Genetic Modifiers of Sickle Cell

Disease

Abdullah Kutlar, MDAbdullah Kutlar, MDAbdullah Kutlar, MDAbdullah Kutlar, MD

Professor of MedicineProfessor of MedicineProfessor of MedicineProfessor of Medicine

Director, Sickle Cell CenterDirector, Sickle Cell CenterDirector, Sickle Cell CenterDirector, Sickle Cell Center

SICKLE CELL DISEASE

� Homozygous state for Hb S

(β6 Glu→Val; GAG→GTG)

� 8-12% of African-Americans carry the Hb S gene

� 1/400 African-American births homozygous for Hb S

� Increased gene frequency in the “malaria belt”

(Africa, Mediterranean, Middle East, India)

� Associated with significant morbidity and mortality

SICKLE CELL DISEASE

� Chronic Hemolytic Anemia

� Vaso-occlusion

� Chronic inflammatory state

� Organ damage

Sickle Cell Disease

Infection/SepsisSplenic SequestrationIschemic StrokeDactylitis

VOEACS

GallstonesPriapism

Fe OverloadAplastic Crisis

Pulmonary HypertensionNephropathyAVNLeg UlcersChronic Lung DiseaseMulti Organ FailureHemorrhagic Stroke

Pediatrics Adults

SICKLE CELL DISEASE: LIFE EXPECTANCY

Male Female

SS 42 48

SC 60 68

Platt, et al. NEJM, 1994;330(23):1639-44

Sickle Cell Disease: Life Expectancy over 3 decades

01020304050

1970 1980 1990 2000

Smith LA, Oyeku SO, Homer C, Zuckerman B. PEDIATRICS. 117(5):1763-1770, 2006

SICKLE CELL DISEASE: PHENOTYPIC HETEROGENEITY

Heterogenity of SCD

� Stroke/CVA – 11% of SS patients up to 20 years of age

� Retinopathy – 3% of SS patients

� Acute Chest Syndrome – 30-50% of SS patients

� Pulmonary hypertension – 20-40% of adult SS patients

� Gallstones – 30-36% of SS patients

� Splenic sequestration – 5-6% up to 6 years of age

� Nephropathy/microalbuminuria – 30%

� Priapism – 6-42% of males with SS

� Avascular Necrosis – 50% at age 35

� Leg ulcers – 4-6% of SS patients > 10 years of age

MODIFIERS OF DISEASE SEVERITY

� α- thalassemia

� High Hb F determinants� Linked ( β-globin haplotypes)

� Unlinked (X-chromosome, others)

� Other genetic factors� Coagulation related polymorphisms

� Endothelial cell function

� Inflammatory mediators

� RBC membrane function

� NO bioavailability

� Others

PHENOTYPIC MODULATION OF SCD: Methodologic Approaches

� Analyses of polymorphisms in candidate genes (SNPs) by high throughput methods

� Genome-wide association studies (GWAS)

� Study of differential gene expression with cDNA microarrays in:� Blood cells (retics, neutrophils, monocytes)

� Bone marrow

� Endothelial cells

� Liver tissue

PHENOTYPIC MODULATION OF SCD

APPROACH:

� Analysis of candidate genes as risk factors for complications of disease

� Comparative study of phenotype in different populations and correlations with genetic factors

� Microarray technology to study differential gene expression

� Design of new therapies based upon new genetic information

APPROACH TO STUDY GENETIC MODIFIERS IN SS

� Study sickle cell patients from distinct populations around the world:

� African-American

� Mediterranean (Italy, Greece,Turkey)

� Asian (Kuwait, India)

� Africa (Nigeria and Kenya)

� South America (Brazil, Caribbean)

APPROACH TO STUDY GENETIC MODIFIERS IN SS

� Phenotypic characterization of distinct sickle cell populations in terms of the frequency of complications/organ damage:

� Stroke risk/CNS disease

� VOE

� Acute chest syndrome

� Avascular necrosis

� Gallstones and cholecystectomy

� Leg ulcers

� Pulmonary hypertension

� Priapism

� Renal disease

EFFECTS OF α-THALASSEMIA INSCD

CELLULAR:

� Decreased MCHC

� Decreased Hb S polymer

� Decreased RBC cation loss

� Decreased RBC density

� Increased RBC deformability

EFFECTS OF α-THALASSEMIA INSCD

HEMATOLOGICAL:

� Reduced Hemolysis

� Decreased Reticulocyte Count

� Increased Hb/Hct

� Decreased MCV

EFFECTS OF α-THALASSEMIA IN

SCD

CLINICAL:

� Increased:� Osteonecrosis

� Splenic sequestration

� Painful episodes (?)

� Decreased:� Cerebrovascular accidents

� Leg ulcers

� Nephropathy/proteinuria

� Longevity

FACTORS INFLUENCING HB F LEVELS IN SCD

� Age

� Gender (F>M)

� βs-globin gene cluster haplotype

� F-cell Production locus (FCP) (Xp22.2)

� Others (Chromosome 6?)

Baseline Hb F levels in SCD

� ~50% variation:

� FCP on Xp22

� -158 C→T at Gγ (Xmn 1 polmorphism)

� ~20-30% variation(12 SNPs in 6q22.2-23.2)

� Phosphodiesterase 7 (PDE7)

� Microtubule associated protein 7 (MAP7)

� Peroxisomal biogenesis factor 7 (PEX7)

� Mitogen activated protein kinase 5 (MAP3K5)

Association Studies in

SCD

AVASCULAR NECROSIS IN SICKLE CELL

DISEASE

AVASCULAR NECROSIS (AVN) OF FEMORAL HEAD IN SCD

RISK FACTORS:

� Alpha thalassemia

� Age

� High hematocrit

� Frequent vaso-occlusive episodes

Ballas et al, Hemoglobin, 13:649,1989

Milner et al, NEJM, 321:1476, 1991

ASSOCIATION STUDIES IN SCD

Phenotype SNP Reference

Osteonecrosis MTHFR Kutlar et al,2001

KL (klotho) Baldwin et al, 2005

BMP6

TGFBR2, 3

EDN1, ECE1

P <0.05

Group

Means & Std Devs

6/6 B 6/7 B 7/7 B

0.0

2.5

5.0

7.5

10.0

Std Dev Mean

Bilirubin Levels and UGT1A1 Polymorphism

p<0.05

Phenotype SNP Reference

Hyperbilirubinemia/ UGT1A1 7/7 Passon et al 2001

Gallstones Fertrin et al, 2003Haverfield et al, 2005

Adekile et al, 2005

ASSOCIATION STUDIES IN SCD

UGT1-A1 POLYMORPHISM IN SCD

� Haverfield et al (Blood; 2005)

� 541 SS patients from Jamaica

� 7/7 genotype associated with higher bilirubin levels

� 7/7 associated with symptomatic gallstones except for younger patients

� Adekile et al (Eur. J. Haematol, 2005)

� 67 SS patients from Georgia (age 2-44)

� Bilirubin levels significantly correlated with Hb F levels and 7/7 genotype in multivariate analyses

� No effect of UGT1-A1 genotype in younger (<10) patients

� No correlation between bilirubin levels and βS-haplotypes and α-thalassemia

ACS

Phenotype SNP Reference

Acute Chest NOS3 T-786C Sharan et al, 2004

NOS1 AAT rep. Sullivan et al, 2001

ASSOCIATION STUDIES IN SCD

Priapus:

In Greek mythology, the god of fertility,

son of Dionysus and Aphrodite,

represented as grotesquely ugly, with

an exaggerated phallus. He was later a

Roman God of gardens,

where his image was frequently

used as a scarecrow.

ASSOCIATION STUDIES IN SCD

Phenotype SNP Reference

Priapism Klotho (KL) Nolan et al, 2004

Pgm: \\Cre_Figure1_MORT.sas Data Lock Date: 31MAR06 Run: 30JUN06

Cumulative Event Rate (%)

0

10

20

30

40

Years Since Randomization

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Phenotype SNP ReferenceSurvival/longevity Klotho (KL) Ashley-Koch et al, 2005

TGFBR2NOS2A

ASSOCIATION STUDIES IN SCD

LEG ULCERS IN

SICKLE CELL

DISEASE

Phenotype SNP Reference

Leg ulcers Klotho Steinberg et al, 2005

TEK

SMAD1

SARA1

ASSOCIATION STUDIES IN SCD

Pulmonary Hypertension

Pulmonary Hypertension in SCD

� Prevalence rate – 20-40% in adults

� TR jet velocity ≥ 2.5 m/sec = PASP >30 mm Hg

� Echo results equivalent to right heart cath

� Lower PAP and higher cardiac output than primary PHTN

� Significantly higher mortality rate - 50% in two years

Pulmonary Hypertension in SCD

ASSOCIATION STUDIES IN SCD

Phenotype SNP Reference

Pulmonary Hypertension BMPR2 Koch et al, 2004

ADCY6

Nephropathy in SCD

ASSOCIATION STUDIES IN SCD

Phenotype SNP Reference

Estimated GFR BMPR1B Nolan et al, 2007

ASSOCIATION STUDIES IN SCD

Phenotype SNP Reference

Infection/Bacteremia IGF1R Adewoye et al, 2006

BMP6

TGFBR3

BMPR1A

SMAD6

SMAD 3

Stroke Risk

CONCURRENT STUDIES FOR STROKE RISK

� Taylor, et al. (Blood 2002)� Protective role for VCAM G1238C

(p=0.04, OR = 0.35)

� Hoppe, et al. (Blood, 2004)� IL4R503, TNF-308, ADRB227 (large vessel

stroke)� VCAM1594, LDLR (small vessel stroke)

� Steinberg, et al. (2003)� Klotho (KL) (p=0.006, OR = 2.8)� ECE1 (p<0.0001, OR = 5.2)� TGFBR3 (p<0.0001, OR = 7.0)

CONCURRENT STUDIES FOR STROKE RISK

� Ware, et al. (2003)� CSSCD pediatric patients

� 226 patients, 70 with stroke

� Increased risk: � PAI-1, 4G/4G (p=0.04)

� FVII 37bp repeat (p=0.07)

� Protection:� α-thal (p=0.0019)

� Angiotensin 196 bp allelle (p=0.0084)

STROKE RISK IN SCD

� Sebastiani et al (Nature Genetics, 2005)

� 1398 patients with SCD from CSSCD

� 92 with stroke, 1306 without stroke

� 108 SNPs in 80 candidate genes

� Bayesian networks to analyze gene-gene interactions and their interplay with clinical variables including Hb F and α-thalasemia

STROKE RISK IN SCD

� Sebastiani et al (Nature Genetics, 2005)

� 11 genes were found to interact with Hb F levels to modulate the risk of stroke

� SNPs in 4 genes highly significant

� TGFBR2

� TGFBR3

� BMP6

� SELP (P-selectin)

� Model validated in a different population with a 98.2% predictive accuracy

ASSOCIATION OF SNPs WITH STROKE IN SCD

SNP EFFECT REFERENCE

VCAM1 G1238C Protective Taylor et al,2002

VCAM1 T-1549C Permissive Hoppe et. al, 2004

IL4R S503P Permissive Hoppe et al, 2004

TNFα -308 Protective Hoppe et al, 2004

LDLR/Nco1 Protective Hoppe et al, 2004

ADRB2 Q27E Protective Hope et al, 2004

HLA loci Both Hoppe et al, 2002

BMP6 Permissive Sebastiani et al, 2005

TGFBR2 Permissive Sebastiani et al, 2005

TGFBR3 Permissive Sebasiani et al, 2005

P-Selectin Permissive Sebastiani et al, 2005

TCD

GENETIC RISK FACTORS FOR STROKE

� Phenotype: High TCD (>200 cm/sec)

� Sample Size:

� 230 high TCD (high risk)

� 400 normal TCD (<170 cm/sec, low risk)

� Candidate gene approach (28 polymorphisms)

� Pooled DNA analysis for genome wide SNPs

� Method: High throughput genotyping with MALDI-TOF

Stroke Risk in SCDSNP ODDS RATIO

(95% CONFIDENCE LIMITS)P

adcy9 1.074 (0.723, 1.596) 0.72

apoe 0.943 (0.626, 6.416) 0.77

esell554p 2.741 (0.857, 8.768) 0.084

esels128a 0.699 (0.310, 1.578) 0.38

eselg98t 0.699 (0.310, 1.578) 0.38

fibc148t 0.810 (0.484, 1.357) 0.42

fibg455a 0.652 (0.317, 1.339) 0.24

fva485l 0.948 (0.639, 1.406) 0.79

fviia353g 1.117 (0.694, 1.799) 0.65

fxiiiv34l 0.952 (0.625, 1.451) 0.82

glyc807t 1.065 (0.722, 1.571) 0.75

gp1bt161m 0.716 (0.483, 1.061) 0.095

glyl33p 0.657 (0.397, 1.086) 0.098

icamg241a 0.550 (0.196, 1.543) 0.25

lselp206l 0.730 (0.476, 1.119) 0.15

metht 1.609 (0.956, 2.709) 0.073

pai1 0.846 (0.568, 1.259) 0.41

tgfbr3 0.735 (0.498, 1.086) 0.12

tnfg308 1.126 (0.729, 1.742) 0.59

throma455v 0.775 (0.380, 1.579) 0.48

vcamg1238c 0.601 (0.375, 0.962) 0.032

Catheter Induced

Thrombosis

R485K POLYMORPHISM AND CATHETER INDUCED THROMBOSIS

61-/-

23-/+

26+/+

CONTROL

n=10

THROMBOSIS

n=10R485K

p=0.006 Odds Ratio=4.9

PITFALLS OF GENETIC ASSOCIATION STUDIES

� Small sample size

� Borderline p values or OR

� Unclear phenotypes

� Errors in Genotyping

� Volume of data and what does it all mean?

Ioannidis, et al. Nature Genetics 2001

Lohmueller et al. Nature Genetics 2003

RECOMMENDATIONS FOR

ASSOCIATION STUDIES

� Clearly defined phenotype

� Large sample size (collaborative, multicenter studies)

� Two studies with p<0.01 or a single study with p<0.001

Ioannidis, et al. Nature Genetics 2001

Lohmueller et al. Nature Genetics 2003

IS THERE CLUSTERING OF CLINICAL COMPLICATIONS IN SCD?

� Study of Jamaican patients with a Bayesian model

� 244 subjects (78%) classified into two groups with 95% confidence interval

� Group I – Frequent VOEs, dactylitis, meningitis/sepsis, ACS, and stroke

� Group II – Frequent leg ulcers and anemia

� Splenic sequestration, hypersplenism, and gallstones not linked to either group

� Hb F lowest in Group I; Hb lower in Group II

Alexander, et al. BJH, 126, 606-611, 2004

ARE THERE TWO DISTINCT SUBPHENOTYPES OF SCD?

� Hemolysis/Endothelial cell dysfunction:� Pulmonary Hypertension

� Priapism

� Leg ulcers

� Stroke?

� Vaso-occlusion/hyperviscosity:� Painful episodes

� Osteonecrosis

� Acute chest syndrome

Gladwin & Steinberg

SUBPHENOTYPES OF SCD

Kato, Gladwin, and Steinberg.Blood Reviews, 21, 37-47, 2007

DOWNSTREAM EFFECTS OF HEMOLYSIS IN SCD

Kato, Gladwin, and Steinberg.Blood Reviews, 21, 37-47, 2007

ASSOCIATION STUDIES IN SCD: CONCLUSIONS

� TGFβ/BMP pathway has emerged as an important modifier of SCD severity

� This pathway regulates a wide range of cellular functions (cellular proliferation, differentiation, tissue repair, immune regulation)

� Klotho (KL) has also emerged as a recurrent theme in genetic modulation of SCD

� KL encodes a transmembrane protein with actions on a wide range of cellular processes (antioxidative defense, atherosclerosis, NO metabolism, aging)

TGF-β SUPERFAMILY

� Major signal transduction pathway present in many tissues and cell types

� Comprises ~30 proteins in vertebrates� Regulates diverse cellular functions:

� Cell proliferation� Apoptosis� Response to tissue injury� Bone homeostasis� Inflammation� Immune regulation

DISEASES ASSOCIATED WITH MUTATIONS IN TGF-β PATHWAY

� Hereditary hemorrhagic telangiactasia

� Primary pulmonary hypertension

� Hereditary nonpolyposis colon cancer

� Juvenile polyposis syndrome

� Persistant mullerian duct syndrome

� Diabetic nephropathy

� Sickle cell disease?

CONCLUSIONS

� Genetic modulation of SCD is complex and likely involves multiple gene-gene interactions

� Emerging picture of the phenotypic heterogeneity in SCD is similar to that of complex “multigenic”diseases (CVD, Cancer)

� Elucidation of the mechanisms and pathogenesis of the phenotypic heterogeneity will be possible with a better understanding of the functional effects of the genetic polymorphisms

� Only with this information will it be possible to design novel therapies targeting genetic alterations

MCG Sickle Cell Center: FACULTY AND STAFF

� Adult ClinicAbdullah Kutlar, MD

Kavita Natarajan, MBBS

Celalettin Ustun, MD

Lisa Daitch, PA-C

Sabine Fields, RN

Chakira Brown

Kelvin Jackson

Marva Hall, RN

Chartara Gilchrist, BSW

� Pediatric ClinicVirgil C. McKie, MD

Kathleen M. McKie, MD

Beverly Blanchard, LPN

Atlee Goodwin, RN

� Research Coordinator

Leigh Wells, MSN

Pritam Bora

� Sickle Cell Center LaboratoryFerdane Kutlar, MD

Niren Patel, MBBS

Brian Zhang, MD/PhD

Tao Li, MD

Chinwe Obiaga

� AdministrationAbdullah Kutlar, MD Betsy Clair