Longitudinal genome-wide DNA methylation changes in response to kidney failure replacement therapy.

Chronic kidney disease (CKD) is an emerging public health priority associated with high mortality rates and demanding treatment regimens, including life-style changes, medications or even dialysis or renal transplantation. Unavoidably, the uremic milieu disturbs homeostatic processes such as DNA methylation and other vital gene regulatory mechanisms. Here, we aimed to investigate how dialysis or kidney transplantation modifies the epigenome-wide methylation signature over 12 months of treatment. We used the Infinium HumanMethylation450 BeadChip on whole blood samples from CKD-patients undergoing either dialysis (n = 11) or kidney transplantation (n = 12) and 24 age- and sex-matched population-based controls. At baseline, comparison between patients and controls identified several significant (PFDR < 0.01) CpG methylation differences in genes with functions relevant to inflammation, cellular ageing and vascular calcification. Following 12 months, the global DNA methylation pattern of patients approached that seen in the control group. Notably, 413 CpG sites remained differentially methylated at follow-up in both treatment groups compared to controls. Together, these data indicate that the uremic milieu drives genome-wide methylation changes that are partially reversed with kidney failure replacement therapy. Differentially methylated CpG sites unaffected by treatment may be of particular interest as they could highlight candidate genes for kidney disease per se.

Treatment Persistence in Patients Cycling on Subcutaneous Tumor Necrosis Factor-Alpha Inhibitors in Inflammatory Arthritis: A Retrospective Study.

INTRODUCTION: Biologic treatments including subcutaneous tumor necrosis factor-alpha inhibitors (SC-TNFis) have greatly improved disease management of rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS) (collectively inflammatory arthritis, IA). Nevertheless, some patients discontinue their first-line treatment; for them, one option may be a subsequent line of the same treatment class (i.e., cycling). The aim of this study was to assess treatment persistence between first- and second-line therapy in Swedish IA patients cycling on SC-TNFis. METHODS: Using data from the Swedish Health Data Registers, adult IA patients filling prescriptions between May 1, 2010, and October 31, 2016, for a SC-TNFi (adalimumab, etanercept, certolizumab and golimumab) were included. Treatment persistence was derived based on information from filled prescriptions and a 60-day grace period. Unadjusted and adjusted marginal Cox proportional hazards models were fitted to estimate the relative risk of discontinuation across treatment lines, using robust sandwich covariance matrix estimates to account for intrapatient dependence (i.e., multiple treatment lines per patient). The analysis was restricted to the first two lines of treatment. RESULTS: Of the eligible patients, 3181 were identified as cyclers. Among these, most were female (68%), and 46%, 28% and 26% were diagnosed with RA, AS and PsA, respectively. Both the unadjusted and adjusted analyses showed that the relative risk of discontinuing SC-TNFi treatment was significantly lower in second compared to first line (hazard ratio; 0.60 [0.57, 0.63] and HR; 0.59 [0.56, 0.62]). This finding was also consistent across IA indications. CONCLUSIONS: In this study of patients cycling on SC-TNFis in IA, persistence was greater in second- compared to first-line treatment. The finding was consistent across all IA indications. Hence, patients who discontinue their first-line treatment may still benefit from treatment with an alternative SC-TNFi as a second-line therapy in IA.

Healthcare-Related Costs Associated with Switching Subcutaneous Tumor Necrosis Factor-α Inhibitor in the Treatment of Inflammatory Arthritis: a Retrospective Study.

INTRODUCTION: Subsequent lines of subcutaneous tumor necrosis factor alpha inhibitor (SC-TNFi) treatment may be well motivated in the management of rheumatoid arthritis (RA), ankylosing spondylitis (AS), and psoriatic arthritis (PsA)-collectively named inflammatory arthritis (IA). However, the costs associated with switching SC-TNFis are largely unknown. The objective of this retrospective observational study was to explore costs of healthcare resource utilization (HCRU) associated with switching SC-TNFi treatment among biologic-naïve Swedish patients with IA. METHODS: Using population-based register data, adult patients filling prescriptions between May 6, 2010 and December 31, 2014 for an SC-TNFi (adalimumab, etanercept, certolizumab, and golimumab) were included. Patients switching treatment (cyclers) were matched to treatment persistent patients on the basis of propensity score and follow-up time. HCRU-associated costs were captured and compared 12 months before and 12 months after the index date (defined as the date of the switch). RESULTS: A balanced cohort of 594 matched pairs was derived. Prior to the index date, cyclers had significantly higher non-treatment HCRU costs compared to persistent patients ($3815 [3498-4147] vs. $2900; 95%CI [2565-3256]). However, 12 months after the index date, cyclers had significantly increased their non-treatment HCRU costs while persistent patients lowered theirs ($822 [232-1490] vs. $- 313 [- 664-36]). This resulted in a statistically significant difference in difference of $1135 between the groups. CONCLUSIONS: In biologic-naïve patients treated with SC-TNFi for IA, cyclers significantly increased their non-treatment HCRU costs 12 months after switching treatment while persistent patients lowered their costs during the same time period. As these findings indicate that differences in treatment persistence may have an impact on costs, further research utilizing more comprehensive data sources in alternate settings is warranted.

Real-World Patient Experience of Switching Biologic Treatment in Inflammatory Arthritis and Ulcerative Colitis - A Systematic Literature Review.

PURPOSE: To obtain an up-to-date overview of the measurement of patient experience of switching biologic treatment in patients with inflammatory arthritis (IA) or ulcerative colitis (UC). Secondary objectives included summarizing the types of patient-reported outcomes (PROs) used (if any), and related findings; and summarizing medical and non-medical reasons for treatment switch and/or discontinuation. METHODS: A systematic literature review (SLR) was performed, searching Medline and Embase for relevant publications. RESULTS: In total, 70 relevant publications were identified. While the majority of these reported reasons for switching and/or discontinuing treatment, only four provided information explicitly regarding patient-reported experience of switching biologic treatment. All four utilized ranking tools to assess patient experience of switching biologic treatment. The most common reason for switching and/or discontinuing treatment was loss of efficacy, while the least common reason was patient preference. CONCLUSION: Although the number of available treatments in IA and UC have increased, there is a sparsity of information regarding patient-reported experience of switching biologic treatment. Further research regarding patient preference and/or experience would benefit this therapeutic area and help guide treatment choices.

The value of persistence in treatment with subcutaneous TNF-alpha inhibitors for ankylosing spondylitis.

OBJECTIVE: To estimate the impact of persistence on cost-effectiveness of subcutaneous tumor necrosis factor-α inhibitors (SC-TNFis) from healthcare and societal perspectives in a United Kingdom ankylosing spondylitis (AS) population using a recently published Markov cohort model. METHODS: A recently published cost-effectiveness model developed for a National Institute for health and Care Excellence appraisal was extended to fit the current study; in brief, it is a Markov cohort model where treatment responders continue from the trial period with maintenance SC-TNFi treatment, while non-responders transition to conventional care. Costs and effects were modeled for a hypothetical SC-TNFi with average efficacy and price. Model outcomes included quality-adjusted life-years (QALYs), total direct and indirect lifetime costs, and incremental cost-effectiveness ratios (ICERs). The cost-effectiveness of SC-TNFi persistence was estimated by decreasing the annual discontinuation probability in five percentage point increments from 25 to 5% per annum. RESULTS: From a health care perspective, the ICERs for the modeled discontinuation rates compared to the baseline annual discontinuation rate (25%) ranged between GBP 17,277 and GBP 18,161. From a societal perspective, increased discontinuation rates resulted in decreased total costs and higher QALYs; hence, lower discontinuation rates dominated higher discontinuation rates from a societal perspective. CONCLUSION: In conclusion, this study shows that, all else equal, higher SC-TNFi treatment persistence in AS is cost effective from a health care perspective and dominant from a societal perspective. Hence, all else equal, prescribing the SC-TNFi with the highest persistence may be considered a cost-effective strategy.

Real-world treatment persistence of golimumab in the management of immune-mediated rheumatic diseases in Europe: a systematic literature review.

OBJECTIVES: To summarise real-world data from studies reporting golimumab persistence in European immune-mediated rheumatic disease (IMRD) populations and to report pooled estimates. DESIGN: Systematic literature review. DATA SOURCES: Relevant literature was identified through searching Medline and Embase via Ovid as well as the conference databases of European League Against Rheumatism and American College of Rheumatology-Association of Rheumatology Health Professionals. ELIGIBILITY CRITERIA: We screened records using predefined patients, interventions, comparators, outcomes and study design criteria. Eligible studies included reports of persistence among adult IMRD patients in Europe receiving treatment with subcutaneous golimumab. Clinical trials, randomised controlled trials, literature reviews, editorials, guidelines and studies with <20 patients receiving golimumab were excluded. DATA EXTRACTION AND SYNTHESIS: Following double screening by two independent reviewers, 27 studies out of 578 identified records were selected for inclusion and subsequent data extraction. Persistence was most commonly reported at 12and 24 months; hence, pooled persistence estimates were calculated for these two time points and reported according to indication. RESULTS: Persistence ranged between 58.1% (psoriatic arthritis (PsA) patients regardless of treatment line) and 75.7% (biological-naïve rheumatoid arthritis patients) at 12 months; at 24 months, the range was 43% (axial spondyloarthritis (AxSpA) patients regardless of treatment line) and 69.6% (biological-naïve PsA patients). On the basis of data from 12 studies, persistence with golimumab treatment was either significantly higher or not significantly different from other tumour necrosis factor inhibitors (TNFi). CONCLUSIONS: Golimumab persistence at 24 months approximates 50%, with a lower persistence among AxSpA (43%) patients. However, as the number of studies in these populations was low, they warrant further research. In 12 studies comparing various TNFi treatments, golimumab was shown to have significantly better or equal persistence to its comparators.

CDKN2A/p16INK4a expression is associated with vascular progeria in chronic kidney disease.

Patients with chronic kidney disease (CKD) display a progeric vascular phenotype linked to apoptosis, cellular senescence and osteogenic transformation. This has proven intractable to modelling appropriately in model organisms. We have therefore investigated this directly in man, using for the first time validated cellular biomarkers of ageing (CDKN2A/p16INK4a, SA-ß-Gal) in arterial biopsies from 61 CKD patients undergoing living donor renal transplantation. We demonstrate that in the uremic milieu, increased arterial expression of CDKN2A/p16INK4a associated with vascular progeria in CKD, independently of chronological age. The arterial expression of CDKN2A/p16INK4a was significantly higher in patients with coronary calcification (p=0.01) and associated cardiovascular disease (CVD) (p=0.004). The correlation between CDKN2A/p16INK4a and media calcification was statistically significant (p=0.0003) after correction for chronological age. We further employed correlate expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2) as additional pathognomonic markers. Higher expression of CDKN2A/p16INK4a, RUNX2 and MGP were observed in arteries with severe media calcification. The number of p16INK4a and SA-ß-Gal positive cells was higher in biopsies with severe media calcification. A strong inverse correlation was observed between CDKN2A/p16INK4a expression and carboxylated osteocalcin levels. Thus, impaired vitamin K mediated carboxylation may contribute to premature vascular senescence.

Increased Telomere Attrition After Renal Transplantation-Impact of Antimetabolite Therapy.

BACKGROUND: The uremic milieu exposes chronic kidney disease (CKD) patients to premature ageing processes. The impact of renal replacement therapy (dialysis and renal transplantation [RTx]) or immunosuppressive treatment regimens on ageing biomarkers has scarcely been studied. METHODS: In this study telomere length in whole blood cells was measured in 49 dialysis patients and 47 RTx patients close to therapy initiation and again after 12 months. Forty-three non-CKD patients were included as controls. RESULTS: Non-CKD patients had significantly (P ≤ 0.01) longer telomeres than CKD patients. Telomere attrition after 12 months was significantly greater in RTx patients compared to dialysis patients (P = 0.008). RTx patients receiving mycophenolate mofetil (MMF) had a greater (P = 0.007) degree of telomere attrition compared to those treated with azathioprine. After 12 months, folate was significantly higher in RTx patients than in dialysis patients (P < 0.0001), whereas the opposite was true for homocysteine (P < 0.0001). The azathioprine group had lower levels of folate after 12 months than the MMF group (P = 0.003). CONCLUSIONS: The associations between immunosuppressive therapy, telomere attrition, and changes in folate indicate a link between methyl donor potential, immunosuppressive drugs, and biological ageing. The hypothesis that the increased telomere attrition, observed in the MMF group after RTx, is driven by the immunosuppressive treatment, deserves further attention.

Genotypic and phenotypic predictors of inflammation in patients with chronic kidney disease.

BACKGROUND: In complex diseases such as chronic kidney disease (CKD), the risk of clinical complications is determined by interactions between phenotypic and genotypic factors. However, clinical epidemiological studies rarely attempt to analyse the combined effect of large numbers of phenotype and genotype features. We have recently shown that the relaxed linear separability (RLS) model of feature selection can address such complex issues. Here, it is applied to identify risk factors for inflammation in CKD. METHODS: The RLS model was applied in 225 CKD stage 5 patients sampled in conjunction with dialysis initiation. Fifty-seven anthropometric or biochemical measurements and 79 genetic polymorphisms were entered into the model. The model was asked to identify phenotypes and genotypes that, when combined, could separate inflamed from non-inflamed patients. Inflammation was defined as a high-sensitivity C-reactive protein concentration above the median (5 mg/L). RESULTS: Among the 60 genotypic and phenotypic features predicting inflammation, 31 were genetic. Among the 10 strongest predictors of inflammation, 8 were single nucleotide polymorphisms located in the NAMPT, CIITA, BMP2 and PIK3CB genes, whereas fibrinogen and bone mineral density were the only phenotypic biomarkers. CONCLUSION: These results indicate a larger involvement of hereditary factors in inflammation than might have been expected and suggest that inclusion of genotype features in risk assessment studies is critical. The RLS model demonstrates that inflammation in CKD is determined by an extensive panel of factors and may prove to be a suitable tool that could enable a much-needed multifactorial approach as opposed to the commonly utilized single-factor analysis.

Global Analysis of DNA 5-Methylcytosine Using the Luminometric Methylation Assay, LUMA.

The study of epigenetic alterations of the genome is becoming increasingly important in order to understand how environment and genetic background interact to build and regulate the functional genome. There are several types of epigenetic modifications to both DNA and histone proteins in eukaryotic cells; chiefly studied among these are changes to cytosine, where methylation of the 5-carbon position is the most prominent. Although this has many consequences for gene regulation and cell differentiation, other modifications have recently emerged as biologically relevant. Since global DNA methylation states may be used as a general measure of the methylome, cost-effective, rapid, and specific analytical tools are wanted.This protocol described here focuses on the Luminometric Methylation Assay (LUMA), a method which analyzes global DNA 5-methylcytosine (5mC) through the use of restriction enzymes and detection with Pyrosequencing(®). Up to 96 samples can be simultaneously analyzed. In contrast to the majority of other methods focused on 5mC analysis, with appropriate enzymes, LUMA does not appear to detect 5-hydroxymethylcytosine (5hmC) and is therefore more specific than most 5mC techniques.

Selection of genetic and phenotypic features associated with inflammatory status of patients on dialysis using relaxed linear separability method.

Identification of risk factors in patients with a particular disease can be analyzed in clinical data sets by using feature selection procedures of pattern recognition and data mining methods. The applicability of the relaxed linear separability (RLS) method of feature subset selection was checked for high-dimensional and mixed type (genetic and phenotypic) clinical data of patients with end-stage renal disease. The RLS method allowed for substantial reduction of the dimensionality through omitting redundant features while maintaining the linear separability of data sets of patients with high and low levels of an inflammatory biomarker. The synergy between genetic and phenotypic features in differentiation between these two subgroups was demonstrated.

Global DNA hypermethylation is associated with high serum levels of persistent organic pollutants in an elderly population.

Dioxin exposure has experimentally been associated with changes in DNA methylation, an epigenetic change that is associated with disease. The present study aims to investigate if serum levels of dioxin and other persistent environmental pollutants are related to global DNA methylation in a human sample. In the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (all aged 70), global DNA methylation was measured by the Luminometric Methylation Assay in 524 subjects. Twenty-three different POPs, including 16 PCBs, five pesticides, one dioxin (OCDD) and one brominated flame retardant (BDE47) were analysed by HRGC/HRMS. Ten single nucleotide polymorphisms (SNPs) in the Aryl hydrocarbon (Ah)-receptor were analysed by mini-sequencing. High levels of toxic equivalency (TEQ) for PCBs and dioxin were associated with DNA hypermethylation (p=0.030). This was mainly attributed to coplanar non-ortho PCBs. While no significant associations were found between DNA methylation and SNPs in the Ah-receptor, an interaction was found between the SNP rs2237297 and TEQ so that TEQ was associated with hypermethylation (p=0.009) only in subjects with one G-allele (n=103). Also high levels of the PCB126 congener, the OCDD, and the pesticide metabolite p,p'-DDE were related to DNA hypermethylation (p=0.01, 0.03 and 0.003, respectively). In conclusion, in a sample of elderly subjects, high TEQ including PCBs and the dioxin OCDD and high serum levels of PCB126, OCDD, and p,p'-DDE were related to global DNA hypermethylation in a cross-sectional analysis.

Genetic studies in chronic kidney disease: interpretation and clinical applicability.

The tools of modern molecular biology are evolving rapidly, resulting in vastly more efficient approaches to illuminating human genetic variations and their effects on common multifactorial disorders such as chronic kidney disease (CKD). Indeed, candidate gene association studies and genome-wide association studies (GWASs) have generated novel genetic variants in previously unrecognized biological pathways, highlighting disease mechanisms with a potential role in CKD etiology, morbidity and mortality. Nephrologists now need to find ways to make use of these advancements and meet the increasingly stringent requirements for valid study design, data handling and interpretation of genetic studies. Adding to our prior article in this journal, which introduced the basics of genotype-phenotype association studies in CKD, this second article focuses on how to ascertain robust and reproducible findings by applying adequate methodological and statistical approaches to genotype-phenotype studies in CKD populations. Moreover, this review will briefly discuss genotype-based risk prediction, pharmacotherapy, drug target identification and individualized treatment solutions, specifically highlighting potentially important findings in CKD patients. This increased knowledge will hopefully facilitate the exciting transition from conventional clinical medicine to gene-based medicine. However, before this can be accomplished, unsolved issues regarding the complex human genetic architecture as well technical and clinically oriented obstacles will have to be overcome. Additionally, new policies and standardized risk evaluations for genetic testing in the clinical setting will have to be established to guarantee that CKD patients are provided with high-quality genotype-guided counseling that will help to improve their poor outcomes.

DNA hypermethylation and inflammatory markers in incident Japanese dialysis patients.

BACKGROUND/AIMS: Inflammation is an established mortality risk factor in chronic kidney disease (CKD) patients. Although a previous report showed that uremic Caucasian patients with inflammation had signs of global DNA hypermethylation, it is still unknown whether DNA hypermethylation is linked to inflammatory markers including a marker of bacterial infections in Japanese CKD patients. METHODS: In 44 consecutive incident dialysis patients (26 males, mean age 59 ± 12 years) without clinical signs of infection, global DNA methylation was evaluated in peripheral blood DNA using the HpaII/MspI ratio by the luminometric methylation assay method. A lower ratio of HpaII/MspI indicates global DNA hypermethylation. Procalcitonin (PCT), a marker of inflammation due to bacterial infections, was measured using an immunochromatographic assay. RESULTS: The patients were divided into hyper- and hypomethylation groups based on the median value of the HpaII/MspI ratio 0.31 (range 0.29-0.37). Whereas patients in the hypermethylation group had higher ferritin levels [133.0 (51.5-247.3) vs. 59.5 (40.0-119.0) ng/ml; p = 0.046], there were no significant differences in age, gender, diabetes, smoking, anemia or serum albumin levels. However, the HpaII/MspI ratio showed significant negative correlations with PCT (ρ = -0.32, p = 0.035) and ferritin (ρ = -0.33, p = 0.027) in Spearman's rank test. In a multiple linear regression analysis, PCT and ferritin were associated with a lower HpaII/MspI ratio (R(2) = 0.24, p = 0.013). CONCLUSION: In this study, global DNA hypermethylation was associated with ferritin and, most likely, PCT, suggesting that inflammation induced by subclinical bacterial infection promoted DNA methylation.

Genetic studies in chronic kidney disease: basic concepts.

In spite of extensive research resulting in major advances in renal care including technological improvements of dialysis, the poor outcome of chronic kidney disease patients has only marginally been improved since the 1980s. It has thus become clear that new strategies are needed to move forward. There are now great expectations that increased knowledge about genetic characteristics combined with other biological markers will identify pathophysiological pathways involved in the initiation and progression of renal damage and that this in turn will help define tools for early disease intervention and personalized treatment strategies. Already, new methodologies have made it possible to study the heritable component of many kidney diseases, and it is probable that DNA-based diagnostics will be performed on a regular basis for many conditions in the near future. This article discusses basic genetic concepts and highlights some of the novel approaches available for genome-wide genetic analyses. We hope that it may serve as an introduction to the research field of what we call "nephrogenetics." A second article in this series will focus on the interpretation and evaluation of genetic association studies and how to make use of this information to improve patient care and outcomes.

Global DNA methylation analysis using the Luminometric Methylation Assay.

Epigenetic alterations regulate the utilization of the genome by permitting or inhibiting access of transcription factors and associated complexes. Although there are several different types of epigenetic alterations, such as acetylation and methylation of histone tails, the one which has been the most extensively studied is DNA-methylation, wherein the cytosine residue in a CpG dinucleotide is methylated. Luminometric Methylation Assay (LUMA) enables researchers to study global methylation by using methylation-sensitive restriction enzymes followed by Pyrosequencing(®) which quantitates the number of cuts in the genome relative to an internal standard. The relative measurement of global methylation levels is simple and enables up to 96 samples to be analyzed at the same time.

Genetic loci influencing kidney function and chronic kidney disease.

Using genome-wide association, we identify common variants at 2p12-p13, 6q26, 17q23 and 19q13 associated with serum creatinine, a marker of kidney function (P = 10(-10) to 10(-15)). Of these, rs10206899 (near NAT8, 2p12-p13) and rs4805834 (near SLC7A9, 19q13) were also associated with chronic kidney disease (P = 5.0 x 10(-5) and P = 3.6 x 10(-4), respectively). Our findings provide insight into metabolic, solute and drug-transport pathways underlying susceptibility to chronic kidney disease.

Genetics/Genomics in chronic kidney disease--towards personalized medicine?

The progression rate of chronic kidney disease (CKD) to its terminal stage, end-stage renal disease (ESRD), and the development and severity of various complications, are at least indirectly influenced by genetic--and epigenetic--factors. For years, scientists have held out hope that the rapidly evolving field of genetics could transform medical diagnosis and treatment, moving beyond a trial-and-error approach towards "personalized medicine." Indeed, there are now signs that the role of genetics and the pursuit of "personalized medicine" in medical care will be a priority for governments during years to come. But the vision of individualized treatment based on a patient's genetic makeup and other biological markers has yet to materialize in the field of CKD and ESRD. As the toxic uremic environment may render CKD patients more sensitive to the effects of genetic variants, it is likely that genetic factors could be of special importance in this high-risk population. Therefore, outcome in the CKD population may be improved by establishing individual genetic/epigenetic profiles, thus enabling physicians to design an individualized therapeutic strategy. Personalized medicine based on a more individualized therapy could be applied in, for example, pharmacotherapy (CYP genes), dialysis therapy, and nutritional and lifestyle modifications.