Chack-ng Yu

Biomarkers of lupus nephritis determined by serial urine proteomics

Lupus nephritis is a frequent and serious complication of systemic lupus erythematosus (SLE), the treatment of which often requires the use of immunosuppressives that can have severe side effects. Here we determined the low-molecular weight proteome of serial lupus urine samples to uncover novel and predictive biomarkers of SLE renal flare. Urine from 25 flare cycles of 19 patients with WHO Class III, IV, and V SLE nephritis were obtained at baseline, pre-flare, flare and post-flare. Each sample was first fractionated to remove proteins larger than 30 kDa, then applied onto weak cation exchanger protein chips for analysis by SELDI-TOF mass spectrometry. We found 176 protein ions of which 27 were differentially expressed between specific flare intervals. On-chip peptide sequencing by integrated tandem mass spectrometry positively identified the 20 and 25 amino-acid isoforms of hepcidin, as well as fragments of alpha1-antitrypsin and albumin among the selected differentially expressed protein ions. Hepcidin 20 increased 4 months before renal flare and returned to baseline at renal flare, whereas hepcidin 25 decreased at renal flare and returned to baseline 4 months after the flare. These studies provide a beginning proteomic analysis aimed at predicting impending renal relapse, relapse severity, and the potential for recovery after SLE nephritis flare.

The complex nature of serum C3 and C4 as biomarkers of lupus renal flare

To assess the relationship between serum C3 or C4 levels and lupus renal flare, C3 and C4 levels were measured bimonthly in 71 lupus nephritis patients for a mean of 35 months, during which time 70 renal flares were identified. Comparing baseline, pre-flare, and at-flare values indicated that neither C3 nor C4 levels decreased pre-flare, but both decreased on average significantly at flare. However, sensitivity/specificity for C3 (75%/71%) and C4 (48%/71%) were low. To account for other influencing factors, multiple regression was performed that included bimonthly values of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and genotype data on C3 (S/F), CRP (1846G > A), and the complement regulator factor H (Y402H). This analysis revealed that reduced levels of C4, but not C3, were independently associated with the two-month pre-flare period. Conversely, reduced levels of C3, but not C4, were independently associated with the flare visit. Significant pro-flare interactions included low C3 levels with the factor H 402HH-encoding genotype, and low CRP levels with the C3 F allele. Together these data suggest that C4 activation is critical for initiating renal flare while C3 activation is involved in the actual tissue damage, and that these effects are influenced by genetic variability in complement activation and regulation.

Phenotypes, genotypes and disease susceptibility associated with gene copy number variations: complement C4 CNVs in European American healthy subjects and those with systemic lupus erythematosus.

A new paradigm in human genetics is high frequencies of inter-individual variations in copy numbers of specific genomic DNA segments. Such common copy number variation (CNV) loci often contain genes engaged in host-environment interaction including those involved in immune effector functions. DNA sequences within a CNV locus often share a high degree of identity but beneficial or deleterious polymorphic variants are present among different individuals. Thus, common gene CNVs can contribute, both qualitatively and quantitatively, to a spectrum of phenotypic variants. In this review we describe the phenotypic and genotypic diversities of complement C4 created by copy number variations of RCCX modules (RP-C4-CYP21-TNX) and size dichotomy of C4 genes. A direct outcome of C4 CNV is the generation of two classes of polymorphic proteins, C4A and C4B, with differential chemical reactivities towards peptide or carbohydrate antigens, and a range of C4 plasma protein concentrations (from 15 to 70 mg/dl) among healthy subjects. Deliberate molecular genetic studies enabled development of definitive techniques to determine exact patterns of RCCX modular variations, copy numbers of long and short C4A and C4B genes by Southern blot analyses or by real-time quantitative PCR. It is found that in healthy European Americans, the total C4 gene copy number per diploid genome ranges from 2 to 6: 60.8% of people with four copies of C4 genes, 27.2% with less than four copies, and 12% with more than four copies. Such a distribution is skewed towards the low copy number side in patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disease with complex etiology. In SLE, the frequency of individuals with less than four copies of C4 is significantly increased (42.2%), while the frequency of those with more than four copies is decreased (6%). This decrease in total C4 gene copy number in SLE is due to increases in homozygous and heterozygous deficiencies of C4A but not C4B. Therefore, it is concluded that lower copy number of C4 is a risk factor for and higher gene copy number of C4 is a protective factor against SLE disease susceptibility.

Clinical significance of fever in the systemic lupus erythematosus patient receiving steroid therapy

BACKGROUND Active systemic lupus erythematosus (SLE) can cause fever. Steroids (glucocorticoids) suppress SLE fever; however, the extent to which steroid therapy affects SLE fever not previously been rigorously studied. METHODS Study A is a prospective study of recurrently active SLE patients (N= 92, 60 renal SLE and 32 nonrenal SLE) who recorded daily oral evening temperatures while participating in a longitudinal study of risk factors for SLE flare. Study B is a retrospective study of consecutive febrile SLE patients (N= 22) who received steroids initially because SLE was suspected. At final analysis 11 had SLE fever and 11 had infection fever. RESULTS In study A during a mean follow-up of 13.2 +/- 8.1 months, 51 of the 92 patients experienced 73 SLE flares. In only one patient was SLE fever associated with SLE flare. In the other 50 patients who flared, there was no significant trend to develop fever prior to or at the onset of SLE flare. Prednisone, median dose 10 mg, was being received at 82% of the study visits at which an SLE flare was declared. In study B, prednisone 28 mg (range 20 to 40 mg) completely suppressed SLE fever, usually within 24 hours. In contrast, infection fever persisted despite prednisone 35 to 300 mg/day. Of those with infection fever, three developed fatal sepsis when high-dose steroid therapy was continued. CONCLUSION In SLE patients receiving prednisone at maintenance doses or greater, SLE fever is rare. When fever does develop, it is usually due to infection. Continuing high steroid dose steroid therapy in those with infection fever may increase the risk of severe sepsis.

Evidence that abnormally large seasonal declines in vitamin D status may trigger SLE flare in non-African Americans.

Cross-sectional studies have shown that low vitamin D (25-hydroxyvitamin D (25(OH)D)) is associated with increased systemic lupus erythematosus (SLE) activity. This study is the first to assess the temporal relationship between 25(OH)D levels and onset of SLE flare. This assessment was made possible because of the specimen bank and database of the Ohio SLE Study (OSS), a longitudinal study of frequently relapsing SLE that involved regular bimonthly patient follow-up. We identified for this study 82 flares from 46 patients that were separated by at least 8 months from previous flares. Serum 25(OH)D levels were measured at 4 and 2 months before flare, and at the time of flare (a flare interval). We found that for flares occurring during low daylight months (LDM, Oct-Mar), 25(OH)D levels were decreased at the time of flare, but only in non-African American (non-AA) patients (32% decrease at flare, compared to 4 months prior, p < 0.001). To control for seasonal effects, we also measured 25(OH)D levels in the LDM “no-flare” intervals, which were intervals that matched to the same calendar months of the patients’ LDM flare intervals, but that didn’t end in flare (n = 24). For these matches, a significant decrease occurred in 25(OH)D levels during the flare intervals (18.1% decrease, p < 0.001), but not during the matching no-flare intervals (6.2% decrease, p = 0.411). For flares occurring during high daylight months (HDM), 25(OH)D levels changed only in non-AA patients, increasing slightly (5.6%, p = 0.010). Analysis of flare rates for the entire OSS cohort (n = 201 flares) revealed a tendency for higher flare rates during LDM compared to HDM, but again only in non-AA patients (p = 0.060). Flare rates were lower during HDM for non-AA patients compared to AA patients (p = 0.028). In conclusion, in non-AA SLE patients, unusually large declines in 25(OH)D during LDM may be mechanistically related to SLE flare, whereas relatively high 25(OH)D levels during HDM may protect against flare.

Random Spot Urine Protein/Creatinine Ratio Is Unreliable for Estimating 24-Hour Proteinuria in Individual Systemic Lupus Erythematosus Nephritis Patients

Background: Recently the American Rheumatologic Association (ARA) recommended random spot urine protein/creatinine ratio (P/C) to monitor systemic lupus erythematosus (SLE) glomerulonephritis (GN). Shortly afterward, 2 works were published, designated Study 1 and Study 2, which are the only studies to test spot P/C in SLE GN. Here we evaluate Study 1 and Study 2, which came to different conclusions. Methods: Study 1 compared spot P/C to the P/C of intended 24-hour collections >50% complete, which reliably estimates 24-hour proteinuria. Study 2 compared spot P/C to the protein content of intended 24-hour collections >80% complete. To compare studies, Study 2 data were converted to P/C ratios. Results: Study 1 and Study 2 were found to be in agreement. Both showed that spot P/C and 24-hour P/C were highly correlated, but only when compared over the entire P/C range (0–8.0) (r = 0.842). Over the P/C range 0.5–3.0 (the most common P/C range encountered in SLE GN), correlation was present, but concordance was poor, rendering random P/C ratio unreliable. Conclusions: Random spot P/C ratio is unreliable for detecting moderate proteinuria change. For example, random spot P/C would not reliably diagnose British Isles Lupus Assessment Group (BILAG) Category A or B proteinuric flares.

Consumption of erythrocyte CR1 (CD35) is associated with protection against systemic lupus erythematosus renal flare

Erythrocyte complement receptor type one (E‐CR1) is thought to protect against immune complex (IC) disease through interactions that lead to E‐CR1 consumption, and low E‐CR1 levels are characteristic of systemic lupus erythematosus (SLE). The purpose of this study was to test the hypothesis that E‐CR1 consumption can predict or mark SLE flare. Recurrently active SLE patients [n = 43; 28 with past or present major renal manifestations (SLER) and 15 without (SLENR)], were evaluated every 2 months by detailed protocol testing (mean follow‐up 22 months), including direct measurements of E‐CR1 levels using a radioimmunoassay. In all patients, detectable E‐CR1 levels fluctuated widely through acute periods of consumption and regeneration, preventing the use of any single value as a baseline. However, when individual chronic baseline values were used, determined as the mean of all E‐CR1 values 4 months or more from a flare, a clear trend was observed. In 16 of 16 instances of non‐renal flare in SLER patients, E‐CR1 levels decreased at flare (mean decrease 34%, P < 0·0001). In contrast, no consistent difference was observed for flare in SLENR patients or for renal flare in SLER patients. Changes in E‐CR1 levels did not correlate with plasma CR1 levels. In conclusion, single occurrences of E‐CR1 consumption did not generally predict or mark SLE flare. However, compared to the average E‐CR1 levels measured during no‐flare intervals, E‐CR1 consumption in SLER patients at flare was strongly associated with freedom from signs of renal involvement. We postulate that E‐CR1 consumption reflects E‐CR1 function that includes protecting against SLE nephritis.

D-Dimer Level and the Risk for Thrombosis in Systemic Lupus Erythematosus

BACKGROUND AND OBJECTIVES Patients who have systemic lupus erythematosus (SLE) and manifest antiphospholipid antibodies (APA) are at increased risk for thrombosis; however, it is difficult to predict who will clot. This study tested the hypothesis that peak D-dimer level measured routinely during follow-up identifies whether a hypercoagulable state is developing and, therefore, the patient is at increased risk for thrombosis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS One hundred consecutive patients who had SLE with recurrent activity (71% renal SLE) and were evaluated for or enrolled in the Ohio SLE Study were studied. D-dimer testing was done annually and usually at SLE flare or other serious illness. When D-dimer was elevated, evaluation for thrombosis (large vessel, small vessel, or Libman-Sacks) was undertaken. Mean follow-up was 37.5 +/- 15 SD months. RESULTS Of those with peak D-dimer <0.5 microg/ml (n = 46), 0% thrombosed, 33% had APA. Of those with peak D-dimer 0.5 to 2.0 microg/ml (n = 19), 6% thrombosed, 44% had APA. Of those with peak D-dimer >2.0 microg/ml (n = 36), 42% thrombosed, 76% had APA. The most common causes of elevated D-dimer in the absence of demonstrable thrombosis were SLE flare and systemic infection. D-dimer levels were usually elevated for several months before thrombosis. CONCLUSIONS Patients with SLE and normal D-dimer levels are at low risk for thrombosis, irrespective of APA status. Those with persistent unexplained elevated D-dimer levels, particularly when >2.0 microg/ml, are at high risk for thrombosis.

Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays

BackgroundArray-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.ResultsWe present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.ConclusionA novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.

Gene CNVs and protein levels of complement C4A and C4B as novel biomarkers for partial disease remissions in new‐onset type 1 diabetes patients

To determine the roles of complement C4A and C4B gene copy‐number variations and their plasma protein concentrations in residual insulin secretion and loss of pancreatic β‐cell function in new‐onset type 1 diabetes (T1D) patients.