Association between Haptoglobin Gene Variants and

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Original Paper Nephron Exp Nephrol 2007;105:e75–e79 DOI: 10.1159/000098563

Received: January 27, 2006 Accepted after revision: March 21, 2006 Published online: January 12, 2007

Association between Haptoglobin Gene Variants and Diabetic Nephropathy: Haptoglobin Polymorphism in Nephropathy Susceptibility Bryan R. Conway a David A. Savage a Hugh R. Brady b Alexander P. Maxwell a a Nephrology Research Group, Queen’s University Belfast, Belfast, UK; b Conway Institute, University College Dublin, Dublin, Ireland

Abstract Background/Aims: The Hp1/Hp2 DNA polymorphism has previously been implicated in susceptibility to diabetic nephropathy in some but not all studies. In an attempt to clarify these conflicting findings, we conducted a case-control association study in a Caucasian population. Methods: We recruited 224 and 285 type 1 diabetic patients with (cases) and without (controls) nephropathy, respectively, from 2 centres based in Northern Ireland and the Republic of Ireland. Hp1/Hp2 genotyping was performed using a combination of long-range and multiplex PCR. Allele and genotype frequencies in cases and controls were compared using the "2 test. Results: There was a statistically significant increase in the frequency of the Hp2 allele in cases compared to controls (65.6 vs. 58.6%, OR = 1.35, 95% CI: 1.04–1.76, p = 0.03). The distributions of genotypes were in Hardy-Weinberg equilibrium for both cases and controls, and the overall frequency of the Hp1 allele was 38.3%, which is similar to that found in other Western European populations. Conclusions: The results suggest that the Hp2 allele may confer susceptibility to nephropathy in patients with type 1 diabetes. Copyright © 2007 S. Karger AG, Basel

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Introduction

Diabetic nephropathy is arguably the most serious of the microvascular diabetic complications, resulting in a significant increase in morbidity and mortality for individuals with diabetes. It is now the most common cause of end-stage renal disease in the Western world [1]. While environmental factors such as glycaemic and blood pressure control influence the rate of progression of diabetic nephropathy, epidemiological and familial studies have indicated that genetic factors play an important role in conferring susceptibility to nephropathy [2, 3]. Oxidative stress is increasingly recognised as a major intermediary in the pathogenesis of diabetic nephropathy [4]. Erythrocyte destruction results in the release of free plasma haemoglobin, which can cross the glomerular filter and promote the generation of free radicals within the kidney [5]. The plasma protein haptoglobin binds with free haemoglobin to form a complex that is not filtered at the glomerulus, but is transported to the liver for clearance. In humans there are 3 common haptoglobin phenotypes, 1-1, 1-2 and 2-2, which are determined by 2 alleles, Hp1 and Hp2, located on chromosome 16q22 [5]. The Hp2 allele contains an internal 1.7-kb duplication resulting in an !-chain of 142 amino acids as compared to 83 amino acids in the Hp1 variant. The 1-1 phenotype, encoded by Dr. Bryan Conway Renal Unit, Tower Block Belfast City Hospital Belfast, BT9 7AB (UK) Tel. +44 77 9077 8526, Fax +44 28 9026 3535, E-Mail [email protected]

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Key Words Diabetic nephropathy ! Genetic susceptibility ! Gene polymorphism ! Oxidative stress

333 bp

1

2

Fig. 1. Partial structure of the Hp and Hp

alleles illustrating the 1.7-kb duplication in the Hp2 allele that includes exons 5 and 6. Unenclosed arrowheads below each allele represent the 2,067- and 3,781-bp fragments resulting from the Hp1 and Hp2 alleles, respectively, during long-range PCR using primers A and B. Unenclosed arrowheads above each allele illustrate that while the Hp1 allele results in a single 333-bp fragment, the Hp2 allele produces an additional 492-bp fragment following multiplex PCR using primers A, C and D.

Hp allele

Exon 3

C

B

Exon 4 2,067 bp

333 bp

Hp 2 allele

A

the Hp1/Hp1 genotype, is characterised by a single lowmolecular-weight heterodimeric band on starch gel electrophoresis. The 2-2 variant, encoded by the Hp2/Hp2 genotype, produces a series of higher molecular weight bands, and the Hp1/Hp2 genotype encodes an intermediate 1-2 phenotype. There is a progressive reduction in the haemoglobin binding and antioxidant capacity of haptoglobin from the 1-1 phenotype through the 1-2 and 2-2 variants [5], and therefore diabetic individuals with the Hp1/Hp1 genotype may be less susceptible to nephropathy. This has been demonstrated in some [6, 7] but not all studies [8, 9]. To investigate whether the Hp1/Hp2 polymorphism influences susceptibility to diabetic nephropathy in our population, we performed a case-control association study, comprising type 1 diabetic patients derived from 2 independent centres in Ireland.

Subjects and Methods Ethical approval was obtained from the respective University Research Ethics Committees and written, informed consent was obtained from all the subjects prior to recruitment. All the cases (n = 224) and controls (n = 285) were Caucasians and were diagnosed as having type 1 diabetes before 35 years of age. They were recruited, employing identical criteria, from nephrology and diabetic clinics in Northern Ireland and the Republic of Ireland. The patients with nephropathy (cases), who had diabetes for at least 10 years prior to the onset of proteinuria (1 0.5 g/24 h), had diabetic retinopathy and exhibited no clinical, serological or radiological evidence of non-diabetic renal disease. The control patients, who had diabetes for more than 15 years, had no evidence of microalbuminuria on repeated testing and were not receiving

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D

Nephron Exp Nephrol 2007;105:e75–e79

492 bp

D Exon 3

D’

C Exon 4

Exon 5

C’

B

Exon 6

3,781 bp

anti-hypertensive medication. A detailed description of the patients recruited to the study has been reported previously [10]. The haptoglobin polymorphism was genotyped using a combination of long-range PCR followed by multiplex PCR. All the samples were initially genotyped by long-range PCR employing primers A: 5"-TCT TTG GTT GGG TAG GAG TG-3" and B: 5"TGT CAC TGC TGC GTA AAG TA-3" (fig. 1). Each 50 #l reaction contained 125 ng of DNA, 1 times PCR buffer, 2.25 mM of Mg2+, 2 mM of each dNTP, 0.2 #M of each primer and 2.62 units of Expand Long Range PCR (Roche Diagnostics, Lewes, UK). PCR was performed under the following conditions: 94 ° C for 2 min; followed by 30 cycles of 94 ° C for 10 s, 54 ° C for 30 s and 68 ° C for 3 min, with the length of hold at 68 ° C increasing by 20 s/cycle from cycles 11–30; and finally 68 ° C for 5 min. The PCR products were electrophoresed on a 3% agarose gel, with bands of 2,067 and 3,781 bp corresponding to the Hp1 and Hp2 alleles, respectively (fig. 2). As there was preferential amplification of the smaller Hp1 allele in the long-range PCR (fig. 2), an additional PCR was required for accurate genotype assignment. All samples producing a single 2,067-bp band on long-range PCR were re-typed by multiplex PCR in order to exclude the presence of the Hp2 allele. In addition to primer A above, this reaction included primers C: 5"-ATC CAT GGA AGC CTA GCA GG-3" and D 5"-GAG TGC TCC ACA TAG CCA TG-3". Each 25 #l reaction contained 50 ng of genomic DNA, 1 times PCR buffer, 0.2 mM of each dNTP, 0.5 #M of each primer and 0.5 units of Taq Polymerase (Qiagen, Crawley, UK). The PCR was performed under the following conditions: 95 ° C for 5 min; followed by 35 cycles of 95 ° C for 45 s, 55 ° C for 45 s, 72 ° C for 1 min; and finally 72 ° C for 7 min. The Hp1 allele produced a single 333-bp fragment, whereas the Hp2 allele produced 2 fragments of 333 and 492 bp, following electrophoresis on a 1.5% agarose gel (fig. 3). An additional 2,057-bp fragment from primers A and D was not seen under these conditions. For both the longrange and multiplex PCR, genotypes were ascertained by 2 independent researchers. The clinical characteristics of the cases and controls were compared using the Student t test. The allele and genotype frequencies in cases and controls were compared using the "2 test,

Conway /Savage /Brady /Maxwell

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1

A

3,781 bp 2,067 bp

Fig. 2. The products of the long-range PCR following electrophoresis on agarose gel, alongside a 1-kb ladder (Invitrogen, Paisley, UK). From the relatively greater intensity of the Hp1 fragments it can be seen that there is preferential amplification of the shorter Hp1 allele.

Fig. 3. Products of the multiplex PCR

492 bp

following electrophoresis alongside a $X174RF DNA/Hae III ladder (Invitrogen). Presence or absence of the Hp2 allele could be determined accurately in samples previously genotyped as Hp1/Hp1 by longrange PCR.

Results

We recruited 137 cases and 179 controls from Northern Ireland and 87 cases and 106 controls from the Republic of Ireland. As anticipated there was a significant difference in both the systolic and diastolic blood pressures and in the glycaemic control between the cases and controls (table 1). Furthermore a significantly greater proportion of cases were male (55 vs. 42%). There were no significant differences between the 2 groups in either the age of onset of type 1 diabetes or in the duration of diabetes prior to recruitment to the study. By the time of recruitment 31.2% of patients with nephropathy had already developed end-stage renal disease. In both the Northern Ireland and Republic of Ireland sample groups, the distributions of genotypes were in Hardy-Weinberg equilibrium for both cases and controls. Association between Haptoglobin Gene Variants and Diabetic Nephropathy

There was a statistically significant increase in the frequency of the Hp2 allele in cases compared to controls (65.6 vs. 58.6%, OR = 1.35, 95% CI: 1.04–1.76, p = 0.03; table 2). The overall frequency of the Hp1 allele was 38.3%, which is similar to that found in other Western European populations.

Discussion

Our results suggest that the Hp2 allele may confer susceptibility to nephropathy in patients with type 1 diabetes in the Irish population. This is consistent with the results of a smaller Israeli study that included patients with both type 1 and 2 diabetes [7]. In the Israeli study the Hp2 allele was significantly more common in diabetic individuals who had either micro- or macroalbuminuria than in normoalbuminuric diabetic controls (83 vs. 61%, OR = 3.05, 95% CI: 1.37–6.96). However, caution should be exercised when interpreting this data due to the small sample sizes employed (29 cases, 81 controls). In contrast, no association was detected between Nephron Exp Nephrol 2007;105:e75–e79

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with the level of significance set at p ! 0.05. This provided 80% power to detect association with an odds ratio (OR) 61.5 and an Hp2 allele frequency of 60% (similar to that reported in other Western European populations) [5].

333 bp

Table 1. Clinical characteristics of the cases and controls

% male Age of onset of diabetes, years Duration of diabetes at time of recruitment, years HbA1c, % Systolic BP, mm Hg Diastolic BP, mm Hg PU/CKD/ESRD, %

Cases (n = 244)

Controls (n = 285)

p value

55.0 17.0 (88.6)

42.3 16.8 (88.2)

0.004 0.80

26.6 (88.4) 9.0 (81.7) 148.0 (817.7) 85.5 (89.4) 23.7/41.4/31.2

26.3 (88.8) 8.3 (81.2) 125.2 (812.8) 76.3 (86.7)

0.71