Comprehensive genetic analysis of complement and coagulation genes in atypical hemolytic uremic syndrome

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy caused by uncontrolled activation of the alternative pathway of complement at the cell surface level that leads to microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. In approximately one half of affected patients, pathogenic loss-of-function variants in regulators of complement or gain-of-function variants in effectors of complement are identified, clearly implicating complement in aHUS. However, there are strong lines of evidence supporting the presence of additional genetic contributions to this disease. To identify novel aHUS-associated genes, we completed a comprehensive screen of the complement and coagulation pathways in 36 patients with sporadic aHUS using targeted genomic enrichment and massively parallel sequencing. After variant calling, quality control, and hard filtering, we identified 84 reported or novel nonsynonymous variants, 22 of which have been previously associated with disease. Using computational prediction methods, 20 of the remaining 62 variants were predicted to be deleterious. Consistent with published data, nearly one half of these 42 variants (19; 45%) were found in genes implicated in the pathogenesis of aHUS. Several genes in the coagulation pathway were also identified as important in the pathogenesis of aHUS. PLG, in particular, carried more pathogenic variants than any other coagulation gene, including three known plasminogen deficiency mutations and a predicted pathogenic variant. These data suggest that mutation screening in patients with aHUS should be broadened to include genes in the coagulation pathway.

Figure 1.

Diagnostic age distributes similarly in male and female aHUS patients. Although a trend toward higher diagnostic age was observed in women, this difference was not significant (Wilcoxon rank sum test P=0.067).

Figure 2.

Application of hard filtering uncovers pathogenic variants. Variants were filtered by focusing on nonsynonymous, frame shift, and indel changes. Rare variants are defined as variants with a minor allele frequency<1% in European-American and African-American populations based on data from the EVS database. Predicted deleterious mutations or rare variants are stop-gain mutations, indels, or missense variants with a high pathogenicity score (PS) (>4). PS for missense variants was calculated using PhyloP, SIFT, PolyPhen2, LRT, MutationTaster, and GERP++, and it ranged from zero to six. The mean PS of reported DRVs was 3.41; the mean PS of predicted deleterious missense variants was 4.57. *DRVs have been previously reported to be disease-causing or -associated. DRVs may or may not be recorded in public single nucleotide polymorphism databases. QC, quality control; UTR, untranslated region.

Figure 3.

Distribution of reported and predicted deleterious variants per gene implicates PLG in aHUS pathogenesis. The second greatest number of deleterious variants was identified in PLG. This enrichment of deleterious variants is statistically significant (P<0.05). A detailed description of the variants is given in Supplemental Table 3.

Figure 4.

Copy number of complement factor H related (CFHR) genes negatively correlates with number of deleterious variants in aHUS patients. Less frequent copy number variation of CFHR genes was observed in patients with more deleterious variants. nsRV, deleterious nonsynonymous rare variant; hom, homozygous; het, heterozygous; del, deletion; dup, duplication.

Figure 5.

Deleterious variants of PLG are conserved. The four identified deleterious variants in PLG localize to different domains of plasminogen. The amino acids are highly conserved across multiple species, suggesting that these variants may impair normal function of the protein.

Figure 6.

Modified model implicates dysregulation of both the complement and coagulation pathways in the pathogenesis of aHUS. This model suggests three stages in the development of aHUS. (1) The disease is initiated by trigger events, such as nonenteric bacterial infections, viruses, drugs, malignancies, transplantation, pregnancy, and medical conditions like scleroderma, antiphospholipid syndrome, and systemic lupus erythematosus. The complement cascade is activated, and/or the coagulation pathway leads to thrombus formation. (2) A dysregulation loop develops, which is driven by overactivation of complement that causes endothelial cells stimulation and lysis. Lysis leads to thrombi formation, with altered function of the coagulation pathway caused by genetic variants in coagulation genes, such as PLG, PLAT (encoding tissue plasminogen activator, the main plasminogen activator), PLAU (encoding urokinase plasminogen activator, primarily responsible for pericellular plasmin activation), F12, ADAMTS13, and VWF. The consequence is enhanced formation or decreased degradation of thrombi, promoting thrombosis. Thrombi in small vessels cause mechanical damage to erythrocytes releasing peptides (such as heme) or overexpression of other unidentified factors that activate the complement system. Genetic variants in complement genes and/or acquired factors like CFH autoantibodies lead to inadequate/ineffective complement regulation. (3) This dysregulation loop induces the clinical onset of aHUS.