Catherine Leiendecker-Foster

Expressing the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) Cystatin C Equations for Estimating GFR With Standardized Serum Cystatin C Values

Assays traceable to high-level a reference materials are critical factors in ensuring accuracy of GFR estimates1. Serum cystatin C (Scys) is being proposed as a filtration marker that can be used with or as an alternative to serum creatinine (Scr) in equations to estimate GFR. The Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) published equations for estimating GFR from Scys in 2008 using serums assayed by the Cleveland Clinic Research Laboratory (CCRL) in 2003 using the Siemens-Dade-Behring (SDB) particle-enhanced immunonephelometric assay (PENIA) with the SDB BN II nephelometer2. At that time, Scys assays traceable to an internationally accepted reference material were not available. To foster consistent Scys results, the International Federation for Clinical Chemists (IFCC) Working Group for the Standardization of serum cystatin C and the Institute for Reference Materials and Measurements (IRMM) have collaborated for the production and characterization of a certified reference material (CRM)3, 4. This material, ERM-DA471/IFCC, was made available to laboratories by IRMM in fall 2010. Here we report on the re-expression of previously reported equations to estimate GFR from Scys for use with Scys values traceable to the new IRMM CRM The CKD-EPI Central Laboratory moved to the University of Minnesota (UMN) in 2009. At the time, comparisons between Scys measurement results at CCRL and UMN were made using serum calibration panels, which had been created in 2003 at CCRL and stored frozen at −70°5. The calibration panel included 40 reference sera pooled from at least 10 mixed-sex donors known to have serum creatinine values covering the range of 0.5 to 5.0 mg/dL. The panel was assayed for Scys in triplicate at CCRL in 2003 using the SDB measurement procedure as described above, and at the UMN in 2009 on a panel with one previous freeze thaw cycle using the same SDB PENIA reagent on a SDB ProSpec nephelometer. Conversion factors between the laboratories were determined using Deming linear regression6,7. To ensure stability over time, the calibration panel was retested on a previously unthawed panel at UMN in January 2011. ERM-DA471/IFCC CRM was reconstituted at UMN as instructed in its Certificate of Analysis yielding an aqueous buffer solution with a cystatin C concentration 5.48 mg/L (uncertainty 0.15 mg/L)3. Lower concentration solutions of the reference material were prepared by dilution of the neat reconstituted reference material with volumetric additions of PENIA reagent buffer to create target values of 1.37 mg/L, 2.74 mg/L, and 4.11 mg/L. The instrument PENIA reagent buffer was considered to have zero concentration. Accuracy of volumetric additions was checked by weight. The materials were assayed in duplicate on two separate days. Regression equations were used to compare the mean concentrations assigned by the SDB PENIA reagents on the ProSpec at UMN compared to the calculated values based on the certified value of the reference material. Intercepts that were very small and non-significant (p >0.05) were dropped from the regression. The calibration factor converts Scys results from UMN in 2009 (Scys-UMN’09) reported values to ERM-DA471/IFCC traceable values, which was then incorporated into the GFR estimating equation. Mean (SD, range) level of Scys measured in the calibration panel was 2.01 (0.74, 0.78-3.64) mg/L in CCRL in 2003, 1.67 (0.58, 0.68-2.97) mg/L in UMN in 2009, and 1.67 (0.59, 0.65-2.98) mg/L in UMN 2011. The mean (SD) difference between Scys values from CCRL in 2003 (Scys-CCRL’03) and Scys-UMN’09 was -0.34 (0.16) mg/L (p-value <0.01). The regression of the Scys-UMN’09 on Scys-CCRL ’03 showed a slope (SE) of 0.789 (0.009) and intercept of 0.083 (0.019) mg/L (Table 1, Equation 1 and Supplemental Figure 1). Table 1 List of equations The SDB ProSpec PENIA method resulted in lower values for Scys for the ERM-DA471/IFCC CRM than calculated based on the materials certified value. For all samples combined, the intercept (SE) was 0.043 (0.031) mg/L (p=0.3), and the slope was 1.109 (p<0.001), R2 =0.9996. After dropping the non- significant intercept, the slope was 1.120 (P<0.001), R2=0.9999 (Table 1, Equation 2). The Scys-CCRL’03 were expressed as standardized Scys values (Table 1, Equations 3-4), and the published CKD-EPI Scys equations (Table 1, Equations 5-7) were re-expressed (Table 1, Equations 8-10). Table 2 compares the assigned values for Scys-CCRL’03, Scys-UMN’09 and standardized Scys and corresponding estimated GFR. Using the Scys-UMN’09 values in the original estimating equations resulted in substantially higher values for estimated GFR than would have been obtained using Scys-CCRL’03 values. The eGFR values calculated standardized Scys and the re-expressed equations are nearly identical to those that would have been obtained using Scys-CCRL’03 values and the original equation. Table 2 Values for Cystatin C and Estimated GFR before and after standardization of the Cystatin C assay and re-expression of the GFR estimating equation In conclusion, drift in the Dade Behring Scys assay was observed between CCRL in 2003 and UMN in 2009. Similar drift has been observed in other laboratories using SDB measurement procedures, suggesting that this is due to subtle changes in their reagents or calibrators (personal communication Russell Tracy, PhD), Availability of CRM should facilitate better comparability of Scys results among laboratories and measurement procedures manufactured by different invitro diagnostic manufacturers. Re-expression of the CKD-EPI Scys equations for use with ERM-DA471/IFCC-traceable Scys measurements will allow more consistent and accurate GFR estimates.

Hemochromatosis and Iron Overload Screening (HEIRS) study design for an evaluation of 100,000 primary care-based adults.

BackgroundThe HEIRS Study will evaluate the prevalence, genetic and environmental determinants, and potential clinical, personal, and societal impact of hemochromatosis and iron overload in a multiethnic, primary care-based sample of 100,000 adults over a 5-year period. Participants are recruited from 5 Field Centers. Laboratory testing and data management and analysis are performed in a Central Laboratory and Coordinating Center, respectively. MethodsParticipants undergo testing for serum iron measures and common mutations of the hemochromatosis gene (HFE) on chromosome 6p and answer questions on demographics, health, and genetic testing attitudes. Participants with elevated values of transferrin saturation and serum ferritin and/or C282Y homozygosity are invited to undergo a comprehensive clinical examination (CCE), as are frequency-matched control subjects. These examinations provide data on personal and family medical history, lifestyle characteristics, physical examination, genetic counseling, and assessment of ethical, legal, and social implications. Primary and secondary causes of iron overload will be distinguished by clinical criteria. Iron overload will be confirmed by quantification of iron stores. Recruiting family members of cases will permit DNA analysis for additional genetic factors that affect iron overload. ResultsOf the first 50,520 screened, 51% are white, 24% are African American, 11% are Asian, 11% are Hispanic, and 3% are of other, mixed, or unidentified race; 63% are female and 37% are male. ConclusionsInformation from the HEIRS Study will inform policy regarding the feasibility, optimal approach, and potential individual and public health benefits and risks of primary care-based screening for iron overload and hemochromatosis.

Pharmacogenetic Association of the NPPA T2238C Genetic Variant With Cardiovascular Disease Outcomes in Patients With Hypertension

CONTEXT The NPPA gene codes for the precursor of atrial natriuretic polypeptide, suggesting that NPPA may modulate the efficacy of some antihypertensive drugs. OBJECTIVE To test whether participants with minor NPPA alleles in the T2238C or G664A variants had different rates of cardiovascular disease or blood pressure (BP) changes than common allele homozygotes when treated with a diuretic vs other antihypertensive medications. DESIGN, SETTING, AND PATIENTS Post hoc analysis of 38,462 participants with hypertension from ALLHAT, a multicenter randomized clinical trial conducted in the United States and Canada. Genotyping was performed from February 2004 to January 2005. INTERVENTION Participants were randomly assigned to receive a diuretic (chlorthalidone; n = 13,860), a calcium antagonist (amlodipine; n = 8174), an angiotensin-converting enzyme inhibitor (lisinopril; n = 8233), or an alpha-blocker (doxazosin; n = 8195). MAIN OUTCOME MEASURE The primary outcome measure was coronary heart disease (CHD), defined as fatal CHD or nonfatal myocardial infarction (mean follow-up, 4.9 years). Secondary outcomes were stroke, all-cause mortality, combined cardiovascular disease outcomes, and 6-month systolic and diastolic BP changes. Genotype x treatment interactions were tested where genotypes were modeled additively and dominantly. RESULTS Depending on genotype, the event rates per 1000 person-years were 15.3 to 19.7 for CHO, 9.6 to 15.4 for stroke, and 27.4 to 30.7 for all-cause mortality. For the NPPA T2238C variant, lower event rates were found for the C allele carriers than for the TT homozygous individuals when comparing chlorthalidone and amlodipine (CHD: CC = 0.86; TC = 0.90; TT = 1.09; P = .03 [dominant model]; stroke: CC = 1.18; TC = 0.82; TT = 1.26; P = .01 [additive and dominant models]; all-cause mortality: CC = 0.87; TC = 0.98; TT = 1.12; P = .05 [dominant model]). Combined end points yielded similar results. Consistent with these clinical findings, 6-month changes in systolic BP for those with the CC genotype showed larger reductions with chlorthalidone (-6.5 mm Hg) than with amlodipine (-3.8 mm Hg), lisinopril (-2.4 mm Hg), or doxazosin (-3.8 mm Hg). Among those with the TT genotype, systolic BP differences between drugs were less (range, -5.4 to -7.5 mm Hg; P value, <.001 to .003 for interaction); diastolic BP showed similar results. We found no pharmacogenetic associations with the NPPA G664A variant. CONCLUSIONS The NPPA T2238C variant was associated with modification of antihypertensive medication effects on cardiovascular disease and BP. Minor C allele carriers experienced more favorable cardiovascular disease outcomes when randomized to receive a diuretic, whereas TT allele carriers had more favorable outcomes when randomized to receive a calcium channel blocker.

Genome-Wide Association Study Identifies Genetic Loci Associated with Iron Deficiency

The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. To identify new genomic locations associated with iron deficiency, a genome-wide association study (GWAS) was performed using DNA collected from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤ 12 µg/L (cases) and iron replete controls (SF>100 µg/L in men, SF>50 µg/L in women). Regression analysis was used to examine the association between case-control status (336 cases, 343 controls) and quantitative serum iron measures and 331,060 single nucleotide polymorphism (SNP) genotypes, with replication analyses performed in a sample of 71 cases and 161 controls from a population of white male and female veterans screened at a US Veterans Affairs (VA) medical center. Five SNPs identified in the GWAS met genome-wide statistical significance for association with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the transferrin (TF) gene region; rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed P<1.51×10−7 for all). An association between total iron binding capacity and SNP rs3811647 in the TF gene (GWAS observed P = 7.0×10−9, corrected P = 0.012) was replicated within the VA samples (observed P = 0.012). Associations with the C282Y mutation in the HFE gene also were replicated. The joint analysis of the HEIRS and VA samples revealed strong associations between rs2698530 on chr. 2p14 and iron status outcomes. These results confirm a previously-described TF polymorphism and implicate one potential new locus as a target for gene identification.

Leptin Is Associated With Blood Pressure and Hypertension in Women From the National Heart, Lung, and Blood Institute Family Heart Study

Leptin is a key neuroendocrine hormone regulating food intake, metabolism, and fat accumulation, and it may also affect blood pressure and contribute to hypertension through sympathetic activation in the vasculature or at the renal level. Although previous studies have shown that the distribution of leptin is significantly different between males and females, as is the risk of hypertension between males and females, results regarding the role of leptin in the gender-specific regulation of blood pressure are controversial. Thus, we performed family-based association analyses in the National Heart, Lung, and Blood Institute Family Heart Study to test the hypothesis that LEPTIN gene variants and the plasma leptin level influence variability in blood pressure and the risk of hypertension differently by gender. We identified significant associations between LEPTIN single nucleotide polymorphisms with blood pressure and hypertension, but in postmenopausal women only. We also identified significant associations between plasma leptin levels and both blood pressure and hypertension in women. The current study supports a role for LEPTIN and plasma leptin levels in blood pressure regulation in women. It also provides insight into the gender differences in hypertension, as well as the differential distribution and activity of leptin in men and women.

HFE C282Y homozygotes have reduced low-density lipoprotein cholesterol: the Atherosclerosis Risk in Communities (ARIC) Study

Recent studies have raised questions about the long-term health risks for individuals with mutations in the HFE gene, although previous studies may have been plagued by selection bias or lack of population-based comparison groups. We examined cardiovascular disease risk factors and iron and liver biomarkers, as well as morbidity and mortality associated with the C282Y and H63D variants of HFE in the Atherosclerosis Risk in Communities (ARIC) study, which is a population-based cohort of nearly 16,000 U.S. white and black men and women who were 45-64 years old at baseline. Subjects were followed for an average of 15 years for death, incident coronary heart disease, stroke, and heart failure, and an average of 8 years for incident diabetes. The prevalence of C282Y homozygosity was 0.42% (45/10,800) in whites, which is similar to other North American population-based studies. C282Y homozygotes had significantly lower mean low-density lipoprotein (LDL) cholesterol and fibrinogen as well as higher mean levels of iron (ferritin, transferrin saturation) and liver biomarkers (alanine aminotransferase, Hepascore) compared with HFE wild-type subjects. Rates of all-cause mortality, cardiovascular disease, and diabetes were similar across HFE genotypes. These prospective, population-based data indicate higher serum iron indices and possible mild liver dysfunction or disease in some C282Y homozygotes, but they provide little evidence that HFE C282Y or H63D mutations are related to all-cause mortality, cardiovascular disease, or diabetes. Reduced LDL in C282Y homozygotes may be because of effects of excess iron on cholesterol metabolism and lipoprotein formation in the liver.

Interactions between the single nucleotide polymorphisms in the homocysteine pathway (MTHFR 677C>T, MTHFR 1298 A>C, and CBSins) and the efficacy of HMG-CoA reductase inhibitors in preventing cardiovascular disease in high-risk patients of hypertension: the GenHAT study.

Background High homocysteine blood concentrations predispose to coronary artery disease and statins influence homocysteine levels. Aim To study whether genes that regulate homocysteine metabolism interact with statins to modify the risk of coronary heart disease (CHD) and other cardiovascular outcomes. Methods The Genetics of Hypertension Associated Treatment is an ancillary study of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). The genotyped population in the Lipid-Lowering Trial of Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial included 9624 participants randomly assigned to pravastatin or to usual care. The efficacy of pravastatin in reducing risk of all-cause mortality and CHD was compared among genotype strata (MTHFR 677 CC, CT, and TT, MTHFR 1298 AA, AC, and CC, CBSins DD and I+) by examining an interaction term in a proportional hazards model. Results No evidence existed of a pharmacogenetic effect on statins with the MTHFR 1298 A>C genotype for CHD risk. However, in persons with the CC variant for the MTHFR 677 C>T genotype, a significantly protective effect against CHD [0.71 (95% CI 0.58–0.87)] was shown, although in the CT [1.25 (95% CI 0.97–1.61)] and TT groups [0.80 (95% CI 0.50–1.28)] there were no such effects (interaction hazard ratio P=0.004). The CBSins, I+ variant was associated with a significantly reduced risk for CHD among those on statin treatment [0.58 (95% CI 0.44–0.78)] whereas the DD genotype showed no effect of statin therapy [1.01 (95% CI 0.84–1.20; P=0.002 for interaction]. For the endpoint all-cause mortality, no significant differences in efficacy were noted. Conclusion Polymorphisms in genes in the homocysteine pathway (MTHFR 677 C>T and CBSins) appear to modify the efficacy of pravastatin in reducing risk of cardiovascular events.

Associations between Single Nucleotide Polymorphisms in Iron-Related Genes and Iron Status in Multiethnic Populations

The existence of multiple inherited disorders of iron metabolism suggests genetic contributions to iron deficiency. We previously performed a genome-wide association study of iron-related single nucleotide polymorphisms (SNPs) using DNA from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤12 µg/L (cases) and controls (SF >100 µg/L in men, SF >50 µg/L in women). We report a follow-up study of white, African-American, Hispanic, and Asian HEIRS participants, analyzed for association between SNPs and eight iron-related outcomes. Three chromosomal regions showed association across multiple populations, including SNPs in the TF and TMPRSS6 genes, and on chromosome 18q21. A novel SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p = 3.7×10−6) and replicated in African Americans (p = 0.0012).Twenty SNPs in the TF gene region were associated with total iron-binding capacity in whites (p<4.4×10−5); six SNPs replicated in other ethnicities (p<0.01). SNP rs10904850 in the CUBN gene on 10p13 was associated with serum iron in African Americans (P = 1.0×10−5). These results confirm known associations with iron measures and give unique evidence of their role in different ethnicities, suggesting origins in a common founder.

Pharmacogenetic Association of NOS3 Variants with Cardiovascular Disease in Patients with Hypertension: The GenHAT Study

Nitric oxide synthase 3 (NOS3) catalyzes production of NO in the endothelium and may play a role in cardiovascular disease (CVD). We assessed the pharmacogenetic associations of three NOS3 polymorphisms and three antihypertensive drugs with CVD outcomes. Hypertensive subjects (n = 30,280) from a multi-center, double-blind clinical trial were randomized to chlorthalidone, amlodipine, or lisinopril treatment (mean follow up, 4.9 years). Outcomes included coronary heart disease (CHD: fatal CHD and nonfatal myocardial infarction); stroke; heart failure (fatal, requiring hospitalization, or outpatient treatment); all-cause mortality; and end-stage renal disease (ESRD). Main effects of NOS3 variants on outcome and genotype-treatment interactions were tested. For NOS3 −690 C>T (rs3918226), a higher hazard ratio (HR) was found in minor allele carriers for CHD (CC = 1.00, CT+TT = 1.12 (95% confidence interval (CI) = 1.00–1.26), P = 0.048). For NOS3 −922 A>G (rs1800779), a higher HR was found in minor allele carriers for heart failure (AA = 1.00, AG+GG = 1.10 (CI = 1.00–1.21), P = 0.046). Significant pharmacogenetic findings were observed for stroke and all-cause mortality. For −690 C>T, a lower HR was observed for stroke in minor allele carriers when treated with amlodipine versus lisinopril (CC = 0.85 (CI = 0.73–0.99), CT+TT = 0.49 (CI = 0.31–0.80), P = 0.04). For glu298asp G>T (rs1799983), a lower HR was observed for all-cause mortality in minor allele carriers when treated with amlodipine versus lisinopril (GG = 1.01 (CI = 0.91–1.13), GT+TT = 0.85 (CI = 0.75–0.97), P = 0.04). We observed significant associations with NOS3 variants and CHD and heart failure and significant pharmacogenetic effects for stroke and all cause mortality. This suggests that NOS3 variants may potentially provide useful clinical information with respect to treatment decisions in the future.

Screening for iron overload: Lessons from the Hemochromatosis and iron Overload Screening (HeirS) Study

BACKGROUND The HEmochromatosis and IRon Overload Screening (HEIRS) Study provided data on a racially, ethnically and geographically diverse cohort of participants in North America screened from primary care populations. METHODS A total of 101,168 participants were screened by testing for HFE C282Y and H63D mutations, and measuring serum ferritin concentration and transferrin saturation. In the present review, lessons from the HEIRS Study are highlighted in the context of the principles of screening for a medical disease as previously outlined by the World Health Organization. RESULTS Genetic testing is well accepted, with minimal risk of discrimination. Transferrin saturation has high biological variability and relatively low sensitivity to detect HFE C282Y homozygotes, which limits its role as a screening test. Symptoms attributable to HFE C282Y homozygosity are no more common in individuals identified by population screening than in control subjects. CONCLUSIONS Generalized population screening in a primary care population as performed in the HEIRS Study is not recommended. There may be a role for focused screening in Caucasian men, with some debate regarding genotyping followed by phenotyping, or phenotyping followed by genotyping.