Human genetics studies reveal that all genes comprising the renin-angiotensin system (RAS) have several forms of polymorphism, raising the possibility that RAS activity may vary among individuals according to their genetic makeup. One such polymorphism is the deletion/insertion (I/D) polymorphism of the ACE gene.
- Study focused on ACE gene in Chinese subjects with igA nephritis.
- Patients treated with ACEI/ATRA experienced progressive retardation of renal failure.
- Patients with D allelle have a higher incidence of ESRF.
Professor Keng-Thye Woo, Senior consultant, Department of renal medicine, Singapore General Hospital looks at ACE gene polymorphism and its influence on disease progression in glomerulonephritis.
Human genetics studies reveal that all genes comprising the renin-angiotensin system (RAS) have several forms of polymorphism, raising the possibility that RAS activity may vary among individuals according to their genetic makeup. One such polymorphism is the deletion/insertion (I/D) polymorphism of the ACE gene. Individual antiproteinuric response to ACEI/ATRA therapy varies depending on ACE gene polymorphism. In this study we investigated the complete genomic sequence of the ACE gene in Chinese subjects with IgA Nephritis (IgA Nx) to determine its role and its influence on response to ACEI/ATRA therapy.
There is a higher chance of detecting rare functional variants (mutation) in diseased than in healthy individuals. It is more cost-effective to do complete gene sequences in a small number of patients in the initial study, and then proceed to determine specific single nucleotide polymorphism (SNP) in large numbers using rapid technique of real-time PCR. Hence we enlisted 40 IgA Nx patients and four healthy Chinese subjects. Of the SNPs detected, interesting SNPs were selected for determination in 100 Chinese with IgA Nx and another 100 healthy Chinese subjects.
Genomic DNA was extracted from peripheral leukocytes, and PCR amplification of the DNA sequence containing the polymorphism was performed using sense and anti-sense primers. Amplification products were digested with restriction enzyme. DNA fragments were then separated and viewed on 2 per cent agarose ethidium bromide gel. Genotypes were identified by the absence or presence of DNA fragments obtained. There are 3 ACE genotypes: DD, II and ID.
For ACE gene sequencing, EDTA blood samples were collected for extraction of genomic DNA, using QIA amp DNA blood-extraction kit. Genomic DNA was subjected to PCR amplification in an automated thermocycler. PCR amplicons were sequenced using the Big Dye Terminator cycle. All sequence data from every primer were aligned using DNA STAR software for visually inspecting sequence variants, and all variants were reconfirmed by additional PCR and sequencing.
A cohort of 40 patients with IgA Nx, 20 on treatment with ACEI/ATRA and 20 not on treatment (control group) were followed up for five years and the longterm outcome to continuing ACEI/ATRA therapy was correlated with genotype (ACE gene pattern, Haplotype, SNPs),to ascertain if the genetic profile plays a role in determining the response to ACEI/ATRA therapy and long-term renal outcome after five years. Patients entered the randomised control trial – 20 in treatment and 20 in control group from Dec 1997 to Dec 1999. Entry criteria were proteinuria >1gm per day and or serum creatinine 1.6mg/dl or more. In the control group, hypertension was treated with atenolol, propranolol, hydrallazine and methyldopa. In the treatment group, patients were treated with 5mg of Enalapril and/or 50mg of Losartan, increasing every three months to reduce proteinuria to less than 1gm a day.
Table 1 compares the clinical data of the treatment and control groups. ACE gene was sequenced (24,000 basepairs) in IgA Nx patients and normal subjects. There were five nucleotide variants, which differed significantly between patients with IgA Nx with renal impairment and those with ESRF in both genotype and allele frequencies. Using Clark’s subtraction algorithm, five locihaplotypes were constructed from these five nucleotide variants. Comparing patients with renal impairment versus those with ESRF, only two haplotypes were distinct, haplotypes 3 and 5. Haplotype 3, associated with a high odds ratio (14.0, p<0.02), indicated association with a high risk of ESRF. In contrast, haplotype 5, with a low odds ratio (0.07, p<0.02), was protective against ESRF.
In the treatment group there were significantly more patients with the II genotype with renal impairment compared with ESRF (p<0.001). There were significantly more patients with ID/DD genotype in the ESRF group compared with the renal impairment Group (p<0.05) and all those with renal impairment have haplotype 5, except for one patient (p<0.05). Those with ESRF tend to have more haplotype 3 (p<0.01).
In the control group, irrespective of ACE genotype, whether it was the Alu I/D variant, haplotype 3 or haplotype 5, there was no relationship to clinical outcome regarding final renal status, normal renal function, renal impairment or ESRF. In contrast, for patients being treated with ACEI/ATRA, the Alu I/D variant, haplotype 3 and 5 influence the response of patients and clinical outcome – those patients with II genotype and haplotype 5 continue in renal impairment whereas the ID/DD genotype and haplotype 3 patients progress to ESRF.
In the treatment group, patients with renal impairment treated with ACEI/ ATRA continue to have progressive retardation of renal failure, with three patients reverting to normal renal function. Except for one patient, all patients had proteinuria reduced to <1 gm/day after five years’ therapy. Patients with II genotype and haplotype 5 when they develop renal failure have slow progressive course (up to 35 years); those with ID or DD genotype and haplotype 3 reach ESRF within six years (p<0.01). Those with renal impairment with haplotype 3 are at risk of ESRF (p<0.01). Those with renal impairment with haplotype 5 are protected (p<0.05) from ESRF. However for patients in the control group the Alu variant ID polymorphism, haplotype 3 and 5 did not influence progression of renal failure. This supports our earlier hypothesis that individual response to ACEI/ATRA therapy is
Intra-group paired t test: a..a p<0.02, b..b p<0.002, c..c p<0.001 influenced by ACE gene polymorphism and its other linked haplotypes. Present data suggest that in IgA Nx, regarding ACE gene polymorphism, the Alu variant (ID) as well as the two linked haplotypes do influence the response of patients to ACEI/ATRA therapy.
This evidence corroborates the genetic influence of the ACE gene on the response of patients with IgA Nx to ACEI/ATRA therapy. This confirms our earlier suspicions that the response of patients with IgA Nx to ACEI/ATRA therapy depends on their ACE gene profile. Also, contrary to what earlier workers have postulated – that patients with the D allelle of the ACE gene respond best to ACEI therapy – our data has shown that those with the D allelle do poorly, have a higher incidence of ESRF and progress to ESRF much faster than those with the II genotype. Other workers have also shown that the DD genotype was associated with progression to ESRF, but the present data also show that the ID polymorphism and its linked haplotypes appear to influence the response of patients with IgA Nx to ACEI/ATRA therapy and progression to ESRF.
