Prediagnostic Appearance of Thrombospondin Type-1 Domain 7A Autoantibodies in Membranous Nephropathy

Abstract

Key Points:

1. The entire extracellular domain of thrombospondin type-1 domain 7A (THSD7A) in the luciferase immunoprecipitation system immunoassay was required to detect autoantibodies with high sensitivity in membranous nephropathy (MN).

2. In THSD7A-seropositive MN patients, changes in antibody levels precede changes in clinical status.

3. Seropositive THSD7A antibodies were detected in some patients with MN considered to be secondary to autoimmunity or cancer.

Background: Pathogenic autoantibodies against thrombospondin type-1 domain 7A (THSD7A) are present in approximately 3% of patients with membranous nephropathy (MN). Compared with PLA2R antibodies, less is known about THSD7A autoantibodies (ABs) because of the relative rarity and the lack of a commercially available quantitative immunoassay.

Methods: In this study, we describe the development and validation of a highly quantitative luciferase immunoprecipitation system (LIPS) assay for detecting THSD7A ABs and used it to study dominant THSD7A epitopes, disease associations, and monitoring disease activity. The Department of Defense Serum Repository (DODSR) was then used to analyze THSD7A AB in 371 longitudinal serum samples collected before clinical diagnosis of MN from 110 PLA2R-negative MN subjects.

Results: LIPS analysis demonstrated that a near full-length THSD7A (amino acids 1–1656) detected robust autoantibody levels in all known seropositive MN patients with 100% sensitivity and specificity compared with ELISA and/or Western blotting. Most of the THSD7A-seropositive subjects in our pilot cohort had evidence of coexisting autoimmunity or cancer. Moreover, three THSD7A-seropositive patients undergoing immunosuppressive therapy showed longitudinal autoantibody levels that tracked clinical status. Additional epitope analysis of two smaller protein THSD7A fragments spanning amino acids 1-416 and 1-671 demonstrated lower sensitivity of 32% and 44%, respectively. In the DODSR cohort, THSD7A seropositivity was detected in 4.5% of PLA2R-negative MN patients. In one primary and in one secondary MN-associated with cancer, THSD7A ABs were detectable <1 month before biopsy-proven diagnosis. In addition, three patients with lupus membranous nephropathy had detectable THSD7A ABs years before hypoalbuminemia and biopsy-proven diagnosis.

Conclusions: Although further studies are needed to explore the significance of THSD7A ABs in lupus membranous nephropathy, this study describes a novel, highly sensitive LIPS immunoassay for detecting THSD7A ABs and adds to the existing literature on THSD7A-associated MN.

Clinical Trial registry name and registration number:: NCT00977977; registration date: September 16, 2009.

Graphical abstract

Figure 1

Characteristics of the three cohorts screened for THSD7A AB by LIPS. This study screened 502 patient serum samples for THSD7A AB from three different cohorts. The characteristics of each cohort are shown above. THSD7A AB status was assigned for each sample before unblinding. Three THSD7A-seropositive patients from the pilot cohort who were treated were also monitored longitudinally for changes in autoantibody levels and clinical response. LIPS, luciferase immunoprecipitation systems; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 2

LIPS detection of THSD7A AB in subjects with MN and kidney disease controls. Kidney disease controls (Kidney CTRLS) and PLA2R-seropositive and seronegative MN subjects were tested by LIPS with the near full-length THSD7A-Gaussia fusion protein. Each circle represents the antibody level in light units (LU) in individual serum sample and is plotted on the y-axis using a log₁₀ scale. The cutoff value for the assay for determining THSD7A seropositive and seronegative status is shown by the dotted line. THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 3

Validation of the LIPS assay for detecting THSD7A AB in an independent cohort. LIPS detection of THSD7A AB was evaluated in a validation cohort of blinded samples from known seronegative and seropositive MN patients. Classification status of seronegative (−) or seropositive (+) was based on THSD7A ELISA antibody testing and/or Western blot analysis. Each circle represents antibody levels in light units (LU) derived from individual subjects plotted on the y-axis using a log₁₀ scale. The cutoff value is shown by the dotted line, and LIPS testing showed 100% sensitivity and specificity. LIPS, luciferase immunoprecipitation systems; MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 4

Detection of patient seropositivity against multiple epitopes of THSD7A. (A) Schematic of THSD7A-Δ1, THSD7A-Δ2, and near full-length THSD7A (FL) chimeric Gaussia luciferase (Luc) fusion proteins used for testing the different MN serum samples. The location of the transmembrane region (amino acids 1608-1628) of THSD7A is shown in THSD7A-FL by the blue box. (B) A heat map is shown of the autoantibody responses to the three different THSD7A proteins from the validation cohort, in which each row represents a different MN sample. To construct the heatmap, the autoantibody values for the 10 seronegative controls (not shown) and the THSD7A-seropositive MN subjects were color-coded based on a Z-score scale shown on the right representing the number of standard deviations above the mean of the controls for that antigen. Coloring in the heat map indicates that the relative antibody levels are at least greater than the mean of the controls plus three standard deviations. The signal intensities range from white to dark red indicating low and high autoantibody levels, respectively. MN, membranous nephropathy; THSD7A, thrombospondin type-1 domain 7A.

Figure 5

Association between THSD7A AB and clinical disease activity in three MN subjects receiving immunosuppressive treatment. THSD7A AB levels (blue circles and lines) and proteinuria (black squares and lines) are shown over time in three different MN patients (A-C). The red arrow denotes the start of treatment, in which all three patients had sustained high levels of THSD7A AB and proteinuria for months before treatment. The x axis is the time in months after initiation of immunosuppressive treatment. The left y axis represents the scale of the autoantibody levels in LU determined by LIPS with the near full-length protein. The right y axis denotes the level of urinary protein excretion expressed as grams per day. The blue dotted line represents the seropositive cutoff value for LIPS; the black dotted line represents complete remission status (proteinuria <300 mg/d). MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 6

Prediagnostic appearance of THSD7A AB before diagnosis. Autoantibody analysis of the DODSR cohort identified five THSD7A-seropositive subjects. The five patients included one with primary MN (A), one cancer-associated MN (B), and three with lupus membranous nephropathy (C–E). THSD7A autoantibody levels in LU derived from serial samples of the seropositive patients are plotted in blue. The blue, horizontal dotted line is the cutoff for determining THSD7A seropositivity. The right y-axis denotes the serum albumin levels, and the cutoff value for determining hypoalbuminemia is shown by the black dotted line. Time zero represents biopsy-proven diagnosis of MN. MN, membranous nephropathy; THSD7A AB, thrombospondin type-1 domain 7A autoantibody.

Figure 7

Prediagnostic appearance of THSD7A AB relative to appearance of Ro52 and Ro60 ABs. From testing the three subjects with lupus membranous nephropathy, two (Pt 8 and Pt 9) were also seropositive for Ro52 and Ro60 ABs. THSD7A autoantibody levels are plotted in blue. The Ro52 and Ro60 ABs are shown in black, and the approximate cutoff value for seropositivity is shown by the red dotted line. The cutoff value for THSD7A AB seropositivity is shown with the dotted blue line. THSD7A AB, thrombospondin type-1 domain 7A autoantibody.