27 bp Insertiondeletion Polymorphism in Intron 4 of eNOS Gene in Turkish Patients with Systemic Scle

1
27 bp Insertion/deletion Polymorphism in Intron 4
of eNOS Gene in Turkish Patients withSystemic
Sclerosis
Incilay Sinici1, Enver Atalar2, Alper Kepez2, Ali
Akdogan4, Mutlu Hayran3, Ferhan Özmen2, Meral
Çalgüneri4 Department of Biochemistry1,
Department of Cardiology2 , Department of
Preventive Oncology3, Department of Internal
Medicine Rheumatology Division4 Hacettepe
University Faculty of Medicine, 06100
Ankara-Turkey BCLF 2007 Antalya-TURKEY

• Incilay Sinici (MD, PhD)
• Hacettepe University Faculty of Medicine
• Department of Biochemistry
• Ankara- TURKEY

2
Systemic Sclerosis (SSc)

• Connective tissue disease characterized by
  generalized microangiopathy and culminating in
  systemic fibrosis.
• The pathogenesis of SSc is poorly understood, but
  the vasospastic abnormalities may result from
  impaired nitric oxide endothelial production.
• Nitric oxide produced by endothelial nitric oxide
  synthase (eNOS) physiologically regulates basal
  vascular tone and vascular function.

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eNOS Gene

• L-arginine NO citrulline

eNO synthase
The eNOS gene is located on chromosome 7q3536
and consists of 26 exons spaning 21 kB
4
Aim

• Whether the three polymorphisms of the eNOS gene
• 27 bp deletion (a)/insertion (b) in intron 4,
• T786C in the promoter region
• G894T in exon 7
• affects the susceptibility to and the clinical
  course of Systemic Sclerosis

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Study Population

• Twenty-eight patients with Systemic Sclerosis
  (mean age 49 13 years, 26 female) and age, sex
  matched 28 control subjects were studied

6
Characteristic of patients with SSC (n28)
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Genotype Analysis

• PCRs were carried out for T786C in the promoter
  region, G894T in exon 7, 27 bp deletion (a)/
  insertion (b) in intron 4 by using specific
  primers and conditions.
• Restriction digestions were carried out with
  MboI and MspI for G894T and T786C polymorphisms,
  respectively.
• Restriction sites were confirmed in 3 NuSieve
  agarose gel.

Tanus-Santos JE et al. Pharmacogenetics. 2002
Jul12(5)407-13
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Genotype Distribution
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Genotype Distribution of eNOS Polymorphisms
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Study groups by haplotype
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Systemic Sclerosis risk associated with eNOS
Polymorphisms
Introduced by presence of heterozygous or
homozygous polymorphisms for exon and promoter,
and presence of deletion for intron
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Discussion and Conclusion

• This is the first and only report to date for
  eNOS gene polymorphisms in SSc in Turkish
  population.
• C allele (786C) in the promoter region of eNOS
  gene is a risk factor for Systemic Sclerosis in
  Turkish patients.
• Current evidence suggests that the eNOS G894T
  polymorphism in exon 7 and 27 bp deletion
  (a)/insertion (b) polymorphisms play no role as a
  risk factor for SSc in Turkish patients

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eNOS gene polymorphism studies in Turkish
population
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• Lack of association of eNOS(G894T) and p22phox
  NADPH oxidase submit (C242T) polymorphisms with
  systemic sclerosis in a cohort of French
  Caucasian patients
• Tikly M et al, Clin Chim Acta. 2005
  Aug358(1-2)196-7.
• Lack of association of eNOS (G894T) and p22phox
  NADPH oxidase subunit (C242T) polymorphisms with
  systemic sclerosis in a cohort of French
  Caucasian patients.
• Yannick Allanorea et al, Clin Chim Acta. 2004
  Dec350(1-2)51-5
• High prevalence of polymorphisms of
  angiotensin-converting enzyme (I/D) and
  endothelial nitric oxide synthase (Glu298Asp) in
  patients with systemic sclerosis .
• Fatini C et al, Am J Med. 2002
  May112(7)540-4.

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Promoter Effect

• It has been shown that the level of eNOS mRNA is
  the lowest for 786C/C promoter genotype
• Using transfection studies, it was shown that
  promoter activity was significantly reduced by
  50 with mutant allele.
• Lower eNOS mRNA and serum nitrite/nitrate levels
  have been found in individuals with the 786C
  variant

Victor E. Dosenko et al. Acta Bioichimica
Polonica. 2006 53299-302.
Nakayama M et al. Circulation. 1999 992864-70.
Miyamoto Y et al. hum Mol Genet. 2000 92629-37.
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• Further studies using larger samples of eNOS gene
  polymorphisms in combination with other relevant
  genes could provide more insight into
  microvascular process in SSc.
• Ultimately identification of genes responsible
  for increased or decreased SSc risk should
  provide opportunities for early intervention and
  possible individually optimized therapies.

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Thank you for your attention