Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next‐generation sequencing

Abstract Essentials The differential diagnosis of acute thrombotic microangiopathy (TMA) is challenging. To the ADAMTS13 activity < or >10% was added a next‐generation sequencing (NGS) gene panel. The ADAMTS13 mutation p.Cys754Arg was frequent in hereditary thrombotic thrombocytopenic purpura. We identified novel complement gene mutations and this procedure improved our diagnostic strategy. Background The 2 main forms of thrombotic microangiopathy (TMA) are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). Deficiency of ADAMTS13 and dysregulation of the complement pathway result in TTP and aHUS, respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging. Objectives and Methods We aimed to develop a TMA diagnosis workflow based on ADAMTS13 activity and screening of ADAMTS13 and complement genes using a custom next‐generation sequencing (NGS) gene panel. Patients For this, from a cohort of 154 Portuguese patients with acute TMA, the genotype‐phenotype correlations were analyzed in 7 hereditary TTP (ADAMTS13 activity <10%, no inhibitor), 36 acquired TTP (ADAMTS13 activity <10%, presence of an inhibitor), and in 34 presumable aHUS. Results In total, 37 different rare variants, 8 of which novel (in ADAMTS13,CFH, and CD46), were identified across 7 genes. Thirteen TTP patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1‐3 deletion. Conclusions Our study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMA, and is a valuable asset in clinical practice to discriminate between TTP and aHUS.


| INTRODUCTION
Thrombotic microangiopathies (TMAs) are characterized by microvascular occlusion, thrombocytopenia, and non-immune hemolytic anemia. The 2 main forms of TMAs are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). These 2 types of TMA have overlapping clinical phenotypes. Conceptually, in TTP, neurological manifestations are more common, whereas, in aHUS, renal involvement is more pronounced. However, this rule is not always reliable; some aHUS patients have neurological complications and some TTP patients have renal failure. [1][2][3][4] Over the last decade, noteworthy progress has been made to improve the clinical and laboratory approaches to predict the expected signs and symptoms, patient outcome, and genotype-phenotype correlations. Despite this, the differential diagnosis of TTP/aHUS remains challenging.
TTP is caused by a severe plasma deficiency (i.e, <10% of the normal) of the cleaving protease of von Willebrand factor (VWF)-ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). A severe ADAMTS13 deficiency is more often due to anti-ADAMTS13 autoantibodies (acquired) or rarely due to homozygous or compound heterozygous ADAMTS13 gene mutations (hereditary, also called Upshaw-Schulman syndrome). 5,6 However, heterozygosity for ADAMTS13 mutations was observed among patients diagnosed with acute acquired TTP and severe ADAMTS13 deficiency. 7,8 In all forms, the predominant pathogenic factor of TTP is the increased number of ultra-large multimers of VWF resulting from the lack of proteolytic regulation by ADAMTS13 and leading to platelet clumping in the microcirculation of various organs. 6 TTP is mainly an autoimmune disorder associated with severe, acquired ADAMTS13 deficiency with the annual incidence rate of 2.17×10 6 people per year, although hereditary TTP accounts for <5% of all ADAMTS13 deficient TTP cases, 9 except in certain geographic locations, e.g, Central Norway, where hereditary TTP seems to be more common than the acquired form. 10 While TTP is characterized by the severe deficiency of ADAMTS13, aHUS is characterized by hyperactivation of the alternative complement pathway resulting from either a loss-of-function mutation in a regulatory gene (CFH, CFI, CD46 (MCP), or THBD) or a gain-of-function mutation in an effector gene (CFB or C3). 1,11 The mutations were mainly found in the heterozygous state, and approximately 5% of patients have combined mutations, usually in CFH with either CD46 or CFI. Homozygosity for risk haplotypes of CFH (rs3753394, c.1-332C>T and rs1065489, c.2808G>T, p. Glu936Asp) that tag the disease risk haplotype CFH-H3 and 1 polymorphism in MCP (rs7144, c.*897T>C) that tags the MCPggaac risk haplotype have been shown to significantly increase disease penetrance and severity. [12][13][14] Additional genetic risk factors include a deficiency of CFHrelated proteins 1 (CFHR1) and 3 (CFHR3), due CFHR1-3 deletion in homozygous state caused by non-allelic homologous recombination of CFHR3 and CFHR1. 15,16 Finally, recessive mutations in DGKE, which encodes diacylglycerol kinaseε and is expressed in endothelial cells, platelets, and podocytes, were identified in children with the onset of aHUS in the first year of life. 17 However, detectable complement abnormalities have been described in only approximately 50%-60% of patients; therefore, the aHUS diagnosis is based on clinical criteria and the exclusion of a severe ADAMTS13 deficiency. 18 Poor penetrance is a common clinical feature, and adult onset occurs in patients with severe deficiencies. In addition, both deficiencies predispose patients to a TMA after a triggering event, such as pregnancy, bacterial and viral infections, neoplasia, autoimmune disorders, and exposure to certain drugs. [19][20][21] Severe ADAMTS13 deficiency (<10%) is an important indicator in the differential diagnosis of TTP/aHUS. However, several studies in aHUS patients have shown reduced ADAMTS13 activity that may predispose patients to the TMA phenotype. 22,23 Indeed, ADAMTS13 single-nucleotide polymorphisms (SNPs) associated with partial deficiency of ADAMTS13 in the presence of a primary trigger or when co-inherited with a mutation in a complement gene have been described in these patients. 23 These findings suggest that TTP and aHUS exhibit overlap not only in their clinical characteristics but also in their pathophysiological mechanisms.
To better understand this clinical variability, which is found even within families, it is necessary to characterize the mutational profile.
Nevertheless, up to now the molecular analysis of genes implicated in TMA was not affordable by diagnostic laboratories because of the high cost involved in the study of multiple genes by conventional Sanger DNA sequencing. The advent of next-generation sequencing (NGS) is changing this paradigm because NGS allows simultaneously sequencing of large gene panels and generates competitive results at a lower cost and in a shorter amount of time.
From this perspective, we conducted a study with 2 main objectives. First, to better understand the genotype-phenotype correlations, we detailed phenotypic characterization in our cohort of patients with TMA who had mutations in ADAMTS13 and/or in complement genes.
Second, we designed and validated an NGS-based gene panel to facilitate genetic testing in TTP and aHUS. This approach allowed the implementation, within our Department of Hematology, of an efficient methodology to establish the diagnosis and prognosis of these 2 rare diseases that exhibit phenotypic similarities.

Essentials
• The differential diagnosis of acute thrombotic microangiopathy (TMA) is challenging.
• To the ADAMTS13 activity < or >10% was added a next-generation sequencing (NGS) gene panel.
• The ADAMTS13 mutation p.Cys754Arg was frequent in hereditary thrombotic thrombocytopenic purpura.
• We identified novel complement gene mutations and this procedure improved our diagnostic strategy. The child probands (n=30) had a median age of 9 years with a range of 1-18 years, and infants included in this study (n=7) ranged in age from 1 week to 11 months.

| Patients and controls
Forty-two healthy volunteers acted as a control group for ADAMTS13 assay measurements and were also investigated for gene variants with the NGS panel. The control group included unrelated individuals (32 females and 10 males) without an individual or family history of excessive bleeding, thrombosis or hemolytic anemia, with a mean age of 33.5±11.12 years.
We also investigated 10 asymptomatic relatives of 5 patients in whom an ADAMTS13 mutation had been identified. In accordance with the Declaration of Helsinki, informed consent was obtained from all patients or from their family members and from the healthy controls.

| Samples and sample processing
ADAMTS13 assays were performed on blood collected into vacuum tubes containing 3.2% sodium citrate (at a ratio of 9:1 vol/vol) and centrifuged within 15 minutes at room temperature for 20 minutes at 2500 g. The obtained platelet-poor plasma was then separated into aliquots and kept frozen at −80°C until further use. Genomic DNA was extracted from EDTA whole blood by automatic isolation on an iPrep instrument using a gDNA Blood Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The DNA concentration was adjusted to a range of 25-50 ng/μL.

| Strategy for mutation analysis
Two different direct sequencing methodologies were used for the mo- Regarding to comparison between laboratories on the basis of coverage and diagnostic yield of NGS, these samples were analyzed to ADAMTS13 ROIs using other NGS panel, as described in the Supplementary Materials.

| Library preparation and NGS
The sequencing libraries were prepared using the AmpliSeq Library

| Bioinformatic analysis
The NGS pipeline output, after QC/QA filtering, was analyzed using Torrent Suite (v3.6; Thermo Fisher Scientific), and sequences were aligned to the human genome version 19 (hg19) using T-MAP (version 3.6.58977). Variants were called using the Torrent Variant Caller (version 3.6.59049, with Germ Line -Low Stringency settings) and annotated using Ion Reporter (Thermo Fisher Scientific).
The workflow procedure of in silico analysis, the genetic databases consulted for validation of rare variants, the criteria for assessment of the pathogenic mutations, and, finally, their validation by Sanger sequencing were in accordance with those previously described in our recent work 26,27 and explained in detail in the Supplementary Materials.

| Characteristics of the patients studied
The present study reviewed 146 patients from different regions of Portugal for whom ADAMTS13 activity levels were measured; 60 patients had a level of ≤10% (severe deficiency) with a residual activity sus) that were not included in the present study. However, it would be highly interesting to see whether complement mutations are in fact more common in aHUS cases than in the other TMAs, as previously described in patients with HELLP syndrome. 28 Considering this, the molecular study of this last patients group is currently in progress and the results will be presented in the near future.
Based on these findings, 3 groups of patients were selected for this analysis (n=77) and underwent a detailed phenotypic study and mutational analysis: (i) hereditary TTP patients (n=7); (ii) acquired TTP patients (n=36), 4 of which have had ADAMTS13 activity of ≈40% after an acute episode, but no detected anti-ADAMTS13 IgG antibodies; and (iii) aHUS patients (n=34) (Figure 1). The demographic, laboratory, and clinical data are summarized in Table S1.

| Phenotype-genotype analysis in TTP patients
Four putative pathogenic ADAMTS13 variants (1 novel) were identified in the 7 hereditary TTP patients of this cohort (5 with disease onset in childhood and 2 with disease onset in pregnancy): 6 patients were homozygous and 1 patient was compound heterozygous (Table 1; Figure 2). One pathogenic mutation, p.Cys754Arg, occurred repetitively and knowing that ABO blood group gene locus lies in the close neighborhood of the ADAMTS13 gene on chromosome 9q34, the blood groups were analyzed, which revealed that all patients homozygous for this mutation were of genotypic blood group 0 1 0 1 (Table 1). Likewise, the analysis of 12 frequent SNPs in ADAMTS13 showed that the most prevalent pathogenic mutation (c.2260T>C, p.Cys754Arg, chr=9) were associated with the same haplotype (Table   S4). The fact that the 9 mutation-carrier chromosomes are in com-  Figure 2). The ADAMTS13 rare variants found in these 2 acquired TTP groups, albeit a small number of patients analyzed, differed significantly (Fisher exact test, P<.001) ( Figure S1).
Of the 11 complement genes studied, only 4 rare benign variants in CFI (n=3) and CFB (n=1) were identified in 4 TTP patients (Table S3). Therefore, these data indicate that the phenotypic variability in these TTP patients was not possibly explained by complement gene variants.
The detailed phenotype-genotype analysis of patients P1-P11 and the family pedigrees (Figure 2A, B) are presented in the Supplementary Results.

| Phenotype-genotype analysis in aHUS patients
In 17 out of 34 (50%) aHUS patients' mutations were identified, 13 patients had mutations in heterozygous state and 4 patients were compound heterozygous. Two patients presented 3 variants in a single gene (CFH) and the remaining 2 patients had combined variants in 2 different complement genes (C3/CFI and C3/CD46). In total, 23 rare variants (7 non-previously described) were identified in 6 complement genes that were located mostly in CFH (12), with 4 in CD46, 4 in C3, 1 in CFI, 1 in CFB, and 1 in CFHR5 (Table 2; Figure 2). Eleven variants were putative pathogenic, 3 likely were pathogenic, 1 was of uncertain significance, 3 were benign, and another 4 were likely benign (Tables 2 and S3). The detailed phenotype-genotype analysis of these 17 patients is presented in the Supplementary Results. In the remaining 17 aHUS patients, although they also present a phenotype according to an overactivation of the alternative complement pathway, no rare genetic variants were found.
Considering all the aHUS patients carrying single or combined complement gene variants, we found that 11/17 (65%) were heterozygous for at least 1 pathogenic mutation and the 3 patients who have had relapses were all carrying pathogenic mutations (CFH, CD46, and C3). On the other hand, however, our data suggest that patients carrying non-pathogenic variants co-inherited more genetic risk factors: patient P12 carrying a single benign CD46 variant and the patient P9 with a single likely pathogenic CFHR5 variant were homozygous for the CFHR1-3 deletion and the patient P14 carrying a single benign C3 variant was homozygous for a block of 4 ADAMTS13 SNPs. These additional genetic risk factors (with impact in the activity and secretion of ADAMTS13 and risk haplotypes of complement genes that significantly increased disease penetrance) could have a cumulative or ADAMTS13 variants ADAMTS13 SNPs mod.

CFHR1-3 alleles Nucleotide Change
Amino Acid Change VC e Zygosity Ref.
c.2074C>T p.Arg692Cys P Homozygous [29] p.  Figure 2). However, these data can only be speculative given the small number of patients involved.

| DISCUSSION
We developed a workflow based on ADAMTS13 activity and screening for genetic TMA susceptibility factors using a successfully de- In agreement with previous studies, only 50% of patients diagnosed with aHUS have rare variants in the complement genes. 47,48 These were predominantly loss-of-function variants in the com- activity was described as a risk factor for the aHUS phenotype. 23 The analyses of ADAMTS13 in our aHUS cohort revealed only common variants (SNPs): Gln448Glu, the most prevalent (20%) and p.Arg7Trp, p.Gln448Glu, p.Pro618Ala, and p.Ala732Val, co-inherited in 1 patient.
The Gln448Glu variant alone had little impact but, conversely, the block of these 4 SNPs has been shown to have a higher impact on ADAMTS13 activity. 39 Nevertheless, this NGS study ended with 17 (50%) genetically unresolved aHUS patients, as described in others aHUS studies. 14,48 Therefore, the expectation that the whole-exome sequencing approaches could identify other genes involved in aHUS is high; however, these studies still remain under investigation. 51 Conversely, NGS-targeted gene panels are being introduced into clinical practice providing substantial benefits for definitive diagnoses in hematological diseases. 26,27,52 The prediction of pathogenicity of the genetic variation 53 has become crucial for understanding the great inter-individual variability.
Therefore, NGS-targeted gene panels have changed the paradigm of routine molecular studies. In the face of the multiple genetic changes found in every patient, the critical challenge was discriminating disease-associated variants from the broader background of variants present in all patients' genomes. [51][52][53][54][55] In conclusion, the study of these 77 TMA Portuguese patients contributes to the better understanding of the molecular genetics of ADAMTS13/complement gene-related phenotypes. Moreover, our study provides evidence of the usefulness of the NGS panel as an excellent advantageous technology that enables more rapid and economic diagnosis of TMA. These findings show that this is a valuable asset in clinical practice given that a correct diagnosis is essential for determining the most effective treatment for each patient with this complex disease.

ACKNOWLEDGMENTS
We are grateful to the Forum Hematológico for the funding for this pro-

RELATIONSHIP DISCLOSURES
None of the authors have any disclosures relevant to this paper.