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Genomewide Linkage Analysis to Serum Creatinine, GFR, and Creatinine Clearance in a Community-Based Population: The Framingham Heart Study

Caroline S. Fox^*,,, Qiong Yang^,||, L. Adrienne Cupples, Chao-Yu Guo, Martin G. Larson^*,, Eric P. Leip^*,, Peter W.F. Wilson^*,¶ and Daniel Levy^*,,¶

*National Heart, Lung, and Blood Institute’s Framingham Heart Study, Framingham, Massachusetts; Department of Endocrinology, Diabetes, and Hypertension, the Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts; ^||Department of Neurology, Boston University School of Medicine, Boston, Massachusetts; ^¶Boston University School of Medicine, Boston, Massachusetts; and the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

Correspondence to Dr. Caroline S. Fox, 73 Mt. Wayte Avenue, Suite #2, Framingham, MA 01702. Phone: 508-935-3447; Fax: 508-626-1262; E-mail: foxca{at}nhlbi.nih.gov

	Abstract

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ABSTRACT. Kidney disease is a risk factor for the development of cardiovascular disease, all-cause mortality, and ESRD. It is not known to what extent genetic factors play a role in the development of kidney disease in the general population. Multipoint variance components linkage analysis was performed using Genehunter on 330 families from the Framingham Heart Study offspring cohort, using a 10-cM genomewide scan for serum creatinine, GFR, and creatinine clearance (CRCL) measured from 1998 to 2001. GFR was estimated using the simplified Modification of Diet in Renal Disease Study equation, and CRCL was estimated using the Cockcroft-Gault equation. Covariates in the adjustment included age, gender, body mass index, diabetes, systolic BP, hypertension treatment, tobacco use, and HDL cholesterol. Overall, 1224 subjects (52% women), mean age 59, were available for analysis. Mean creatinine was 0.87 mg/dl, mean GFR was 87 ml/min per 1.73 m², and mean creatinine clearance was 100 ml/min. The multivariable-adjusted heritability estimates for creatinine, GFR, and CRCL were 0.29, 0.33, and 0.46, respectively. The peak log of the odds ratio (LOD) scores for serum creatinine, GFR, and CRCL were 2.28 at 176 cM on chromosome 4, 2.19 at 78 cM on chromosome 4, and 1.91 at 103 cM on chromosome 3, respectively. In a community-based sample, measures of serum creatinine, GFR, and CRCL are heritable, suggesting an underlying genetic component. These results also provide suggestive evidence for linkage to measures of kidney function. Further research is necessary to identify the genes involved in the development of kidney disease and to understand their roles in this complex process.

	Introduction

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Elevation of serum creatinine is a risk factor for development of cardiovascular disease (1), all-cause mortality (2), and ESRD (3). These risks are evident at even moderate elevations in serum creatinine (1,2). Among patients who develop ESRD, the risk of cardiovascular disease is 10 to 20 times higher than that of the general population (4–6).

Genetic factors are likely to play a role in the progression of renal disease. Familial aggregation of ESRD has been identified (7). Linkage analyses of kidney function have been conducted (8–11), but results are limited because of the use of populations enriched for hypertension (8,11), premature cardiovascular disease (11), progressive renal failure (9), and ESRD (10). Thus, little is known about the genetics of kidney function in the general population. We sought to assess heritability and linkage of measures of renal function in the Framingham Heart Study, a community-based sample.

	Materials and Methods

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The Framingham Heart Study began in 1948 with the enrollment of 5209 men and women who were 28 to 62 yr of age at study entry, with subjects undergoing repeat examinations every 2 yr (12). In 1971, 5124 men and women were enrolled into the offspring cohort of the Framingham Heart Study, which included the children or spouses of the children of the original study cohort. Offspring subjects underwent examinations approximately every 4 yr; the design and methods of the offspring cohort has been previously described (13).

Details regarding the methods of risk factor measurement and laboratory analysis have been described (14). Each examination included an extensive cardiovascular disease assessment, 12-lead ECG, and blood testing. Serum creatinine was measured from 1998 to 2001 using the modified Jaffe method. GFR was estimated using the simplified Modification of Diet in Renal Disease Study equation (15,16), and creatinine clearance (CRCL) was estimated using the Cockcroft-Gault equation (17).

Leukocyte DNA was extracted from 5 to 10 ml of whole-blood or buffy-coat specimens using a standard protocol (18). Aliquots of DNA from members of the largest Framingham Heart Study families were sent in four batches to the Mammalian Genotyping Service Laboratory at the Marshfield Clinic (Marshfield, WI), consisting of 330 pedigrees and 546 sibships, ranging in size from 0 to 7. A 10-cM density genomewide scan was performed. Genotype data cleaning, including verification of family relationships and Mendelian inconsistencies, have been previously described (19).

Statistical Analyses
Using SAS (20), gender-specific residuals were computed. Gender-specific residuals were used to account for gender differences in serum creatinine, GFR, and CRCL measures. Residuals included adjustment for age, body mass index, HDL cholesterol, systolic BP, hypertension treatment, diabetes, and current smoking. A residual value >2.5 SD from the mean was reduced to the 2.5 SD value to improve the skewness and kurtosis of the data (21). In addition, all GFR and CRCL values >200 were considered to be 200.

Variance components linkage analysis (22,23), implemented in Solar (22) and Genehunter (24), were used to perform the heritability and genomewide linkage analyses, respectively. Heritability is defined as the proportion of variability in the trait attributable to the additive effect of genes and represents the contribution of both genes and early common environment. The underlying model assumes that variation in the trait can be partitioned into genetic and random environmental components. It is assumed that the genetic component is polygenic with no variation attributable to dominance components. Variance components linkage analysis uses the genotype information at a locus to decompose the phenotypic variance into a component attributable to the locus (known as a quantitative trait locus [QTL]), a polygenic component, and a random environmental component. Genotype information at a locus is characterized by the probability that two related individuals share 0, 1, or 2 alleles identical by descent.

All variance components were estimated by maximum likelihood. Linkage was tested by a likelihood ratio test in which the hypothesis that the QTL variance component is equal to zero was compared with its being greater than zero. The resulting ² statistic was converted to a traditional LOD score by dividing by 2*ln (10). LOD scores >1.9 were considered suggestive, and LOD scores >3.3 were considered significant (25).

	Results

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Overall, 1224 subjects were available for analysis. The mean age was 59 yr; 52% of the sample was female. Mean serum creatinine, GFR, and CRCL were 0.87 mg/dl, 87 ml/min per 1.73 m², and 100 ml/min, respectively. Baseline characteristics of the study sample are shown in Table 1. Correlations between covariates are presented in Table 2,

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Multipoint LOD scores for serum creatinine, GFR, and creatinine clearance (CRCL) on chromosomes 3, 4, and 11.

	Discussion

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To our knowledge, these data are the first population-based genomewide linkage analyses to demonstrate suggestive linkage to measures of kidney function. Although other linkage analyses for measures of kidney function have been performed, they have been conducted in populations enriched for hypertension (8,11), premature cardiovascular disease (11), progressive renal failure (9), or ESRD (10), limiting their generalizability. In the HyperGEN Study, DeWan et al. (8) reported a LOD score of 3.36 on chromosome 3 at 115 cM among white individuals with a high prevalence of hypertension; this locus is near our peak LOD score for CRCL (1.91 at 103 cM on chromosome 3). However, in the HyperGEN sample, this locus was not substantiated in their subsequent fine-mapping of the region (26). In a Utah pedigree study with subjects ascertained for premature coronary heart disease, stroke deaths, or hypertension (mean age of 40), a maximum LOD score of 2.1 was found on chromosome 10 at 113 cM (11). The full linkage analyses were not presented; thus, we cannot assess whether any areas of overlap with our findings exist. Potential differences between this study and ours include ascertainment for subjects enriched for cardiovascular disease, a 12-h urine sample for the assessment of CRCL, a younger population, larger families, and lack of adjustment for diabetes and hypertension (two key determinants of kidney function). Among black sibpairs with ESRD, a LOD score of 3.4 was reported on chromosome 10 (10), and an autosomal dominant form of kidney failure has been mapped to chromosome 1q21 (9).

Some potentially exciting candidate genes exist under our linkage peaks. Inflammation is known to play a role in kidney disease (27,28). The tumor necrosis factor receptor–activating factor (TRAF6) gene is found on chromosome 11 and is a member of the TNF receptor–associated factor family. TRAF6 has been shown to reside in the human proximal tubule cells and is part of the pathway for activation of IL-8 and monocyte chemoattractant protein-1 (29). The IL-8 gene lies under our peak on chromosome 4 at 75 mB. The protein encoded by this gene is a member of the CXC chemokine family and is an important mediator of the inflammatory response. IL-8 is produced by the proximal tubular epithelial cells (30), and exposure of these cells to albumin has been shown to stimulate IL-8 expression (31). It is interesting that the albumin gene also resides in this genomic region, suggesting a possible role for epistasis among these three genes. In addition to IL-8, the CXC chemokine family is clustered in this portion of the genome, including CXCL6, CXCL1, CXCL5, CXCL3, CXCL2, CXCL9, CXCL10, CXCL11, and CXCL13, suggesting that genes involved in inflammation may play a role in kidney function. The vascular endothelial growth factor C gene also resides on chromosome 4 near our linkage peak for serum creatinine. It is a member of the vascular endothelial growth factor family and is important in angiogenesis and endothelial cell growth (32).

Certain limitations of our study deserve attention. We used estimates of kidney function and did not measure GFR or CRCL directly. However, this is often not feasible in a large population-based study. Although serum creatinine is an imperfect measure of kidney function because it is strongly influenced by age, weight, and gender, it is the only parameter in our study that is measured directly. The predominantly white sample that composes the majority of the Framingham offspring cohort may limit the generalizability of our findings. However, cardiovascular disease risk factor relationships from Framingham have been validated in six ethnically and geographically diverse cohorts and were found to be applicable in other populations, reinforcing the generalizability of our data (33). Because our phenotype information was not collected until 1995, there may have been a survival bias, because subjects with a greater genetic predisposition to more severe kidney disease may have died prematurely. However, this loss would likely attenuate our results. Our peak LOD score of 2.28 is only suggestive of linkage because of the possibility that our results may represent a false-positive finding. Simulations indicate that the false-positive rate for a LOD score of 2.3 in a genomewide scan for our data is 16%. Linkage peaks for serum creatinine, GFR, and CRCL did not always map to the same location, underscoring the need for further studies to replicate our findings. Our sample size and the marker spacing of 10 cM in the genomewide linkage analysis may limit our power to localize disease-influencing QTL in linkage with measures of kidney function. Although there may be additional QTL not detected in our study, simulation studies using finer marker maps have demonstrated that the location error is low in maps that use marker spacing of 10 cM as compared with 0.5 cM (34),

suggesting that this should not have affected our results significantly.

Measures of serum creatinine, GFR, and CRCL are heritable, suggesting an underlying genetic component. We provide evidence for suggestive linkage of novel loci to measures of kidney function. Further research is necessary to identify the genes involved in the development of kidney disease.

	Acknowledgments

 
This work was supported by the National Heart, Lung, and Blood Institute’s Framingham Heart Study (N01-HC-25195).

	References

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