LAL. Limulus amaebocyte lysate. LCAT. Lecithin:cholesterol acyltransferase. LDL ... R2A. Reasoners 2A (nutrient medium). RBP. Retinol binding protein. RO.
Nephrol Dial Transplant (2002) 17 wSuppl 3x: 5–11
Molecular genetics in renal medicine: what can we hope to achieve? L. Nordfors, P. Stenvinkel1, A. Marchlewska, R. Weiss1, O. Heimbu¨rger2, J. Bergstro¨m3, B. Lindholm3 and M. Schalling Department of Molecular Medicine, Neurogenetics Unit, Karolinska Hospital, 2Division of Renal Medicine, Department of Clinical Science, Huddinge University Hospital, 3Baxter Novum, Department of Clinical Science, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden and 1Department of Internal Medicine, Division of Nephrology, University of California Davis, California, USA
Keywords: end-stage renal disease; molecular genetics
Introduction End-stage renal disease (ESRD) is a complex disorder encompassing a large variety of phenotypes; each phenotype is the result of an underlying kidney disease and superimposing environmental and genetic factors. The complexity of the phenotypic make-up of renal diseases makes it difficult to diagnose and predict their progression and to decide on the optimal treatment for each patient. For example, the rate of progression of chronic renal failure and the side effects following dialysis treatment may vary considerably between individuals. Moreover, the prevalence of complications such as malnutrition, inflammation and atherosclerosis varies considerably in different cohorts of dialysis patients. This cannot be explained solely by ethnic, social and environmental factors but is probably also a result of interactions between various genes. Ever since DNA was reported to carry genetic information in 1944, and the structure of DNA was revealed to be a complementary double helix, in 1953, the knowledge about genetic factors and heredity for determining susceptibility to disease has increased steadily. Today, the revelation of the human genomic sequence through the HUGO and Celera projects w1,2x (see special issues of Nature and Science published in February 2001) has given us new tools to use in order to search for genes and genetic variations associated with particular human diseases. The impact of genetic variability on the development of renal failure is becoming clearer and emphasizes the need to elucidate the genetic basis for renal disease and its
Correspondence and offprint requests to: Martin Schalling, MD Professor, Department of Molecular Medicine, Neurogenetics Unit, CMM, L8:00, Karolinska Hospital, S-171 76 Stockholm, Sweden. #
complications. This would lead to a better understanding of the different phenotypes observed in ESRD and would enable us to determine if a patient is, for example, genetically predisposed to such complications as atherosclerosis, inflammation, obesity and malnutrition. A number of genetic variations, or polymorphisms, that play a role in ESRD have already been discovered (Table 1), but new efficient methods for genotyping, together with the fact that the entire human genomic sequence has been revealed, will accelerate the identification of disease genes and the development of treatments for specific diseases. The aim of this paper is to review the possible impact of genetic variability on ESRD and its complications and to discuss the possibilities that will be available with new emerging technologies, in particular as it applies to treatment opportunities or preventive measures.
Single nucleotide polymorphisms (SNPs) A number of different types of polymorphisms (the existence of two or more alleles at significant frequencies in a population) are found in the human genome, e.g. insertionudeletions (IuDs), mini- and microsatellites (di, tri- and tetranucleotide repeats) and single nucleotide polymorphisms (SNPs). These may all serve as markers in genetic analysis. Until recently, screenings of affected families and populations for identifying disease-causing loci and mapping of disease genes generally was performed using panels of microsatellite markers. Today, SNP markers allow a more detailed genetic analysis. SNPs are the most common type of human genetic variation. These single-base sequence variations occur at a frequency of approximately one SNP every 1000 bp throughout the human genome w3x. Largescale identification of SNPs began only recently w3x but, to date, some 3 3 106 SNPs have already been mapped and are now being assembled into large databases,
2002 European Renal Association–European Dialysis and Transplant Association
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Table 1. Some gene polymorphisms that may affect the phenotype observed in ESRD Effects on
Gene
Polymorphism
Effect
Energy expenditure Inflammation
UCP2 IL-1Ra GNB3 ICAM-1
Atherosclerosis
ApoE
Apo(a) eNOs
45 bp IuD exon 8 86 bp VNTR C825T R241 exon 4 E469 exon 6 «4 «2 C677T A1298C Apo(a) isoform 27 bp VNTR
ACE
250 bp IuD intron 16
AGT
M235T
Fat accumulation during PD Association with autoimmune and inflammatory diseases Higher CRP levels and mortality Up-regulation of adhesion molecules in CAF Up-regulation of adhesion molecules in CAF Increased risk of ICVD Possible predisposition to ESRD Increased homocysteine levels Increased homocysteine levels Elevated plasma Lp(a) levels Related to pathogenesis of CHD and ESRD Increased severity of UPE in IgAN Increased p-ACE levels, association and progression with interstitial nephritis Association with interstitial nephritis
MTHFR
Hypertension
ACE, angiotensin I-converting enzyme; AGT, angiotensinogen; APO, apolipoprotein; CAF, chronic allograft failure; CHD, coronary heart disease; eNOs, endothelial nitric oxide synthase; GNB3, G protein b3-subunit; ICAM-1, intercellular adhesion molecule-1; ICVD, ischaemic cerebrovascular disease; IgAN, IgA nephropathy; IL-1Ra, interleukin-1 receptor antagonist; MTHFR, methylenetetrahydrofolate reductase; UPE, urinary protein excretion; UCP2, uncoupling protein 2.
for example the National Center for Biotechnology Information (NCBI) and the Celera SNP databases. The high frequency of SNPs suggests that SNP genotyping will play a key role in future research aimed at identifying genetic variants involved in disease and defining the genetics of disease predisposition as well as inter-individual variability in drug response (pharmacogenetics). However, can existing technologies manage the enormous amounts of genotyping that will be required? Future studies may involve analysis of thousands of SNPs in, for example, clinical testing of large patient materials, or in association studies including several thousands of individuals, which suggests that there is an increasing need for robust and high-throughput allele typing techniques. Today, the main SNP genotyping platforms are based on nucleic acid hybridization on filters w4x or chips w5x, and on primer extension methods w6,7x, but most methods in routine use are too time consuming and expensive to match future needs and expectations.
New methods for SNP analysis Recently, a new straightforward sequencing method, Pyrosequencing2, was introduced. This method is based on sequencing-by-synthesis and on real-time pyrophosphate (PPi) detection, which in several aspects may be advantageous compared with already existing methodologies w8x. The technical platform employed involves a highly automated sequencing instrument, allowing analysis of 96 samples in 10–15 min. The method relies on the fact that PPi is released as a result of nucleotide incorporation during DNA synthesis and, most importantly, for every incorporation event, the release is equimolar to the number of nucleotides incorporated. PPi quantification is performed by
indirect luminometric detection, permitting parallel, real-time analysis of all samples. The sequencing reaction is carried out in a cocktail consisting of four different enzymes as well as luciferin, which is the substrate for firefly luciferase. The PPi is converted to ATP by ATP sulfurylase and the ATP, in turn, promotes the luciferase-mediated conversion of luciferin into visible light. A CCD camera detects the light, and the resulting real-time signals, representing the DNA sequence, are presented in a pyrogram, with peak heights corresponding to the number of identical residues incorporated. The Pyrosequencing2 method is flexible in the sense that it allows either analysis of 96 different SNPs in one individual or analysis of one SNP in 96 different individuals. Also, since the polymerase chain reaction (PCR) amplification is done prior to this procedure in a thermal cycler, the pyrosequencing instrument is occupied only during reading and automatic analysis of the plate. This means that only the number of PCRs that are possible to run in one day limits the maximum throughput. The pyrosequencing process has been fully automated and adapted to screenings of large SNP panels. Also, the simplicity of the method allows SNP genotyping to become accessible for personnel other than specialized researchers, which, from our experience, makes it suitable for clinical genetic testing of large patient materials. Since it is most probable that gene polymorphisms play an important role in the expression of different phenotypes in ESRD patients, we believe that genotyping of polymorphisms is destined to be one of the most important future tools to understand better the progression towards disease as well as the observed outcomes in different ESRD patient populations. In addition, we believe that determination of gene polymorphisms may play an important role in helping clinicians choose which treatment strategy (such as
Molecular genetics in renal medicine
choice of antihypertensive and immunosuppressive drugs and modality of dialysis treatment) to use for the individual ESRD patient. In the following, we will discuss some areas within nephrology in which we believe that gene polymorphisms will play a significant future role for both the understanding and treatment of the observed different phenotypes.
Malnutrition and obesity Malnutrition and wasting is a common and important clinical problem and also a source of concern since parameters of nutritional status (such as serum albumin) have been shown to be among the most powerful predictors of morbidity and mortality in ESRD patients w9x. Whereas a number of dialysisrelated as well as non-dialysis-related factors might contribute to malnutrition in ESRD patients, the main cause for the decline in nutritional status during the course of renal failure is probably anorexia. Leptin is a satiety hormone acting as an afferent signal in a negative feedback loop, decreasing food intake w10x and increasing energy expenditure in animal models w11x. This protein is produced principally in adipose tissue and is secreted into the bloodstream. Since the circulating leptin levels are elevated inappropriately in most, but not all, ESRD patients w12x, hyperleptinaemia has been suggested to be one of the factors contributing to anorexia and weight loss in these patients w13x. Gene expression studies have shown that leptin mRNA levels are significantly lower in patients with ESRD than in healthy body mass index (BMI)matched controls, suggesting a hyperleptinaemiainduced feedback inhibition of leptin expression that is not present in obesity w14x. Consequently, ESRD patients may not be resistant to leptin signalling, which may be the case in obesity. Peritoneal dialysis (PD) and haemodialysis (HD) are both valid methods for treatment of ESRD but, despite optimal dialysis treatment, the nutritional status does not always improve. Studies on eating behaviour have shown that hunger and eating drive are lower in patients treated with PD than in those treated with HD w15x. Whereas several factors, such as the continuous glucose load and a feeling of fullness from the dialysate, may contribute to reduced appetite in PD patients, further research is needed to establish if hyperleptinaemia during PD also contributes. Polymorphisms in the leptin gene are not likely to influence serum leptin levels w16x. However, a silent mutation at codon 25 (CAAuCAG, glutamine) may be linked to morbid obesity in a Japanese population w17x. A significantly higher prevalence of the variant leptin 25CAG allele was found in 53 obese subjects than in 132 non-obese controls and may therefore be a new genetic marker for obesity susceptibility, at least in the Japanese population. Another common and important side effect of dialysis, especially PD treatment, is accumulation of body fat stores. However, marked variations have been
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observed between patients w18–20x that cannot be explained by variations in glucose absorption, suggesting that genetic differences in basal metabolic rate may play a significant role. In this respect, the uncoupling proteins (UCPs), which are inner membrane mitochondrial proteins believed to increase thermogenesis, may be of interest, and it has been speculated that UCP2 may play a role in fat tissue accumulation and obesity w21,22x. Thus, an IuD in the UCP2 gene has been shown to be associated with lower metabolic rate w23x, and we have demonstrated that this polymorphism influences changes in body composition during 12 months of PD w24x. Further studies are needed to investigate if other genotype variants may predict changes in body composition during dialysis treatment. Overweight is also common in HD patients w25–27x and may also be related to genetic factors. Interestingly, obesity in these patients appears to be associated with improved survival w26,27x.
Inflammation The prevalence of inflammation has been reported to be high in ESRD patients, varying between 30 and 50% in different pre-dialysis and dialysis populations w28x. The high prevalence of inflammation in ESRD is of clinical importance not only because inflammation may play an important role in the progression of chronic renal failure (CRF) but also since inflammation, as evidenced by high C-reactive protein (CRP), has been shown to be an independent risk factor for cardiovascular disease (CVD) in dialysis patients w29x. The causes of the high prevalence of elevated CRP levels remain unknown, although it seems conceivable to speculate that both dialysis-related and non-dialysisrelated factors, such as genetic polymorhisms, might contribute. Serum levels of CRP appear to reflect generation of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6 and tumour necrosis factor-a (TNF-a), all of which are increased in ESRD w30x. Since CRP levels vary considerably in ESRD patients, it could be hypothesized that patient-related factors such as polymorphisms in genes encoding proinflammatory cytokines may be involved in determining the individual cytokine production in response to a given insult. In this respect, a number of different cytokine polymorphisms might be of importance. IL-1 is regulated by the IL-1 receptor antagonist IL-1Ra, which is a competitive IL-1 inhibitor and a strong anti-inflammatory agent. An 86 bp VNTR in intron 2 of this gene (allele 2) is associated with a number of autoimmune and inflammatory diseases. No clear association was found between allele 2 and ischaemic heart disease. However, among non-renal patients with myocardial infarction, an increased allele 2 frequency was observed in those of younger age and with higher CRP levels; this finding merits further investigation w31x. On the other hand, a preliminary study including 58 continuous ambulatory peritoneal
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dialysis (CAPD) and 84 HD patients concluded that polymorphisms in the genes encoding TNF and interferon-c (IFN-c) do not play a role in determining CRP levels w32x. Another polymorphism that might contribute to different inflammatory responses is the C825T polymorphism in the GNB3 gene, encoding the ubiquitously expressed b3-subunit of the G proteins, which is involved in immune cell function in humans. In a prospective study including 228 HD patients, higher CRP levels and higher mortality were seen in T homozygotes, suggesting that the C825T polymorphism might influence mortality rate in HD patients w33x. Soluble cellular adhesion molecules wintercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectinx are a group of molecules which are expressed on the surface of vascular endothelial cells in response to pro-inflammatory cytokines. Recent data have shown elevated levels of cellular adhesion molecules in ESRD patients who are inflamed, malnourished and have signs of CVD w34x. In this respect, it is of interest that up-regulation of adhesion molecules has been observed in chronic allograft failure, which is a common cause of late graft loss in renal transplantation. McLaren et al. w35x have shown that two polymorphisms (R241 in exon 4 and E469 in exon 6) in the ICAM-1 gene, which could influence the mechanisms of ICAM-1 action, may represent genetic risk factors for chronic allograft failure. Based on the presently available and very limited information regarding the role of various cytokine polymorphisms, it is obvious that further studies are needed to investigate the importance of cytokines and other gene polymorphism(s) in progression towards ESRD.
Atherosclerosis The significant burden of atherosclerotic CVD in ESRD has been recognized for )25 years w36x. However, the prevalence of atherosclerotic complications varies considerably between different dialysis populations and it seems conceivable to speculate that genetic factors affecting lipid metabolism, inflammation and other possible risk factors, such as elevated homocysteine levels, might explain this. Atherosclerosis, which has itself been described in a recent review as an inflammatory disease w37x, is manifest by the aberrant proliferation of vascular smooth muscle (VSM) cells. There are multiple influences that act upon this cell, and its cousin the renal mesangial cell w38x, to promote cell growth. While the classical peptide growth factors, such as platelet-derived growth factor (PDGF), are clearly important in this process, it is likely that other substances, such as the inflammatory cytokines, play a similar role. As an added complexity, some serum factors, such as transforming growth factor-b (TGF-b), have complex roles in these cells, being at times stimulatory and inhibitory, depending on environmental conditions w39,40x. This biphasic nature of
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growth factors on VSM and mesangial cell growth is reflected in parallel pleiotropic responses observed in their signalling cascades w41,42x and is especially important in the field of nephrology, since TGF-b is a potent inducer of mesangial and vascular matrix proteins w43x and may lead to microvascular and glomerular scarring. The roles of the various growth factors in the process of VSM cell proliferation, which occurs in response to inflammation and atherosclerosis, are only now being elucidated. In the general population, cross-sectional studies have shown that the «4 allele of the apolipoprotein E (ApoE) gene is associated with an increased risk of atherosclerotic CVD w44x. Similarly, in ESRD patients, the prevalence of ischaemic cerebrovascular disease was significantly higher in subjects carrying one or two «4 alleles as compared with patients without this allele w45x. Lipid abnormalities are found frequently in ESRD patients, and abnormal metabolism may contribute to the progression of renal disease w46x as well as accelerated atherosclerosis. It has been reported that ApoE may play an important role in lipoprotein metabolism, and variations at the ApoE locus have been shown to affect low-density lipoprotein (LDL) cholesterol levels in CAPD subjects, suggesting that «4 carriers may be more susceptible to accelerated development of atherosclerosis in this condition w47,48x. Moreover, in 592 Japanese uraemic patients, Oda et al. w49x found a higher frequency of the «2 allele, suggesting that ApoE2 is associated with a possible predisposition to ESRD. In their study, it was also demonstrated that the impact of ApoE2 and ApoE4 on the lipid profile was unique and different from the normal population w49x. However, a recent study w50x including 269 patients indicated that, although ApoE polymorphisms significantly affected serum levels of total cholesterol and LDL cholesterol, it was not associated with the presence of atherosclerosis. Thus, the association between ApoE polymorphisms and atherosclerosis still remains unclear in ESRD patients. Homocysteine is considered to be a risk factor for accelerated atherosclerosis in both the general population w51x and ESRD patients w52x. However, it should be pointed out that not all studies have found an association between hyperhomocysteinaemia and CVD in ESRD patients w53x. The C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene have been shown to influence serum levels of homocysteine in healthy individuals, dialysis patients and kidney graft recipients w54,55x. Consequently, more studies are needed to investigate if these polymorphisms may be associated with an accelerated atherosclerosis in ESRD patients. Nitric oxide is a potent regulator of intrarenal haemodynamics and may play an important role in decreasing renal function and in accelerated atherosclerosis. A 27 bp tandem repeat polymorphism (allele a and b have four and five repeats, respectively) in intron 4 of the endothelial nitric oxide synthase (eNOs) gene is believed to be related to the pathogenesis of coronary heart disease and terminal chronic
Molecular genetics in renal medicine
renal failure. Since the frequency of the a allele was significantly higher in patients with non-diabetic ESRD than in controls, and therefore appears to affect the progression of renal failure w56x, further studies are needed to investigate the role of this polymorphism in ESRD patients. Lipoprotein(a) wLp(a)x is a cholesterol-rich, LDL-like particle whose protein moiety contains Apo(a) and Apo B-100. A number of epidemiological studies have shown that Lp(a) is an independent risk factor for atherosclerotic complications in ESRD w57x as well as in the general population w58x. In normal subjects, the genetic polymorphism of Apo(a) explains a major part (;45–70%) of the variability of plasma Lp(a) concentration, which is closely related to the Lp(a) isoform size w59x. Since most, but not all, ESRD patients have elevated plasma Lp(a) levels, it has been proposed that this may be a factor contributing to accelerated atherosclerosis in this group of patients. However, we w60x and others w61x have demonstrated that the size of the Apo(a) isoform may be more predictive for cardiovascular complications than plasma Lp(a) levels per se, which suggests that genetic components may be one cause of the accelerated atherosclerosis observed in ESRD patients.
Hypertension and progression of renal failure ESRD is preceded by a stage of progressive decline in renal function usually characterized by hypertension and activation of the renin–angiotensin system (RAS). However, there are considerable variations in the rapidity of renal disease progression in individual patients, and it could be speculated that genetic factors contribute to some of the observed differences. It is therefore of interest that a number of polymorphisms are found in genes encoding components of the RAS, which is involved in systemic blood pressure regulation as well as in the regulation of haemodynamics w62–65x. An IuD polymorphism in intron 16 in the angiotensin I-converting enzyme (ACE) gene, where the D allele is associated with higher plasma levels of ACE, has been shown to be associated with susceptibility to interstitial nephritis w66x. In 80 family trios (proband and parents) with interstitial nephritis, the D allele was transmitted significantly more frequently than would have been expected if no association existed. Furthermore, the ID and the DD genotypes were associated with a faster rate of renal function decline. Moreover, a number of studies have shown that the DD genotype implies worse renal prognoses and an increased risk of developing ESRD at an early age w67,68x. However, contradictory results were seen in one study involving patients with autosomal dominant polycystic kidney disease (ADPKD), where no relationship between progression towards ESRD and the ACE IuD polymorphism was detected w69x. In the same 80 family trios, the M235T polymorphism in the angiotensinogen (AGT) gene was associated with interstitial nephritis,
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where the 235T allele was transmitted significantly more frequently than expected w66x. In addition, in 95 Japanese children with IgA nephropathy, the TT genotype was associated with increased severity of proteinuria and may thus play a significant role in the progression of disease in these patients w70x.
Summary and conclusion The landmark publications of the human genome in Nature and Science, February 2001, mark the beginning of a new era in clinical medicine, with entirely new possibilities for identification of the genetic background to, and manifestations of, human diseases. At the same time, new methodology allowing rapid analysis of gene polymorphisms in large patient cohorts with different phenotypes can now be used increasingly in clinical research. Altogether, this is likely to have a powerful and significant impact on clinical practice. The utilization of these new tools for research into the diagnosis and therapy of human disease will require increased collaboration between clinical investigators (as experts on phenotyping) and clinical and molecular geneticists (as experts on genotyping). In addition, these collaborations generate large data sets requiring skills in bio-informatics that need to be complemented by genetic epidemiology to understand gene– environment interaction and the complex mix of populations in relation to disease. As shown in this review, there are already some examples of such a development in the field of molecular genetics and renal disease. During the next few years, application of molecular genetics in renal disease and complications associated with ESRD will almost certainly result in new important discoveries that could benefit our patients in developing optimal treatment selections as well as new and more selective drugs.
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