Peroxisome proliferator activated receptor γ Pro12Ala polymorphism in β thalassemia major patients Peroxisome proliferator activated receptor γ Pro12Ala polymorphism in β thalassemia major patients thesis defence

2. Peroxisome
proliferator
activated receptor γ
Pro12Ala
polymorphism in
β thalassemia
major patients
Marwa Mahmoud
Khalifa
Alex Uni, fac of Med

3. INTRODUCTION

4.  Thalassemia refers to group of blood diseases
characterized by decreased synthesis of α or β
polypeptide chains of haemoglobin.
 The molecular defects in β thalassemia result
in absent or reduced β - chain production.

5. Pathophysiology
Imbalance in
globin chain
production
Excess of α
chains
precipitate in
red cell
precursors
Ineffective
erythropoiesis
Anemia and
hemolysis
Increased
Erythropoietin
Expanded
bone
marrow

6. Inadequately transfused children present
with growth retardation, pallor,
splenomegaly and hypersplenism.

7.  Expansion of bone marrow leads to
deformities of skull with marked bossing
of zygoma giving rise to classical
mongoloid facies.

8. These signs are
associated with
radiologic features that
include lacy and
tubercular pattern of
long bones , phalanges
and typical hair on end
appearance of skull.

10. Bone disease in thalassemia
Osteopenia Osteoporosis Syndrome
(OOS)
 A major cause of bone pains and fractures in
70-80% of adult β thalassemia major patients.
The causes of OOS are multifactorial.

11. 1. Genetic factors
 Collagen type IA1 (major bone matrix
protein) polymorphism. (COLIA 1)
 Vitamin D receptor polymorphism.
 Sequence variation of transforming growth
factor-β1. (TGF-β)
 Calcitonin receptor gene restriction
fragment length polymorphism.
 Estrogen receptor restriction fragment
length polymorphism.
 Interleukin-6 (IL-6) gene restriction
fragment length polymorphism.

12. 2. Hormonal factors
Diabetes.
Thyroid/parathyroid dysfunction.
Hypogonadism (low estrogen and
progesterone levels in females and low
testosterone levels in males).
Insufficiency of the GH-IGF-1 axis.

13. 3. Iron overload and iron
chelators
Iron deposition in the bone impairs osteoid
maturation and inhibits mineralization
locally, resulting in focal osteomalacia.
Desferrioxamine inhibits DNA synthesis,
osteoblast and fibroblast proliferation,
osteoblast precursors differentiation, and
collagen formation.

14. 4. Miscellaneous
Nutritional deficits .
Vitamin C deficiency .
Low vitamin D levels.
Decreased levels of physical activity.

15. Mechanism of
OOS
Increased
osteoclastic
activity
Decreased
osteoblastic
activity

16. Increased osteoclastic
activity
1. Imbalance in the receptor activator of nuclear
factor-kappa B ligand (RANKL)
/osteoprotegerin (OPG) system.
2. Elevated markers of bone resorption such as
n-terminal cross-linking telopeptide of
collagen type-I (NTX), tartrate-resistant acid
phosphatase type 5b (TRACP-5b), Activin-A,
pyridinoline and deoxypyridinoline.

17. Decreased osteoblastic activity
1. Low serum IGF-I.
2. Decreased neoformation phase evidenced by
low levels of osteocalcin.
3. High circulating levels of Dickkopf-1 and
sclerostin.

18. Treatment of thalassemia
Regular blood
transfusion
Iron chelation
therapy

19. Comparison between iron chelators
Property Deferoxamine Deferiprone Deferasirox
Usual dose
(mg/kg/day)
25-60 75 20-30
Route SC,IV (8-12 hours,
5days/ week)
Oral 3 times daily Oral once daily
Half life 20-30minutes 3-4 hours 12-16 hours
Excretion Urinary , fecal Urinary Fecal
Adverse effects Local reactions,
ophthalmologic,
auditory, growth
retardation, allergy
GIT disturbances,
agranulocytosis,
arthralgia
GIT disturbances,
rash, mild non
progressive
creatinine increase

20. Treatment of thalassemia (cont.)
Bone marrow transplant is effective
particularly if carried out before complications
of iron overload develop.
Other experimental approaches:
fetal hemoglobin synthesis stimulation and
gene therapy.

21. Peroxisome Proliferator Activator
Receptor (PPAR)
A member of the nuclear hormone receptor
superfamily.
Regulates genes controlling cell growth,
differentiation, and metabolism in response
to lipophilic hormones, dietary fatty acids,
and their metabolites.
Three isoforms, encoded by separate genes,
have been identified α, β, γ.

22. MECHANISM OF ACTION
Ligand dependent transcription factors.
Bind to specific peroxisome proliferator
response elements (PPREs) in enhancer sites of
regulated genes.
 Each receptor binds to its PPRE as a
heterodimer with a retinoid X receptor (RXR).
The conformation of PPAR is altered and
stabilized such that a binding cleft is created.
The result is an increase in gene transcription.

23.  The PPAR /RXR heterodimer binds to distinct
DNA sequence elements.
 Coactivators interact with nuclear receptors
in an agonist-dependent manner.

24. Various PPAR signaling pathways

25. Peroxisome proliferator activator
receptor gamma (PPARγ)
Located in chromosome 3.
Genomic Span >150kbp.
 9 exons (A1, A2, B, and 1–6).
Target for thiazolidinedines (TZDs).
Pro12Ala mutation in PPARγ2 is the most
common mutation which has the substitution of
proline to alanine at codon 12 in exon B (cytosine
to guanine CCA–to–GCA).

26. Functions of PPAR γ
Macroph-
ages
• Enhance foam cell formation.
• Increase uptake of oxidized LDL.
• Enhance atherogenesis.
Heart
Bone
Adipocyte
• Regulate heart hypertrophy, and high fat diet
induced hypertension.
• Regulate bone homeostasis.
• Regulate differentiation.
• Promote lipid accumulation.
• Maintain viability and normal function.

27. Skeletal
muscle
• Regulate normal glucose metabolism.
• Improve insulin sensitivity.
Liver
• Control systemic glucose and lipid metabolism.
• Improve insulin sensitivity.
Inflamma
tion
• Suppress of inflammatory cytokine production.
• Improve insulin sensitivity.

28. Effect of PPARγ2Pro12Ala
on bone

30. AIM OF THE WORK

31.  To detect the frequency of the Pro12Ala
polymorphism of the peroxisome
proliferator-activated receptor-γ gene in β-
thalassemia major patients and its relation
to osteopenia, dyslipidemia, cardiovascular
complications and diabetes mellitus.

32. SUBJECTS

33. 30 patients with β-thalassemia major and 10
healthy age and sex matched controls.
 From outpatient Hematology clinic in
Alexandria Main University Hospital.
A written informed consent.
Approval of research ethics committee of
Alexandria Faculty of Medicine.

34. Inclusion criteria:
oBeta-thalassemia major patients diagnosed
by hemoglobin electrophoresis.
oSex: both genders.
oAge: ≥ 15 years old.

35. METHODS

36. All patients in this study were
subjected to:
Clinical
examinationHistory
taking
Investigations

37.  Complete blood count (CBC)
 Liver function tests : ALT, AST, Bilirubin (total
and direct)
 Hepatitis viral markers : HCV Ab, HBs Ag.
 Serum calcium.
 Renal function tests : urea, creatinine.
 Fasting blood glucose.
 Lipid profile : total cholesterol, HDL, LDL,
triglycerides .
 Serum ferritin .

38.  Echocardiography
 DEXA scan
 Genotyping by polymerase chain reaction
restriction fragment length polymorphism
(PCR-RFLP)

39. RESULTS

40. The frequency of clinical
findings in patients group (n=30)
Presenting features Number of cases Percentage %
History of fractures 5 16.67
Short stature 8 26.67
Heart failure symptoms 0 0
Diabetes mellitus 1 3.33
History of splenectomy 20 66.67
Splenomegaly 10 33.33
Hepatomegaly 17 56.67

42. Haemoglobin (g/dl)
0
2
4
6
8
10
12
14
Patients Control
P= <0.001

43. Urea (mg/dl) & Creatinine (mg/dl)
26
26.5
27
27.5
28
28.5
29
Patients Control
Urea (mg/dl)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Patients Control
Creatinine (mg/dl)
P= 0.001
P= 0.409

44. ALT (mg/dl)& AST (mg/dl)
0
10
20
30
40
50
60
70
80
90
100
Patients Control
AST (mg/dl)
0
10
20
30
40
50
60
70
80
90
Patients Control
ALT (mg/dl)
P= <0.001
P= <0.001

45. Ferritin (ng/ml)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
PATIENTS GROUP CONTROL GROUP
P=<0.001

46. Fasting blood glucose (mg/dl)
Only one patient was diabetic (3.33%)
82
84
86
88
90
92
94
96
98
100
102
Patients Control
P=0.444

47. Lipid profile (mg/dl)
0
20
40
60
80
100
120
140
160
TG TC HDL LDL
Patients
Control
P=
<0.001
P=
0.577
P=
<0.001
P=
0.397

48. Ejection fraction%
Ejection
fraction %
Cases (n=30) Control (n=10)
Test of
significance
P value
Min. – Max.
Mean ± SD
Median
57.0 – 70.0
62.23 ± 3.46
62.50
58.0 – 70.0
63.80 ± 4.34
63.50
t=1.163 0.252

49. Bone mineral density
by DEXA scan
0
10
20
30
40
50
60
70
80
90
100
higher or -1 -1 to -2.5 -2.5or less
Patients
Control
P=<0.001

50. Genotyping
Genotyping
Cases (n = 30) Control (n = 10)
Test of
sig.
MCp
No. % No. %
Normal 28 93.3 9 90.0
2=1.509 0.585Homozygous 1 3.3 1 10.0
Heterozygous 1 3.3 0 0.0

51. Summation of data of the 2
positive cases:
Parameter Homozygous
Pro12Ala
Heterozygous
Pro12Ala
Age 20 18
Sex male female
Body mass index 19.83 (normal) 20.20 (normal)
Ferritin 4886 4923
FBG 79 87
TG 128 97
TC 130 161
HDL 8 25
LDL 80 71
Ejection fraction 68 70
BMD -6 -2.4

53. 1. Decreased BMD was present in all patients.
Osteopenia was present in 23.3% while
osteoporosis was present in 76.7%.
2.PPARγ Pro12Ala polymorphism was present
in 6.6 % of patients (2 patients) and in 10 %
of control group.
3.Homozygous PPARγ Pro12Ala
polymorphism patient has lower BMD than
heterozygous genotype.
4.Patients with PPARγ Pro12Ala
polymorphism had normal lipid profile.

54. RECOMMENDATIONS

55. 1. Further studies on a larger population of
patients are still needed to precisely detect
PPARγ Ala12Pro polymorphism frequency
and its relation to BMD.
2. PPARγ Ala12Pro polymorphism is not
recommended as a routine investigation for
thalassemic patients and should be conserved
to those who present with pathological
fractures or very low BMD.