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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 19  |  Issue : 5  |  Page : 761-766
Association of Angiotensin-Converting Enzyme Gene Dimorphisms with Severity of Lupus Disease


1 Department of Medicine, The Aga Khan University, Karachi, Pakistan
2 Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan

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   Abstract 

Angiotensin-converting enzyme (ACE) plays an important role in the development of systemic lupus erythematosus (SLE) because its end-product, angiotensin II, plays an integral role in the regulatory system responsible for endothelial control and vascular tone, systems that are commonly affected in patients with SLE. Additionally, ACE inhibitors have been shown to retard the progression of SLE and lupus nephritis. Our goal was to investigate whether ACE gene polymorphisms are associated with increasing severity of SLE. We genotyped 39 SLE patients of varying disease severity from a homogenous Asian population and 79 control subjects for ACE I/D and 2350 G > A dimorphisms. All patients met the American College of Rheumatology (ACR) criteria for SLE and their disease severity was measured using Systemic Lupus Activity Measure (SLAM). The "A" allele was found to be associated with increase in severity of SLE with the AA genotype present only in severe disease. No association with SLE in general, compared to healthy subjects, was found with either dimorphism. We also examined the transmission of haplotypes as defined by these polymorphisms. The D and A alleles were found in strong linkage disequilibrium especially in severe SLE. The DA-haplotype was more frequent in severe SLE, than mild to moderate disease. Our findings suggest that DNA sequence variation in the ACE gene influences disease progression and severity of SLE.

Keywords: SLE, ACE, Polymorphisms, PCR, Asian

How to cite this article:
Rabbani MA, Mahmood M S, Mekan SF, Frossard PM. Association of Angiotensin-Converting Enzyme Gene Dimorphisms with Severity of Lupus Disease. Saudi J Kidney Dis Transpl 2008;19:761-6

How to cite this URL:
Rabbani MA, Mahmood M S, Mekan SF, Frossard PM. Association of Angiotensin-Converting Enzyme Gene Dimorphisms with Severity of Lupus Disease. Saudi J Kidney Dis Transpl [serial online] 2008 [cited 2017 Sep 24];19:761-6. Available from: http://www.sjkdt.org/text.asp?2008/19/5/761/42452

   Introduction Top


Systemic lupus erythematosus (SLE) is a complex autoimmune disorder which shows various clinical manifestations characterized by inflammation in many different organ sys­tems. The cause of SLE is still unclear; how­ever, genetic and immunological abnormalities are considered to be responsible for the patho­genesis of this disorder. [1] Recently, angiotensin converting enzyme (ACE) gene has also been implicated.

ACE is a dipeptidyl carboxypeptidase I, (DCP I; 3.4.15.1) that activates angiotensin I through cleavage of the dipeptide at the carboxy­terminal end into the potent vasoconstrictor angiotensin II (AII) [1],[2] shown to be involved in the thickening and oxidative damage of the vascular wall, [4],[5] eventually leading to the deve­lopment of vascular disease. [2],[3] Thus, it is a strong candidate for the perpetuation of vis­ceral damage, vascular autoimmunity and neph­ritis in SLE.

Circulating ACE levels show extensive inter­individual variability and are highly genetically determined. [6],[7] An insertion/deletion (I/D) dimor­phism, due to the presence or absence of a 287 base pair (bp) alu-type sequence in intron 16 of the ACE gene, has been shown to co­segregate with serum and tissue ACE acti­vities, and the D allele is associated with ele­vated ACE levels. [6],[7],[8] Amongst the 13 poly­morphisms of the ACE gene recently reported, a dimorphism in exon 17, ACE 2350 G> A, has the most significant effect on plasma ACE concentrations. [9]

Thus, the ACE gene is viewed as a quan­titative trait locus (QTL) that modulates circu­lating ACE levels, and the ACE I/D and 2350 G > A polymorphisms are markers thought to be in linkage disequilibrium (LD) with func­tional variants located in the ACE gene [8],[9] that are implicated in vascular disease.

To assess the value of genotyping of ACE, we carried out a retrospective, case-control study of the two dimorphisms for a putative association with SLE amongst Pakistani natio­nals. Our study was aimed at establishing whether these dimorphisms are genetic mar­kers and independent risk factors for SLE.


   Materials and Methods Top


Subjects

In this pilot, retrospective case-control study, we investigated a sample population of 39 SLE patients and 79 control subjects from The Aga Khan University Hospital, Karachi, with a view to identify putative associations between ACE dimorphisms and SLE [Table 1]. Informed con­sent was taken from all study participants and international guidelines for sample collection for genetic studies were followed. [10] All our patients fulfilled four or more of the American College of Rheumatology criteria for the diag­nosis of SLE. [11],[12] The grading of disease seve­rity of SLE was done by Systemic Lupus Acti­vity Measure (SLAM). [13] Seventy-nine healthy subjects unrelated to the patients, without inflammatory or autoimmune disease, matched for age, gender and ethnicity were recruited as controls.

DNA analysis

Blood was collected in 10 mL Na-EDTA tubes and DNA was extracted using standard protocols [14] and stored in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. The ACE I/D dimorphism was genotyped using conditions previously described. [15] Codon 2350 ACE genotypes were visualized by PCR-RFLP as described else­where. [16] Briefly, a 122–bp fragment was am­plified by PCR and restricted with BstU1 (Life Technologies, Beverly, MA) at 60 °C for two hours. Digested fragments were separated by gel electrophoresis on 3% agarose gel and identified by ethidium bromide staining. Allele G2350 was visualized as a 122–bp fragment and allele A2350 as 100–bp and 22–bp frag­ments. [16]

Data analyses

Statistical analyses were carried out using the SPSS® (Statistical Package for Social Scien­ces) Software Version 11.0 for Windows® (Gorinchem, The Netherlands). Distribution differences of ACE I/D and 2350 G > A genotypes in the SLE patients as compared to distribution in the control group were assessed by chi-squared analyses [Table 2],[Table 3]. Esti­mations of departures from Hardy-Weinberg equilibria (D A ) and the tests for statistical significance were calculated as reported by Haviland et al. [14] Allele frequencies were com­pared between patient and control groups and among groups of different disease severity within cases of SLE by using the two-sided Chi-squared Test and Students't-test. For all analyses, statistical significance was considered when p values were lower than 0.05.


   Results Top


This study included 39 Pakistani unrelated SLE patients (nine males) and age- and gender­matched 79 control subjects. The overall mean age of SLE patients was 33 ± 10 years with general clinical features as outlined in [Table 1]. According to the SLAM score, our patients predominantly had moderate disease.

Hypertension developed in 33% patients du­ring the course of their disease. Most had mild hypertension with two cases having severe hypertension (BP > 180/100 mmHg). Echocar­diographic evidence of carditis was found in 20% of the patients. One patient developed severe carditis with hemodynamic compromise. Lupus nephritis was advanced with raised serum creatinine in 33% of the patients and three patients had serum creatinine levels > 4 mg/dL. Frank proteinuria (urine dipstick 3+­4+) was present in 40% of the patients.

Lupus cerebritis was not uncommon amongst our patients. Four patients gave a history of multiple transient ischemic attacks and three patients developed stroke during the course of their disease. Seizures occurred in seven pa­tients during the course of their illness. Four patients developed severe depression with cog­nitive impairment whereas mild depressive symptoms were present in 38% of the patients. Headache of mild to moderate intensity was also common (54%).

Hematological abnormalities were widespread with anemia (67%) and lymphopenia (61%) being most common. Four patients had leuko­penia (white cell count < 4000/microliter). Thrombocytopenia was present in 46% of the patients.

Significant weight loss (> 10%) was found in 18% of the patients at initial presentation. Ray­naud's phenomenon was present in 27% and mild, diffuse maculopapular rash in 41% of the patients, although one patient developed se­vere rash involving > 50% of body surface area. Four patients presented with ophthalmic mani­festations. Mild retinal hemorrhages were found in three patients whereas one patient had se­vere retinal hemorrhages with significant vi­sual impairment and cystoid bodies.

[Table 2] shows the data pertaining to both genotype and allele distributions in the two groups of subjects. Genotypes of both dimor­phisms occurred in Hardy-Weinberg propor­tions in both groups of subjects as shown by D A statistics. Differences in the distributions of the three genotypes according to clinical phe­notype were not statistically significant for ACE 2350 G> A (SLE vs controls: χ 2 = 1.41, 2 df, P= 0.49) and ACE I/D (SLE vs controls: χ 2 = 2.26, 2 df, P= 0.32). Frequency of the "A" allele was 0.29 ± 0.07 amongst the SLE patient group compared to 0.30 ± 0.04 in control subjects. Further analysis however, revealed that the frequency of the "A" allele increased from 17% in mild SLE to 25% in moderate disease to 36% in severe SLE.

[Table 3] shows the genotype distributions according to the disease severity of SLE. The most notable aspect was the presence of AA genotype in only severe SLE and its absence in mild to moderate disease. This was a male pa­tient having the DD genotype as well. There­fore, we calculated the mean SLAM scores for patients having the DD genotype and increa­sing number of "A" alleles. The mean SLAM score for patients having GG-DD genotypes was 16 (n= 3), patients having GA-DD geno­types had a score of 17.5 (n= 7) and the patient having AA-DD genotypes had a SLAM score of 22. This effect however, was not statis­tically significant, due to the small sample size of our study groups.

Examining the transmission of haplotypes as defined by these polymorphisms, the D and A alleles were found in strong linkage disequi­librium [Table 4]. The DA-haplotype was more frequent in severe SLE than mild to moderate disease (χ 2 = 0.87, 2 df, p= 0.35, odds ratio = 2.1 95% confidence interval = 0.34 - 13.23) [Figure 1]. The finding did not achieve statis­tical significance because of the small sample size.


   Discussion Top


ACE plays an important role in the development of vascular pathological states because it results in the formation of AII, which is a vasoactive peptide and growth factor that con­tributes to vascular reactivity, tissue remode­ling and fibrosis. [6],[7] AII is also a pro-inflamma­tory mediator with the ability to augment and perpetuate immune responses in several tissues. [8],[9] Thus, ACE plays an integral role in the regulatory system responsible for endo­thelial control and vascular tone, systems that are commonly affected in lupus. The results of this study suggest that DNA sequence varia­tion in the ACE gene influences disease severity of SLE.

ACE polymorphisms are not involved in the development of SLE as shown by the lack of association of the ACE I/D and G > A dimor­phisms with SLE. These results are consistent with previous studies which demonstrate a lack of association or linkage with ACE I/D. [17],[18] Yet, some studies report an association of the Alu D allele with SLE, [11],[12],[13] others have shown an association with I allele, [14],[15],[16],[17] while associations of the I/D polymorphism have also been demonstrated with clinical subgroups such as lupus nephritis. [11],[18] The associations have been more striking in non-Caucasian populations and there is evidence of ethnic heterogeneity (G & I). Parsa et al, conducted an association study of three polymorphisms in ACE including I/D with lupus nephritis. They demonstrated associations of two of the poly­morphisms with SLE and lupus nephritis. They found a stronger association in the non-Cauca­sian subgroup of patients which was a com­posite of Hispanic, Asian and Pacific popu­lations. The Asian/Pacific subgroup was in fact smaller in size than our sample population.

Keeping in view the findings of previous studies, we selected a more homogenous Asian population with varying disease severity of SLE. We selected ACE 2350 G > A dimor­phism since it is an exonic marker and has also been implicated as the functional mutation in ACE, modulating blood pressure (Zhu, Mah­mood). Since the literature is conflicting with regards to the association of ACE I/D with SLE, we hypothesized that the genetic se­quence variation of ACE may not be causative with regard to SLE but in fact, be involved in some way with disease progression. This assumption was backed by clinical data sup­porting the use of ACE inhibitors in lupus nephritis. [10]

This is the first study demonstrating the involvement of ACE in the disease severity of SLE. More than 40 genes have been impli­cated in conferring lupus susceptibility. Diffe­rent genes may modulate different aspects of SLE. In this study, we present the evidence that ACE might be involved in disease prog­ression of SLE.


   Acknowledgements Top


We are grateful to Dr. M. Ata Khan for the help in recruitment of lupus patients and Ms Hina Zuberi for lab assistance.

 
   References Top

1.Arnett FC, Hamiliton RG, Reveille JD, Bias WB, Harley JB, Reichlin M. Genetic studies of Ro (SS-A) and La (SS-B) autoantibodies in families with systemic lupus erythematosus and primary Sjogren's syndrome. Arthritis Rheum 1989;32(4):413-9.  Back to cited text no. 1    
2.Erdos EG, Skidgel RA. The angiotensin I­converting enzyme. Lab Invest 1987;56(4): 345-8.  Back to cited text no. 2    
3.Ehlers MR, Riordan JF. Angiotensin­converting enzyme: new concepts concerning its biological role. Biochemistry 1989;28(13): 5311-8.  Back to cited text no. 3    
4.Mancini GB, Henry GC, Macaya C, et al. Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease. The TREND (Trial on Reversing Endothelial Dysfunction) Study. Circulation 1996;94(3):258-65.  Back to cited text no. 4    
5.Lonn E, Yusuf S, Dzavik V, et al. Effects of ramipril and vitamin E on atherosclerosis. The Study to Evaluate Carotid Ultrasound Changes in Patients Treated with Ramipril and Vitamin E (SECURE). Circulation 2001;103(7):919-25.  Back to cited text no. 5    
6.Alhenc-Gelas F, Richard JL, Courbon D, Warnet JM, Corvol P. Distribution of plasma angiotensin I-converting enzyme levels in healthy men: Relationship to environmental and hormonal parameters. J Lab Clin Med 1991;117(1):33-9.  Back to cited text no. 6    
7.Cambien F, Alhenc-Gelas F, Herbeth B, et al. Familial resemblance of plasma angiotensin­converting enzyme level: the Nancy Study. Am J Hum Genet 1988;43(5):774-80.  Back to cited text no. 7    
8.Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin-I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86(4):1343-6.  Back to cited text no. 8    
9.Tiret L, Rigat B, Visvikis S, et al. Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I­converting enzyme (ACE) gene controls plasma ACE levels. J Hum Genet 1992;51(1): 197-205.  Back to cited text no. 9    
10.Gostin LO. National health information privacy: Regulations under the Health Insurance Portability and Accountability Act. JAMA 2001;285(23):3015-21.  Back to cited text no. 10    
11.McKenzie CA, Julier C, Forrester T, et al. Segregation and linkage analysis of serum angiotensin I-converting enzyme levels: Evidence for two quantitative-trait loci. Am J Hum Genet 1995;57(6):1426-35.  Back to cited text no. 11    
12.Zhu X, Bouzekri N, Southam L, et al. Linkage and association analysis of angiotensin I­converting enzyme (ACE)-gene polymerphisms with ACE concentration and blood pressure. Am J Hum Genet. 2001;68(5):1139-48.  Back to cited text no. 12    
13.Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual, 2nd ed. Cold Spring: Cold Spring Harbour Laboratory Press, New York, 1989.  Back to cited text no. 13    
14.Haviland MB, Kassling AM, Davingnon J, Sing CF. Estimation of Hardy-Weinberg pairwise disequilibrium in apolipoprotein AI­CIII-AIV gene cluster. Am J Hum Genet 1991;49(2):350-65.  Back to cited text no. 14    
15.Cambien F, Poirier O, Lecerf L, et al. Deletion polymorphism in the gene for angiotensin­converting enzyme is a potent risk factor for myocardial infarction. Nature 1992;359(6396): 641-4.  Back to cited text no. 15    
16.Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25(11):1271-7.  Back to cited text no. 16    
17.Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosu. Arthritis Rheum 1997;40(9):1725.  Back to cited text no. 17    
18.Gladman DD Goldsmith CH, Urowitz MB, et al. Crosscultural validation and reliability of 3 disease activity indices in Systemic Lupus Erythematosus. J Rheumatol 1992;19(4):608-11.  Back to cited text no. 18    

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Correspondence Address:
Malik Anas Rabbani
Associate Professor and Consultant Nephrologist, The Kidney Center, Post Graduate Training Institute, Rafiqui Shaheed Road, Karachi-75530
Pakistan
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PMID: 18711292

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    Tables

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