Diabetologia (2002) 45:933–935 DOI 10.1007/s00125-002-0838-1

Comment to: Pinizzotto M, Castillo E, Fiaux M, Temier E, Gaillard RC, Ruiz J (2001) Paraoxonase 2 polymorphisms are associated with diabetic nephropathy in Type II diabetes. Diabetologia 44: 104–107 To the Editor: Paraoxonase is a HDL-associated enzyme, which is thought to have a protective role in preventing the oxidation of LDL, therefore protecting tissue from oxidative damage. There is increasing evidence to suggest that oxidative stress has an important role in the development of diabetic microvascular complications. We have recently carried out a study of the polymorphisms of the paraoxonase genes (PON1 and PON2) in relation to diabetic microvascular complications in patients with Type I (insulin-dependent) diabetes mellitus (T1DM). The data from this study does not support the letter of Canani et al [1], which suggests that there is no association with PON2 polymorphisms and diabetic nephropathy in Type I diabetes. To investigate the roles of the PON2 and PON1 polymorphisms and the susceptibility to diabetic nephropathy, we looked at DNA samples from 213 British Caucasoid patients with Type I diabetes and a total of 129 DNA samples from normal healthy British Caucasoid subjects. The patients were classified according to their microvascular complications as described previously [2]. These are summarised as follows: Uncomplicated patients. These patients (n=44) had Type I diabetes mellitus for at least 20 years but remained free of retinopathy (fewer than five dots or blots per fundus) and proteinuria (urine Albustix negative on three consecutive occasions over 12 months).

Received: 7 November 2001 / Revised: 4 March 2002 Published online: 9 May 2002 © Springer-Verlag 2002

Nephropaths. These patients (n=72) had Type I diabetes for a duration of 10 years, and proteinuria (3 consecutive Albustix over the past 12 months) and retinopathy. Retinopaths. These patients (n=46) had retinopathy defined as more than five dots or blots for each eye, hard or soft exudates new vessels or fluorescein angiographic evidence of maculopathy or previous laser treatment for perproliferative or proliferative retinopathy, and maculopathy or vitreous hemorrhage. None of these patients had proteinuria. Fundoscopy was performed by both a diabetologist and ophthalmologist. Short duration. These patients (n=51) had a duration of diabetes of less than 20 years but had no evidence of retinopathy, proteinuria, or overt neuropathy. PCR was used to amplify the genomic DNA from the patients and normal controls using previously published primers; PON1 [3] and PON2 [4]. The genotypes for PON1 and PON2 were determined using restriction endonuclease enzymes. To identify the PON2 Ser311Cys variants the restriction enzyme DdeI was used, after digestion fragment sizes of 142 bp and 120 bp corresponded to the base pair (bp) Cys311 – C allele, while 75 bp and 67 bp corresponded to the Ser311 – S allele. The PON1 polymorphism was detected using the restriction enzyme AIWI. If the AIWI cut site was present two fragments of 60 bp and 40 bp were generated – the B allele, if the cut site was absent a fragment of 100 bp in size was generated – the A allele. In the normal control group the PON1 and PON2 genotypes were in Hardy-Weinberg equilibrium (χ2=2.6). A comparison of the PON2 genotype frequencies in all the patients (n=213) compared to the normal control subjects showed them to be nearly identical. When looking at the frequencies of the PON2 genotypes in relation to microvascular complications, we found no significant differences between patients with nephropathy and the uncomplicated group of patients. Although there was an increase in the frequency of the Cys/Cys genotype in the nephropaths compared with the uncomplicated group (41.1% vs 19.2% respectively), this difference was not signifi-

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Table 1 Frequency of PON1 and PON2 alleles and genotypes in patients with Type I diabetes mellitus and normal healthy control subjects

PON1: Genotype AA AB BB Total Allele: A B PON2: Genotype CC CS SS Total Allele: C S

Uncomplicated (n=44)

Nephropaths (n=72)

Retinopaths (n=46)

Short Duration (n=51)

Normal Controls (n=129)

66.6 (22) 24.2 (8) 9.0 (3) 33

44.4 (20) 37.7 (17) 17.7 (8) 45

42.1 (16) 42.1 (16) 15.7 (6) 38

43.5 (17) 43.5 (17) 12.8 (5) 39

30.3 (30) 65.6 (65) 4.0 (4) 99

78.7 (52) 21.2 (14)

63.3 (57) 36.6 (33)

64.1 (50) 35.8 (28)

64.5 (51) 35.4 (28)

63.1 (125) 36.8 (73)

19.2 (5) 50.0 (13) 30.7 (8) 26

41.1 (21) 23.5 (12) 35.2(18) 51

15.3 (6) 58.9 (23) 25.6 (10) 39

29.5 (8) 78.0 (32) 2.4 (1) 41

44.2 (23) 55.7 (29)

43.3 (63) 47.5 (57)

44.8 (35) 55.1 (43)

58.5 (48) 41.4 (34)

20.8 (24) 40.0 (46) 39.1 (45) 115 41.7 (96) 58.2 (134)

The number of subjects is given in parentheses. The number of subjects varied for each patient group and control group from the total looked at ie 44 uncomplicated subjects, as results for PON1 and PON2 were not obtained for each subject

cant. However, we did find an increase in the frequency of the Cys/Cys genotype in the nephropaths when compared to the non-nephropaths (uncomplicated and retinopaths combined) (41.1% vs 16.9% p=0.004, Pc=0.01) as well as the normal healthy control subjects (41.1% vs 20.8%, 7.3, p=0.006, Pc=0.01). The frequency of the carriers of the 311 Cys variant were similar in the uncomplicated, normal healthy control subjects and the nephropaths as found by Canani et al (69.0%, 60.6% and 64.0% respectively) (Table 1). It should be taken into account that this is only a small study of patients with Type I diabetes. However, it is a step closer to determining a role for PON2 in the susceptibility to diabetic nephropathy. Our findings are in agreement with those of Pinizzotto et al that is PON2 could have a role to play in the susceptibility to diabetic nephropathy in Type II diabetes. This is contrary to those of Canani et al who found no association with nephropathy in their population of patients with Type I diabetes. We also found a small association with the PON1 polymorphism Arg/Gln 191 (A and B alleles) in relation to diabetic nephropathy, that has not been found by either Pinizzotto M et al [5] or Araki et al [6]. We found that there was a significant increase in the frequency of the BB genotype in the nephropaths compared to the normal healthy control subjects (17.7% vs. 4.0% χ2=9.8, p=0.005, Pc=0.01). This was accompanied by a subsequent decrease in the AB genotype in the nephropaths compared to the normal healthy control subjects (37.7% vs. 65.6% χ2=7.64, p=0.001, Pc=0.002). An association with the PON1 polymor-

phism Gln192Arg and diabetic nephropathy has been shown previously [7]. However the PON1 genotype shown to be associated with increased urinary albumin excretion was different to the risk allele linked to cardiovascular disease. The genotype we found to be associated in this study is the BB genotype, which has been linked to cardiovascular disease. This has also been found in coronary heart disease in patients with Type II diabetes [8]. It is possible that the B isotype of the paraoxanse gene does not function well against excessive LDL oxidation therefore leading to oxidative tissue damage. Acknowledgements. This work was funded in part by the Diabetes UK, the British Council for the Prevention of Blindness, and the Northcott Devon Medical Foundation.

A. D. Hodgkinson, B. A. Millward, A. G. Demaine

References 1. Canani LH, Araki S, Warram JH, Krolewski AS (2001) Paraoxonase 2 polymorphisms are associated with diabetic nephropathy in Type II diabetes. Diabetologia 44:1062– 1069 2. Heesom AE, Hibberd ML, Millward BA et al. (1997) Polymorphism in the 5′-end of the aldose reductase is strongly associated with the development of diabetic nephropathy in type 1 diabetes. Diabetes 46:287–291 3. Sanghera DK, Saha N, Aston CE, Kamboh I (1997) Genetic Polymorphism of the Paraoxonase and the Risk of Coronary Heart Disease. Arteriosclerosis, Thrombosis, and Vascular Biology 17:1067–1073

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4. Sanghera DK, Aston CE, Saha N, Kamboh I (1998) DNA Polymorphisms in Two Paraoxonase genes (PON1 and PON2) Are Associated with the Risk of Coronary Heart Disease. Am J Hum Genet 62:36–44 5. Pinizzotto M, Castillo E, Fiaux M, Temler E, Gaillard RC, Ruiz J (2001) Paraoxonase 2 polymorphisms are associated with diabetic nephropathy in Type II diabetes. Diabetologia 44:104–107 6. Araki S, Makita Y, Canani L, Ng D, Warram JH, Krolewski AS (2000) Polymorphisms of human paraoxonase 1 gene (PON1) and susceptibility to diabetic nephropathy in Type I diabetes mellitus. Diabetologia 43:1540–1543

7. Jenkins AJ, Klein RL, Zheng D et al. (2000) Paraoxonase genotype (192 Gln-Arg) and serum paraoxonase-arylesterase activity: Relationship with Type 1 diabetes and nephropathy. Diabetes 49 [Suppl 1]:A157 (Abstract) 8. Ruiz J, Blanche H, James RW, Garin MC et al. (1995) GlnArg192 polymorphisms of paraoxonase and coronary heart disease in type 2 diabetes. Lancet 346:856 Corresponding author: A. G. Demaine, Molecular Medicine Research Group, Postgraduate Medical School, University of Plymouth, ITTC Building, Tamar Science Park, Plymouth PL6 8BX, England, E-mail: [email protected]