treatment using CRISPR-Cas9

gene editing from US companies at ClinicalTrials.gov

on August 31. Participants will be adults with transfusion-dependent β- thalassemia, and the

treatment will reactivate production of fetal

hemoglobin. …CTX001 doesn't alter the mutant β globin gene behind both

diseases, but instead switches on the fetal hemoglobin gene.

To do so, CRISPR-Cas9 introduces small deletions into an

"enhancer" gene called BCL11A…

Anaemia/Hypoxia

Erythroferron (ERFE)

Erythroid iron intake

Hemichromes and ROS

Hepcidin

Iron absorption

JAK2 inhibitors

Activin receptor II

trap ligands

Apo-Transferrin

Erythroid cell

replication

Erythroid cell

differentiation

Minihepcidins

and TMPRSS6

inhibitors

Erythroid

progenitor

cells

Epo/Jak2 pathway

Rivella S. Haematologica. 2015 Apr;100(4):418-30.

GDF11

Mahidol University

Systems Biology & Thalassemia

Globin genemutation

PhenotypeGenotype

Red Cell IronClinical

Manifestation

Iron Absorption(Hepcidin, GDF15)FerrokineticOxidative StressAntioxidants

ErythropoiesisApoptosisAutophagy

Anemia

DrugMetabolism

CVSHigh HbF GenesEpoNO

UGTCyt P450

OrganPathology

LiverEndocrine glands

Rapid Targeted Next-Generation Sequencing Platform for Molecular

Screening and Clinical Genotyping in Subjects with Hemoglobinopathies

(Shang X, et al. EBioMedicine 2017; 23: 150-9)

- Targeted the entire protein-coding regions, key regulatory regions, known pathogenic copy number variants (CNVs) regions and single nucleotide variants (SNVs)/insertion and deletion variants (indels) in the non-coding regions of hemoglobin gene clusters (α- and β-globin gene clusters)

- 4 modifier genes (KLF1, BCL11A, HBS1L and MYB)

- 21 autosomal SNP sites and 6 sex chromosomes genes as markers

for identity and sex tracing of the samples.

- The total size of the targeted sequences was 275,234 bp.

NGS Assay

Subjects: Guangxi, Guandong, Yunnan, Guizhou and Hainan

• Analyzed 20,222 subjects by using an NGS assay

• At total of 4840 mutant alleles from 4180 individuals were detected

• Overall variant carrier frequency of 23.93%, including

16.77% for α-thalassemia,

4.70% for β-thalassemia,

1.70% for CNV variants (1.69% associated with the α-globin cluster and 0.01% associated with the β-globin cluster),

0.43% for KLF1 variants and

0.33% for structural hemoglobin variants

Result:

• 12 couples had a negative phenotype in the first step of

hematologic testing,

• Mutations in 7 couples were misdiagnosed and

• Mutations in 16 couples were undetected

• The NGS approach enabled us to identify an additional 586 mutant alleles (/4840 mutant alleles), including

- 343 pathogenic copy-number variations (CNV),

- 114 disease-causing mutations with no phenotypic red cell changes,

- 87 alleles with zinc-finger mutations in KLF1,

- 4 alleles with new variants (- -30.8, -α1.2, HBA1: c.96-1GNC and HBB: c.41CNT) and

- 38 carriers co-inherited with α- and/or β-globin gene defects.

- Traditional screening/molecular testing methods, which failed to

detect 35 at-risk couples but identified by our NGS method due to

additional disease-causing mutations and/or modifier mutations,

may confer small risk for thalassemias in mutation-positive couples

- In this study, the NGS had a throughput of 3000 samples per run.

With the introduction of automated instruments for the entire

workflow, the total run time for 3000 samples was 178 h, and the

cost per sample was approximately $30.

Conclusion:

- Thalassemia diagnosis can be done at the molecular levels.This has been applied for prenatal and preimplantation diagnosis and

prevention of thalassemia.

- Thalassemia can be cured by gene therapy

- Future of precision medicine in thalassemia

Will NGS replace thalassemia screening by conventional technique?

How to make gene therapy available for patients at large?

Conclusion: Precision medicine and thalassemia