A Non-Invasive Laboratory Panel as a Diagnostic and Prognostic Biomarker for Thrombotic Microangiopathy: Development and Application in a Chinese Cohort Study

<div><p>Background</p><p>Thrombotic microangiopathy (TMA) in the kidney is a histopathologic lesion that occurs in a number of clinical settings and is often associated with poor renal prognosis. The standard test for the diagnosis of TMA is the renal biopsy; noninvasive parameters such as potential biomarkers have not been developed.</p><p>Methods</p><p>We analyzed routine parameters in a cohort of 220 patients with suspected TMA and developed a diagnostic laboratory panel by logistic regression. The levels of candidate markers were validated using an independent cohort (n = 46), a cohort of systemic lupus erythematosus (SLE) (n = 157) and an expanded cohort (n = 113), as well as 9 patients with repeat biopsies.</p><p>Results</p><p>Of the 220 patients in the derivation cohort, 51 patients with biopsy-proven TMA presented with a worse renal prognosis than those with no TMA (P = 0.002). Platelet and L-lactate dehydrogenase (LDH) levels showed an acceptable diagnostic value of TMA (AUC = 0.739 and 0.756, respectively). A panel of 4 variables - creatinine, platelets, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats 13) activity and LDH - can effectively discriminate patients with TMA (AUC = 0.800). In the validation cohort, the platelet and LDH levels and the 4-variable panel signature robustly distinguished patients with TMA. The discrimination effects of these three markers were confirmed in patients with SLE. Moreover, LDH levels and the 4-variable panel signature also showed discrimination values in an expanded set. Among patients undergoing repeat biopsy, increased LDH levels and panel signatures were associated with TMA status when paired evaluations were performed. Importantly, only the 4-variable panel was an independent prognostic marker for renal outcome (hazard ratio = 3.549; P<0.001).</p><p>Conclusions</p><p>The noninvasive laboratory diagnostic panel is better for the early detection and prognosis of TMA compared with a single parameter, and may provide a promising biomarker for clinical application.</p></div

Comparison of renal outcome in suspected patients.

<p>After 12 months of follow-up, the patients with TMA had a worse outcome of renal survival than those without TMA (Log rank: P = 0.002).</p

Association of LDH levels and 4-variable panel with renal survival.

<p>(A) Using the cutoff value of 0.248, suspected patients were divided into two groups by more or less than the value and the renal survival of two groups followed up 12 months was significantly different (P<0.001). (B) The pattern of increased LDH concentrations (more than the cutoff point of 289 u/L) associated statistically poorer renal outcome was not observed (P = 0.183).</p

Receiver operating characteristic curves and calibration curve for a diagnostic panel.

<p>(A) The diagnostic panel was developed in the derivation group of 220 suspected patients, with AUC 0.800, P<0.001. (B) This marker was validated in 46 independent patients, with AUC 0.815, P<0.001. (C) Bootstrap validation shows the calibration curve of the diagnostic panel. Cross-validated estimates of the AUC, calibration-curve intercept and slope were 0.777, 0.07 and 0.64, respectively. The loess-smoothed estimates of the cross-validated and unadjusted calibration curves are overlaid on a diagonal reference line representing good model calibration.</p

Receiver operating characteristic curves of laboratory parameters.

<p>(A) The fraction of true positive results (sensitivity) and the fraction of false positive results (1-specificity) for LDH, HGB, SCr, PLT, THBD and ADAMTS13 activity were developed in 220 patients (all P≤0.001), and the levels of platelet and LDH showed acceptable discrimination, with AUC 0.739 and 0.756, respectively. (B) The levels of platelet and LDH could discriminate patients with TMA from those with no TMA in the validation cohort (n = 46), with AUC 0.747 and 0.741, respectively.</p

Clinical laboratory data for suspected patients in derivation cohort (n = 220).

<p>Values are expressed as medians (range), means ± standard deviation or percentages. <i>P</i> values were calculated by Mann-Whitney U test, Fisher’s exact test or chi-square test as appropriate. HUS: hemolytic uremic syndrome; TTP: thrombotic thrombocytopenic purpura; SLE: systemic lupus erythematosus; C3: Complement component 3; C4: Complement component 4; ADAMTS13: A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats 13; NEC: normal endothelial cells; VCAM: vascular cell adhesion molecule; vWF: von Willebrand factor.</p><p>Clinical laboratory data for suspected patients in derivation cohort (n = 220).</p

Primers used in mutation screening of the EXT1 and EXT2 genes.

<p>Primers used in mutation screening of the EXT1 and EXT2 genes.</p

Decay of mutant EXT2 mRNA and protein.

<p>(A) Levels of EXT2 mRNA by RT-PCR. The mRNA levels were higher in the patients than that in the controls (<i>P</i> = 0.016 in a two-sided Student's t-test). (B) Clone sequencing of wild-type and mutant transcripts. Among the 32 randomly picked clones, 27 (84.4%) were identified as wild-type by direct sequencing, while only five (15.6%) were identified as mutant transcript. (C) Western blots of wild-type and mutant proteins. The band for the predicted truncated protein could not be detected in the HME patients. (D) Comparison of EXT2 protein levels in HME patients and controls. The levels were significantly lower in the patients compared with the controls (<i>P</i> = 0.006). (E) Comparison of EXT1 mRNA expression levels in HME patients and controls. Real-time PCR revealed that EXT1 mRNA was more highly expressed in the patients compared with the controls (<i>P</i> = 0.024). (F) Comparison of EXT1 protein levels in HME patients and controls. The EXT1 levels higher in the patients compared with the controls (<i>P</i> = 0.003). (G) Comparison of HS proteoglycan expression in HME patients and controls. A group of HS proteoglycans around 70 kDa (upper box) were detected in patients and controls; while two HS proteoglycans patterns around 40 kDa and 25–30 kDa (middle and lower boxes) were detected only in the patients.</p

Mutation analysis and identification of EXT2.

<p>(A) DNA sequences of the EXT2 gene. Arrows indicate the heterozygous G to A transition site in intron 4 of EXT2 from the proband (upper) and one affected individuals (lower). The mutation was not detected in normal family members or in the healthy controls of the same ethnic origin (control, middle). (B) Alignment of EXT2 gene sequences from 43 species. Conservative character analysis indicated that the G residue (shown in red) at the first position of intron 4 was a highly conserved splicing donor site.</p

Aberrant EXT2 splicing transcripts with premature termination codon.

<p>(A) Screenshot of a CRYP-SKIP output. The input EXT2 sequences included exon 4 (upper) and the flanking intronic sequences (lower). The table on the left lists the summarized values of the predictor variables used in CRYP-SKIP (PESS, putative exonic splicing silencers; NN 5'ss, neural network 5' splice sites; SF2/ASF, the most important SR protein for aberrant splice-site activation, FAS-ESS, ESSs discovered by a fluorescence-activated screen; EIE, exon and intron identity element). PCR-E (shown in light blue) for the mutated sequence is 0.23 in favor of exon skipping. The red vertical mark in the sequence indicates the predicted cryptic donor splice site. The blue vertical mark indicates the predicted acceptor site. (B) BDGP prediction. The red vertical mark indicates the authentic splice sites. The vertical mark indicates the predicted cryptic 5' splice sites with scores >0.90. The blue vertical mark shows the decoy splice site that was confirmed by PCR. (C) Electrophoresis of RT-PCR products derived from the proband, affected individuals, and the controls. Lane 1: DNA marker; Lanes 2, 4, 6: proband and cases; Lanes 3, 5: controls. (D) Direct sequencing of RT-PCR products. Arrow shows the heterozygous insertion of one cryptic splice site 5 bp downstream of the original splice donor site. (E) Clone sequencing of RT-PCR products. The mutant mRNA sequence (c.743+1G>A) has an ATAAG insertion (arrow) compared with the wild-type (WT). (F) Schematic representation of wild-type and aberrant mRNA transcripts. In the wild-type sequence (WT) splicing occurred at the authentic splice sites. In the mutant mRNAs, splicing occurred at the decoy splice site in intron 4 the five additional nucleotides (ATAAG) of intron4 were inserted, which generated the premature termination codon UAA.</p