Altered composition of triglyceride-rich lipoproteins and coronary artery disease in a large case-control study.

BACKGROUND: Traditional beta-quantification of plasma lipoproteins by ultracentrifugation separates triglyceride-rich lipoproteins (TGRL) from higher density lipoproteins. The cholesterol in the TGRL fraction is referred to as measured very low-density lipoprotein cholesterol (VLDL-C) recognizing that other TGRL may be present. The measured VLDL-C to total plasma triglyceride (VLDL-C/TG) has long been considered an index of average TGRL composition with abnormally high VLDL-C/TG ratios (>or=0.30 with TG>150mg/dL) indicative of atherogenic remnant accumulation (type III hyperlipidemia). However, virtually no reports are available which examine potential associations between CAD and VLDL-C/TG at the lower end of the spectrum. METHODS AND RESULTS: We performed ultracentrifugation in 1170 cases with premature-onset, familial CAD and 1759 population-based controls and examined the VLDL-C/TG ratio as an index of TGRL composition. As expected, we found very high CAD risk associated with severe type III hyperlipidemia (OR 10.5, p=0.02). Unexpectedly, however, we found a robust, graded, and independent association between CAD risk and lower than average VLDL-C/TG ratios (p<0.0001 as ordered categories or as a continuous variable). Among those in the lowest VLDL-C/TG category (a ratio <0.12), CAD risk was clearly increased (OR 4.5, 95% CI 2.9-6.9) and remained significantly elevated in various subgroups including those with triglycerides below 200mg/dl, in males and females separately, as well as among those with no traditional CAD risk factors (OR 5.8, 95% CI 1.5-22). Significant compositional differences by case status were confirmed in a subset whose samples were re-spun with measurement of lipids and apolipoprotein B (apo B) in each subfraction. CONCLUSIONS: We found a strong, graded, independent, and robust association between CAD and lower VLDL-C/TG ratios. We consider this a novel, hypothesis-generating observation which will hopefully generate additional future studies to provide confirmation and further insight into potential mechanisms.

A panel of multiple markers associated with chronic systemic inflammation and the risk of atherogenesis is detectable in asthma and chronic obstructive pulmonary disease.

Asthma and chronic obstructive pulmonary disease (COPD) are both lung diseases involving chronic inflammation of the airway. The injury is reversible in asthma whereas it is mostly irreversible in COPD. Both patients of asthma and COPD are known at risk for cardiovascular disease (CVD) and type 2 diabetes (T2DM), nephropathy, and cancer. We measured multiple risk markers for atherogenesis in 55 patients with asthma and 62 patients with COPD. We wanted to know whether risk markers for atherogenesis corresponding to sequence of events of chronic inflammation were also detectable in the airway inflammatory diseases. Elevation of almost all markers involving inflammation of the endothelial cells in the coronary artery were detectable in asthma and COPD involving the inflammation of the epithelial cell lining of the airway. Both the level and % elevation of all markers were found mostly higher in COPD, the more severe form of the lung disease. We believe that these markers are useful for predicting risk of developing clinical complications such as CVD.

Cosmetic liposuction causes only transient elevation of acute inflammatory response and does not advance to oxidative and nitrosative stress.

Cosmetic liposuction is a surgical procedure performed on normal nonobese subjects to remove unwanted fat. We are interested to know whether the impact of acute inflammatory response induced by liposuction differs from that of chronic inflammation and whether acute inflammatory response will also advance further and cause oxidative and nitrosative stress leading to various clinical complications. In our investigation we monitored 15 nonobese women prior to liposuction, and one day and one month after the surgery with multiple markers associated with chronic inflammation and oxidative stress. Our results indicate that liposuction causes only a transient elevation of acute inflammatory markers such as interleukin-6 (IL-6), high sensitive C-reactive protein (hCRP), and serum amyloid A (SAA), and a transient decrease of nitric oxide (NO). Apparently the impact of liposuction for normal subjects did not advance beyond acute inflammatory response; there was little change in the levels of markers corresponding to downstream events of chronic systemic inflammation such as adhesion molecules, urinary microalbumin (uMA), homocysteine (Hcy), uric acid (UA), and markers of oxidative stress, including urinary 8-hydroxydeoxyguanosine (8-OhdG) and 3-nitrotyrosine (3NT). It appears that the acute inflammatory response of cosmetic liposuction does not lead to impaired renal function and oxidative and nitrosative damage, which are frequently associated with chronic inflammation.

Multiple risk markers for atherogenesis associated with chronic inflammation are detectable in patients with renal stones.

Patients with renal stones are known to be at risk of clinical complications such as cardiovascular disease (CVD), nephropathy, and cancer. Recently, it has been realized that almost all risk markers for CVD, nephropathy, etc. are all markers associated with the sequence of reactions of chronic inflammation. It has been reported that chronic inflammation is involved not only in the pathogenesis of nephrolithiasis but also contributes to the development of clinical complications in this condition; therefore, we decided to find out whether these multiple markers are detectable in patients with renal stones so that they can be used to predict the risk of clinical complications in these patients. There were 33 patients with nephrolithiasis included in this study. We found that almost all major markers of chronic inflammation were elevated in patients with renal stones, including proinflammatory cytokine, acute inflammation markers, adhesion molecules, urinary microalbumin (uMA), myeloperoxidase (MPO), 8-hydroxydeoxyguanosine (8-OHdG), 3-nitrotyrosine (3NT), and monocyte chemoattractant protein. It appears that it is possible to assess the risk of clinical complications by monitoring these markers in patients with renal stones.

Establishment of an in-house ELISA and the reference range for serum amyloid A (SAA): complementarity between SAA and C-reactive protein as markers of inflammation.

INTRODUCTION: Serum amyloid A (SAA) and C-reactive protein (CRP) are both acute-phase reactants synthesized by the liver upon stimulation by proinflammatory cytokines reflecting both the acute and chronic inflammatory states. METHODS: We have established a one-step, sandwich ELISA on microplate for SAA using commercial antibodies for coating and detection. RESULTS: This in-house ELISA has a sensitivity of 0.12 mg/l. Both within-day and between-day CVs were <10%. The in-house assay correlated well with the commercial ELISA kit from Anogen (r=0.95). We also established the reference range for apparently healthy Chinese. Statistically higher SAA values were found in those >50 years old. No difference was found between genders. We found only slightly increased levels of SAA in early stage of type 2 diabetics, but highly increased levels of SAA were detected in patients with acute myocardial infarction, generally associated with intense inflammation. At the early stage of type 2 diabetes associated with low inflammation, SAA was found to be complementary to CRP in test sensitivity. CONCLUSIONS: Based on our data and reports from the literature we believe that SAA responds differently than CRP in inflammatory diseases such as in type 2 diabetes and acute myocardial infarction, and is complementary to CRP in test sensitivity.

Establishment of a sandwich ELISA using commercial antibody for plasma or serum 3-nitrotyrosine (3NT). Elevation in inflammatory diseases and complementary between 3NT and myeloperoxidase.

BACKGROUND: Amino acid tyrosine residue of a protein can be nitrated to form 3-nitrotyrosine (3NT), which is now being considered as a marker of inflammation, oxidative and nitrosative stress. METHOD: An in-house ELISA has been established using the same commercial antibody for both binding and detection of 3NT containing proteins. RESULTS: The sensitivity of the in-house ELISA was 1.8 nmol/l. The imprecision was <10% at all concentrations. The in-house assay correlates well with a commercial kit (r=0.89). In addition to EDTA plasma, we found that both heparinized plasma and serum can also be used to quantify 3NT concentration. Using the in-house ELISA we have detected increased concentrations of 3NT in diseases known to be associated with inflammation and also in subjects with polyps. As marker of oxidative stress and inflammation, both 3NT and myeloperoxidase are complementary to each other in test sensitivity. CONCLUSION: This ELISA can be used in the clinical laboratories to monitor the inflammatory disease activity and assess early risks that are associated with inflammation, oxidative and nitrosative stress.

Development of an ELISA for myeloperoxidase on microplate: normal reference values and effect of temperature on specimen preparation.

BACKGROUND: Myeloperoxidase (MPO), a leukocyte enzyme, is implicated in both the pathogenesis and the progression of atherosclerosis. METHODS: We developed a sandwich ELISA on microplate using commercial antibodies for the measurement of plasma MPO. RESULTS: The in-house ELISA has a sensitivity of 15 ng/ml. Both within-day and between-day imprecision were <10%. The in-house assay was well correlated with the commercial kit from Oxis (gamma=0.96). We have established normal reference range for MPO for apparently healthy Chinese. No statistical difference was found between males and females and the various age groups. Because the coating antibodies used by two different kits are different in their affinities for MPO, the analysis by the in-house ELISA that was approximately three times that of the Oxis kit when testing the same specimens. We found that it is necessary to keep the heparinized whole blood on ice before centrifugation in order to prevent further release of MPO from the leukocytes at room temperature. For the same reason, serum is not recommended for MPO measurement. We also found that either pooled human plasma or serum containing MPO can be used as calibrators. CONCLUSIONS: We believe that this ELISA for MPO is useful to assess risk for inflammation, oxidative stress, nitrosative stress, and for predicting cardiac events.

Lack of association of glutamate decarboxylase 2 gene polymorphisms with severe obesity in utah.

Three polymorphisms of the glutamate decarboxylase 2 gene, which encodes the glutamic acid decarboxylase enzyme, have been associated with severe obesity in a large French cohort. One of these polymorphisms was shown to have functional consequences on promoter expression. Another polymorphism was associated with insulin levels and secretion. These associations were examined in 855 severely obese Utah subjects (mean BMI = 48 kg/m(2)) and a normal-weight and normoglycemic subset (N = 130, mean BMI = 22 kg/m(2)) of a random sample of the Utah population (N = 462). Comparisons of the normal-weight random group with the severely obese group did not result in significant genotype or allele frequency differences for any of the three polymorphisms, C61450A, T83897A, or A-243G (all p > or = 0.18). Haplotypes were also not related to severe obesity (p = 0.10). None of the polymorphisms was significantly related to fasting glucose, insulin levels, or homeostasis model assessment insulin resistance or secretion indices. This study of normal-weight and severely obese subjects from Utah does not provide evidence for involvement of the three genotyped polymorphisms in the glutamate decarboxylase 2 gene with obesity or with insulin- and glucose-related measures associated with obesity.

Linking inflammation and atherogenesis: Soluble markers identified for the detection of risk factors and for early risk assessment.

Increasing evidence has shown that atherogenesis is not only caused by hypercholesterolemia. Several risk factors including abdominal obesity, dyslipidemia, hyperglycemia, bacterial and viral infection, hyperhomocysteinemia have been identified recently, all mediated through inflammation, which can lead to atherosclerosis. Several events have also been identified to be involved in the overall inflammation reaction in the blood vessel which include endothelium dysfunction, expression of adhesion molecules, recruitment of leukocytes to the injured endothelium, migration of monocytes to the arterial intima, and transformation of monocytes to macrophages. In order to facilitate the assessment of early risk for atherogenesis we have made an effort in this review to identify soluble markers that will allow the detection of these risk factors and the identification of associated inflammation events. Since early risks for atherogenesis are largely preventable with dietary modification and lifestyle changes, capable of detecting early risks by monitoring soluble risk markers is conceivably important for asymptomatic individuals to avoid serious or fatal consequences of atherosclerosis. These soluble markers should also be useful for monitoring the effectiveness of intervention and for the identification of therapeutic targets.

Sodium bicarbonate cotransporter polymorphisms are associated with baseline and 10-year follow-up blood pressures.

The NaHCO3 cotransporter gene (SLC4A5) on chromosome 2 encodes a protein that transports sodium and bicarbonate across the cell membrane and regulates cellular pH. The National Heart, Lung, and Blood Institute Family Blood Pressure Program found linkage of blood pressure-related traits to the chromosomal region containing SLC4A5 and phenotype associations with single nucleotide polymorphisms (SNPs) in this gene. However, the results were inconsistent over various phenotypes and SNPs. Nevertheless, the evidence was strong enough to propose this gene as a blood pressure-related gene. To extend these findings, SLC4A5 SNPs were genotyped in an independent set of 96 Utah pedigrees of 1040 adult subjects at baseline, 760 of whom were followed longitudinally for 10 years. After adjusting for age, gender, body mass index, and polygenic correlations within pedigrees, SNP hcv1137534 was significantly associated with both systolic blood pressure and diastolic blood pressure (DBP) at baseline (unadjusted P=0.009 and P=0.043; respectively) and at 10-year follow-up (P=0.008 and P=0.007; respectively). In secondary tests of association of baseline-stressed blood pressure, hcv1137534 was borderline or significantly associated with DBP change during an isometric handgrip test (P=0.054), DBP change from supine to standing (P=0.020), and DBP change after a 50 degrees tilt (P=0.034). There was no evidence for compensation of abnormal SLC4A5 sodium transport by genotype-specific differences in sodium-lithium countertransport, lithium-potassium cotransport, altered plasma sodium, chloride, or CO2 levels. Therefore, in these Utah pedigrees, the SLC4A5 gene was significantly associated with blood pressure and persisted after 10 years of follow-up. These results additionally confirm the involvement of SLC4A5 with blood pressure control, although the mechanism is still unclear.

Association of soluble markers with various stages and major events of atherosclerosis.

The purpose of this review is to identify soluble markers from the recent literature that facilitate the early prevention and early detection of atherosclerosis, and that may serve as therapeutic targets. Soluble markers associated with various stages and major events of atherosclerosis were identified. We divided the process of atherosclerosis into several stages, including stages for the early risk, plaque expansion, and stable and unstable angina-though excluding the end stage of myocardial infarction. For major events taking place prior to and during the progression of atherosclerosis we included events such as endothelial dysfunction in the artery, expression of adhesion molecules at the injured endothelium, continued inflammatory responses, oxidative stress, and ischemia. We found that reactions such as cell injury, adhesion, inflammation, and oxidative stress occur not only at the early stage of risk but persist throughout the process of atherosclerosis. Most markers associated with these major events are clustered together at any time of the disease. Few markers are characteristic of individual stages. We noted that reactions such as inflammation are continuously intensified with the progression of the disease. Finally, we underscore the importance of measuring a panel consisting of minimal numbers of multiple markers with the maximal sensitivity for early risk assessment, diagnosis, and prognosis. We envision that patterns characteristic of various stages of atherosclerosis may be identifiable with the use of the multiple markers described in this review.

Plasma triglycerides and type III hyperlipidemia are independently associated with premature familial coronary artery disease.

OBJECTIVES: This study was designed to explore contributions of plasma total triglycerides (TGs) and type III hyperlipidemia to the risk of premature familial coronary artery disease (CAD). BACKGROUND: Although plasma TGs are recognized as a risk factor for CAD, the independence of this association from related risk factors remains controversial. Also, the degree of CAD risk conferred by excess remnants of TG-rich lipoproteins in type III hyperlipidemia remains unclear. METHODS: We analyzed lipids by ultracentrifugation in a series of 653 cases with premature familial CAD (myocardial infarction or revascularization by age 55 years in men or age 65 years in women, with similar onset in at least one other first-degree relative) and in 1,029 control subjects. The relationship of CAD risk to various strata of plasma TGs, high-density lipoprotein (HDL) cholesterol, and type III hyperlipidemia, and interactions among these variables were examined by multiple logistic regression, adjusting for other CAD risk factors. RESULTS: The odds ratio for CAD with elevated plasma TG rose progressively to 11.4 in those with TGs 500 to 799 mg/dl (95% confidence interval 3.4 to 38.0, p < 0.0001) compared with <100 mg/dl, even after correction for HDL cholesterol, other elements of the metabolic syndrome, and other CAD risk factors. Risk of CAD associated with type III hyperlipidemia (found in 3.4% of cases) was also markedly increased independent of other risk factors (odds ratios of 5 to 10 depending on the model, all with p < 0.002). CONCLUSIONS: The association between the plasma TG level and premature familial CAD is strong, graded, and independent. Risk of CAD is also strikingly elevated with type III hyperlipidemia.

Linkage of serum creatinine and glomerular filtration rate to chromosome 2 in Utah pedigrees.

BACKGROUND: Serum creatinine and creatinine clearance are used as indicators of renal function and may indicate a propensity for development of end-stage renal disease. Identifying genes related to future decreases in renal function could be important in assessing risk and defining abnormal mechanisms amenable to preventive measures. Although creatinine clearance is a better measure of renal function than serum creatinine, proper and complete urine collections in large population studies are sometimes problematic. This can lead to a loss in power to detect linkage. Therefore, in this study we also investigated serum creatinine and estimated glomerular filtration rates (GFR), both of which are more reliably measured. METHODS: Linkage was tested in a genome scan using 49 large Utah pedigrees examined three times over 10 years to detect regions harboring genes related to reduced renal function. RESULTS: Heritability of serum creatinine ranged from 25% to 31% across three examinations, and heritability of GFR ranged from 37% to 42%. The highest log of the odds (LOD) score for serum creatinine was found on chromosome 2 at 145 cM on the Marshfield map (D2S1334). Consistent nonparametric linkage for serum creatinine was found for all three examinations (LOD = 3.15, 2.75, and 2.00, respectively). Estimates of GFR also showed linkage to this region. CONCLUSIONS: The consistency of linkage to chromosome 2 over longitudinally repeated measurements increases the likelihood that this region harbors a gene influencing phenotypic variation in serum creatinine and GFR. Identification of this gene could help to predict which individuals are most likely to progress to renal disease.

Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics.

Reactive oxygen species (ROS) produced either endogenously or exogenously can attack lipid, protein and nucleic acid simultaneously in the living cells. In nuclear and mitochondrial DNA, 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Numerous evidences have indicated that urinary 8-OHdG not only is a biomarker of generalized, cellular oxidative stress but might also be a risk factor for cancer, atherosclerosis and diabetes. For example, elevated level of urinary 8-OHdG has been detected in patients with various cancers. In human atherosclerotic plaques, there were increased amounts of oxidatively modified DNA and 8-OHdG. Elevated urinary 8-OHdG and leukocyte DNA were also detected in diabetic patients with hyperglycemia, and the level of urinary 8-OHdG in diabetes correlated with the severity of diabetic nephropathy and retinopathy. We have discussed various methods for determining 8-OHdG in the tissue and urine, including HPLC with and without extraction, and ELISA. Using the ELISA we developed, we found that the normal range of urinary 8-OHdG for females was 43.9 +/- 42.1 ng/mg creatinine and 29.6 +/- 24.5 ng/mg creatinine for males, respectively. We found that the normal value between females and males is significantly different (p < 0.001).

Soluble epoxide hydrolase variant (Glu287Arg) modifies plasma total cholesterol and triglyceride phenotype in familial hypercholesterolemia: intrafamilial association study in an eight-generation hyperlipidemic kindred.

Plasma lipid and lipoprotein in general reflect the complex influences of multiple genetic loci, for instance, even familial hypercholesterolemia (FH), a representative example of monogenic hyperlipidemia, often presents with phenotypic heterogeneity. In the course of investigating familial coronary artery disease in Utah, we studied 160 members of an eight-generation extended family of FH in which 69 members were affected with type IIa hyperlipoproteinemia (HLPIIa; high plasma cholesterol) and ten with type IIb hyperlipoproteinemia (HLPIIb; high plasma cholesterol as well as plasma triglyceride). Soluble epoxide hydrolase ( EPHX2, sEH) plays a role in disposition of epoxides in plasma lipoprotein particles. Intrafamilial correlation analysis of the modifier effect of Glu287Arg substitution in the EPHX2 gene was carried out among 79 LDLR mutation carriers and 81 noncarriers. In the carriers, plasma cholesterol levels were elevated among carriers of the 287Arg allele (mean +/- SD=358 +/- 72 mg/dl) in comparison with 287Glu homozygotes (mean +/- SD=302 +/- 72 mg/dl) (p=0.0087). Similarly, in the LDLR mutation carriers, the plasma triglyceride levels were elevated among carriers of the 287Arg allele (mean +/- SD=260 +/- 100 mg/dl) in comparison with 287Glu homozygotes (mean +/- SD=169 +/- 83 mg/dl) (p=0.020). No such gene-interactive effect was observed among noncarriers of the LDLR mutation. Half of the patients who presented with HLPIIb had inherited a defective LDLR allele as well as an EPHX2-287Arg allele, whereas the majority who presented with HLPIIa had a defective LDLR allele but not an EPHX2-287Arg allele. These results indicate a significant modification of the phenotype of FH with defective LDLR allele by EPHX2-287Arg variation in our studied kindred.

Growth hormone receptor variant (L526I) modifies plasma HDL cholesterol phenotype in familial hypercholesterolemia: intra-familial association study in an eight-generation hyperlipidemic kindred.

Defect of growth hormone receptor (GHR) is classically known to cause Laron syndrome, characterized by short stature, specific facial appearance, elevated serum growth hormone levels, and decreased insulin-like growth factor I levels. In addition, an increased cardiovascular risk due to elevated plasma total and LDL cholesterol levels marks another feature of the disease. Growth hormone (GH) plays an important role in the regulation of lipoprotein metabolism. GH status was found to be an independent determinant of plasma total cholesterol and triglyceride levels in humans. We studied a total of 207 members of eight-generation extended family of familial hypercholesterolemia (FH) in which affected members presented with various lipoprotein phenotypes. Intra-familial correlation analysis of a modifier effect of a Leu526Ile substitution in GHR gene was carried out among 95 carriers for LDL receptor gene (LDLR) mutation and 112 non-carriers. When plasma high-density lipoprotein cholesterol (HDL-c) levels in the LDLR-mutation carriers were compared, a significant lowering effect of HDL-c was observed with the Leu allele; the values were lowest among Leu/Leu homozygotes (mean +/- SD = 37 +/- 2 mg/dl), highest in Ile/Ile homozygotes (50 +/- 4 mg/dl), and intermediate among Leu/Ile heterozygotes (41 +/- 2 mg/dl) (P = 0.0021). The results indicate a significant modification of the phenotype of FH with the defective LDLR allele, by GHR Leu variation in the kindred studied.

Apolipoprotein H variant modifies plasma triglyceride phenotype in familial hypercholesterolemia: a molecular study in an eight-generation hyperlipidemic family.

In the course of investigating familial coronary artery disease in Utah, we studied 196 members of an eight-generation extended family of familial hypercholesterolemia (FH), in which 73 members were affected with type IIa hyperlipoproteinemia (HLPIIa; high plasma cholesterol) and 11 members with type IIb hyperlipoproteinemia (HLPIIb; high plasma cholesterol as well as plasma triglyceride). A splice-site mutation of the LDL receptor (LDLR) gene (IVS14 + G > A) co-segregated with elevated plasma cholesterol among all the members, but not with the elevated plasma triglyceride and VLDL cholesterol levels seen in HLPIIb patients. The apolipoprotein H (apoH) gene plays a role in plasma triglyceride removal and lipoprotein lipase enhancement. Intra-familial correlation analysis of the modifier effect of Val247Leu substitution in the apoH gene was carried out among 84 LDLR-mutation carriers and 112 non-carriers. When plasma triglyceride levels in the LDLR-mutation carriers were compared, the values were lowest among V/V homozygotes (mean +/- SD = 145 +/- 53 mg/dl), highest in L/L homozygotes (277 +/- 177 mg/dl), and intermediate among V/L heterozygotes (191 +/- 102 mg/dl) (p = 0.0015). All eleven patients who presented with HLPIIb had inherited both the defective LDLR allele and an apoH 247Leu allele, whereas all 45 carriers of the defective LDLR allele not carrying the apoH Leu allele presented with HLPIIa but not HLPIIb (p = 0.0001). These results indicate a significant modification of the phenotype of FH with a defective LDLR allele, by apoH Leu variation in our studied family.

Linkage of creatinine clearance to chromosome 10 in Utah pedigrees replicates a locus for end-stage renal disease in humans and renal failure in the fawn-hooded rat.

BACKGROUND: Renal failure is an important health concern for persons with hypertension and diabetes. In the fawn-hooded rat, a renal failure locus, Rf-1, has been identified on rat chromosome 1. A study of African American sibpairs with end-stage renal disease (ESRD) replicated this finding on the orthologous region in humans, chromosome 10, with a maximum logarithm of odds (LOD) score of 3.4. An important question is whether this region can be detected in healthy subjects prior to onset of ESRD by examining creatinine clearance as an indicator of early renal damage. METHODS: We analyzed 49 Utah Caucasian pedigrees and performed quantitative nonparametric linkage analysis using 21 markers spanning chromosome 10. Pedigree members (mean age of 40 +/- 17) were examined up to three different times over 10 years, with creatinine clearance measured at each exam. For examination 1, three overnight, timed, 12-hour urine samples were obtained and averaged. One 12-hour sample was obtained for examinations 2 and 3. RESULTS: Heritabilities of creatinine clearance were 0.33 (N = 1360), 0.36 (N = 1196), and 0.53 (N = 718) for the three examinations, respectively. The nonparametric LOD score for examination 1 was 1.4 at marker D10S677 (approximately 117 cM). The LOD score at examination 2, an average of 21/2 years later, was 1.8 at marker D10S1239 (approximately 123 cM) and 1.9 at marker D10S1425 (approximately 137 cM). The LOD score at examination 3, an average of 10 years from baseline, was 2.1 at marker D10S2470 (approximately 113 cM). Thus, there is consistent evidence of linkage to this region from three different examinations spanning a period of 10 years. CONCLUSIONS: These linkage results confirm the ESRD linkage and the rat renal failure linkage to this region even though the LOD score is somewhat weaker, probably due to the less severe phenotype that was analyzed. It also suggests that there may be a locus on chromosome 10 that leads to reduced renal function that can be detected while subjects are still healthy. Identification of the responsible gene may help in predicting renal disease progression in susceptible patients.