Sickle cell disease

World Cord Blood Congress IV and Innovative Therapies for Sickle Cell Disease Monaco October 2013, 24-27

Sickle cell disease: Genetic etiologies for phenotypic diversity Jacques Elion* & Marc Romana** Inserm U665 Laboratory of Excellence on the Red Cell – GR-Ex French National Reference Centers for Sickle Cell Disease *Robert Debré Mother and Child University Hospital, 75019 Paris, France **Guadeloupe University Hospital, 97139 Les Abymes (French West Indies)

SCD: a monogenic disorder solely due to HbS • SCD phenotype caused by several common and some rare genotypes – Sickle cell anemia (SCA = SS disease) – HbSC disease – HbS-beta thalassemia – HbSDPunjab, HbS/OArab – Rare dominant variants SAntilles, SOman BUT within each SCD genotype, considerable clinical heterogeneity exists making prediction of complications difficult Elion & Romana

Inserm U665

HbS: a single mutation but a polysystemic disease p.Val6Glu → HbS

deoxyHbS polymerisation

two trigerring factors

free Hb NO scavenging

Haemolysis Abnormal adhesion to endothelial cells

Vasoocclusion Acute complications

Steinberg M.H., 1999

Elion & Romana

- painful crises - dactilytis - acute chest syndrome - splenic sequestration Inserm U665


Polysystemic chronic complications



free Hb NO scavenging Nonhemorrhagic Stroke

Haemolysis Haemolysis Cholelithiasis

Pulmonary Hypertension

Renal Failure

Abnormal adhesion to endothelial cells Priapism

Steinberg M.H., 1999

Elion & Romana

Osteonecrosis

Vasoocclusion Acute complications Vasoocclusion Leg - painful crises (VOC) Bacteremia Ulceration - dactilytis - acute chest syndrome - splenic sequestration Inserm U665


Polysystemic chronic complications



free Hb NO scavenging Nonhemorrhagic Stroke

Haemolysis Haemolysis Cholelithiasis

Pulmonary Hypertension

Renal Failure

Abnormal adhesion to endothelial cells Priapism Osteonecrosis It is classical to distinguish a severe presentation in African patients Vasoocclusion from a moderate presentation in Arabia andAcute India,complications but predictive Vasoocclusion Leg individual markers of severityBacteremia are lackingcrises - painful (VOC) Ulceration - dactilytis urgent need of predictive markers to develop - acute innovative chest syndrome Steinberg M.H., 1999 preventive interventions - splenic sequestration

Elion & Romana

Inserm U665

Hematological variability Hemoglobin level Greeks 9.36 vs Jamaicans 7.83, p< 0.001

Saudi 10.27 vs Jamaican 7.83, p< 0.001

Indian 9.36 vs Jamaican 7.83, p< 0.001

Graham Sergeant personnal data

Hematological variability HbF level Greeks 5.58 vs Jamaicans 5.97, NS

Saudi 13.34 vs Jamaican 6.11, p< 0.001

Indian 16.64 vs Jamaican 6.11, p< 0.001

Graham Sergeant personnal data

A monogenic disorder phenotypically multifactorial

SCD High interindividual variability of clinical & hematological expression

a monogenic autosomal recessive disorder

Multifactorial Genetic modifiers

Accessible for evaluation Adapted from R. Krishnamoorthy

Environmental & interventional influences

Difficult to assess/control

Identification of genetic modifiers is important to: • Improve disease management & treatment “personal medicine” including indication for HSC transplantation

• Refine genetic counseling • Get further insight into a complex pathophysiology • Imagine innovative therapies and prevention

Elion & Romana

Established major modulators • Associated -thalassemia • Thalassemic behavior of the S gene in India • Mechanism • Reduces HbS intra-cellular concentration

• HbF concentration and cellular distribution • Mechanism: • Dilutes HbS within the cell • Disrupts HbS polymerization

all act on the primary mechanism all are heritable and under genetic control Elion & Romana

Inserm U665

Alpha-thalassemia reduces hemolysis in SCD Alpha-thal is present in about 30% of patients with SCA (one or 2 alpha-globin gene deletions, rarely point mutations)

Reticulocytes, bilirubin, LDH are reduced Ht is increased Dense RBC are reduced AA

SS-2 SS-3 SS-4  

Associated with decreased incidence of stroke, priapism, leg ulcers and pulmonary hypertension. Associated with increased incidence of ACS and ? VOC Adapted from Carolyn Hoppe

Genetic determinants of HbF expression

and -globin gene haplotypes have been associated to some extent with distinct hematological and clinical phenotypes Senegal/Arab-India Benin Bantu Elion & Romana

HbF highest intermediate lowest

Clinical best intermediate worst

The S mutation has arisen independently at least three times in Africa and once in India chromosomes with Genetic determinants ofonHbF expression different RFLP haplotypes and genetic backgrounds

and -globin gene haplotypes have been associated to some extent with distinct hematological and clinical phenotypes Senegal/Arab-India Benin Bantu Elion & Romana

HbF highest intermediate lowest

Clinical best intermediate worst

Polymorphisms within the -globin gene complex microsatellites LCR - HS2 (AT)xN12(AT)y

 silencer

repeated TG motives



HS4 HS3 HS2 HS1

G

(AT)xTy



A



HinfI RsaI AvaII HinfI

HincII HincII

HindIII TaqI HindIII

XmnI

HincII

HpaI HindIII BamHI

3'

5'

RFLPs

Extended haplotypes

(including regulatory regions)

Inserm U665

Functional polymorphisms in the -globin locus regulatory sequences account for the thalassemic t behavior of the S gene in India



G

A

+





S

5’ -

3’ HS

+

HS1

HS2

HS3

HS5 HS4

LCR

3’

(AT)x Ty BP1

The (AT)9T5 Arab-Indian motif of the  silencer binds BP1 repressor 9 times more strongly than the African motives Inserm U665

Genome-wide association studies have identified three majors genetic determinants accounting for HbF level heritablity

Xmn1 Labie et al 1983 11p globin cluster

Menzel S et al, Nat Gen 2007; Uda M et al, PNAS 2008

Heritability of HbF level Cis-acting elements (within β-globin locus) 11p13

XmnI -158 C>T polymorphism

Candidate gene approach

Trans-acting QTL (outside of β-globin locus) 6q23

MYB-HBS1L low level of MYB: accelerated erythroid differentiation

2p16.1

BCL11A

Elion & Romana

BCL11A: a key regulator of the globin switch

Global approach

Heritability of HbF level Cis-acting elements (within β-globin locus) 11p13

XmnI -158 C>T polymorphism

Candidate gene approach

Trans-acting QTL (outside of β-globin locus) 6q23

MYB-HBS1L low level of MYB: accelerated erythroid differentiation

2p16.1

BCL11A

BCL11A: a key regulator of the globin switch

A success story: the erythroid BCL11A-interacting DNA sequence is now considered as a unique potential target for gene therapy of the hemoglobinopathies via genome editing Elion & Romana

Global approach

Heritability of HbF level key for success Robust data because validated • in replication cohorts with independent samples • across populations African Americans, Africans, Caucasians, Chinese

• across healthy and different disease contexts SCD and β thalassemia

Adapted from R. Krishnamoorthy

Inserm U665

Genetic modulation of SCD complications Complex pathophysiology predicts multiple sites for modulation of the phenotype and subphenotypes

up to now: candidate gene approach Elion & Romana

Many associations of SNPs in candidate genes with specific complications of SCD have been reported Steinberg M, SWJ 2009

, MYH9-APOL1

Elion & Romana

Inserm U665

Many associations in candidate genes with However, most of SNPs these associations have not specific complications SCD have been reported beenofreplicated Steinberg M, SWJ 2009

, MYH9-APOL1

caution is warranted Elion & Romana

Inserm U665

Many associations in candidate genes with However, most of SNPs these associations have not specific complications SCD have been reported beenofreplicated Steinberg M, SWJ 2009

, MYH9-APOL1

only two of them have been replicated and are robust caution is warranted because relevant also in contexts other than SCD Inserm U665

Genetic modifiers of SCD complications despite extensive research, results are disappointing both from: - candidate gene approach - global approach

no individual genetic profile of risk

Elion & Romana

Identification of genetic modifiers by genetic association study design is crucial Phenotype needs to have a consistent definition Phenotype needs to have a genetic component (heritability) Low heritability = a substantial portion of phenotype variation is under the influence of environmental factors Adapted from R. Krishnamoorthy

Inserm U665

Heritability of a phenotype or complication can be estimated by assessing phenotype concordance in families (sib pairs) Rarely evaluated in most published reports


CSSCD Sib pairs study Correlation coefficient was High for

• Hb F level • Pain crises rate • Stroke

= heritability high

Moderate for

• Acute Chest Syndrome • Leg ulcer • Osteonecrosis • Priapism

= environmental/ interventional influence significant

Low for

• Survival

= not a heritable trait


Gobal approaches: what works?  Large studies  Replication (planned and coordinated)  Rigorous, high-quality design, conduct, analysis – Genomics – Statistics – Bioinformatics +++  Data sharing  Accomplished through consortia

Adapted from Carolyn Hoppe

Be ambitious but be modest… Genetic factors are only one piece of the puzzle… Need to consider contribution of non-genetic factors on SCD phenotype: – – – – –

Environmental exposures Nutrition Infection Social structure Lifestyle (cultural practices)

Adapted from Carolyn Hoppe

Inserm U665