Article Characterization of a New DGKE Intronic Mutation in Genetically Unsolved Cases of Familial Atypical Hemolytic Uremic Syndrome Caterina Mele, Mathieu Lemaire, Paraskevas Iatropoulos, Rossella Piras, Elena Bresin, Serena Bettoni, David Bick, Daniel Helbling, Regan Veith, Elisabetta Valoti, Roberta Donadelli, Luisa Murer, Maria Neunha¨userer, Matteo Breno, Ve´ronique Fre´meaux-Bacchi, Richard Lifton, Giuseppe Remuzzi, and Marina Noris Due to the number of contributing authors, Abstract the affiliations are Background and objectives Genetic and acquired abnormalities causing dysregulation of the complement provided in the alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by Supplemental thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a Material. substantial proportion of patients the disease-associated alterations are still unknown. Correspondence: Dr. Giuseppe Remuzzi, Design, setting, participants, & measurements Whole-exome and whole-genome sequencing were performed in IRCCS - Istituto di two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to Ricerche test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) Farmacologiche Mario protein was evaluated with western blotting. Negri, Centro Anna Maria Astori, Science fi and Technology Park Results Whole-exome sequencing analysis with conventional variant ltering parameters did not reveal any Kilometro Rosso, Via obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encoding Stezzano 87, 24126 DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, Bergamo, Italy. E-mail: allowing identification of a novel intronic DGKE mutation (c.888+40A.G) that segregated with disease. Se- giuseppe.remuzzi@ marionegri.it quencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause pro- found abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE-associated aHUS. Conclusions This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next- generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases. Clin J Am Soc Nephrol 10: 1011–1019, 2015. doi: 10.2215/CJN.08520814 Introduction membrane cofactor protein [MCP], and thrombo- Atypical hemolytic uremic syndrome (aHUS) is a rare modulin [THBD]) have been documented in nearly disorder resulting in thrombocytopenia, nonimmune 60% of patients (1–10). These findings paved the way microangiopathic hemolytic anemia, and acute kidney for complement-tailored treatments (1,11) that have led failure (1). It has a poor prognosis with approxi- to impressive improvements in short- and long-term mately 60% of patients progressing to ESRD and a prognosis (12). However, the underlying cause remains mortality rate between 4% and 25% (1,2). Extensive elusive for a substantial proportion of patients. The ad- studies showed that hyperactivation of the comple- vent of next-generation sequencing has resulted in ment alternative pathway is the main pathogenetic progress toward filling these knowledge gaps, allow- effector mechanism leading to endothelial damage ing for a rapid exome-/genome-wide search for path- and microvascular thrombosis in most patients with ogenic mutations (13). Recently, using whole-exome aHUS (1,2). Genetic and autoimmune abnormalities sequencing (WES), Lemaire and colleagues success- affecting complement proteins (complement factor fully identified recessive mutations in DGKE, encod- H [FH], factor H–related proteins [FHRs], factor I ing diacylglycerol kinase epsilon (DGKE), as a novel [FI], factor B [FB], complement component 3 [C3], cause of aHUS (14). Patients showed a specific clinical www.cjasn.org Vol 10 June, 2015 Copyright © 2015 by the American Society of Nephrology 1011 1012 Clinical Journal of the American Society of Nephrology phenotype characterized by onset in infancy, multiple re- aHUS was diagnosed at 10 months in the girl (no. 452) lapsing episodes, and nephrotic-range proteinuria (14). Be- and at 5 months in the boy (no. 1200). Both siblings had sides cobalamin C deficiency–associated aHUS (15), DGKE thrombocytopenia, hemolytic anemia with schistocytes on is the only other gene implicated in aHUS that does not blood smear, and renal impairment (Table 1). Mild pro- encode a complement component (16). teinuria and hypertension were also documented. C3 lev- Patients carrying DGKE mutations did not show con- els were lower than normal, and C4 was normal. Complete sumption of serum complement components, suggesting remission was achieved for both patients with supportive the existence of a novel pathogenetic mechanism for throm- therapy, which only included correction of anemia with botic microangiopathy (14). However, this assumption has packed erythrocytes and antihypertensive therapy. Both been challenged by the report of a DGKE-truncating muta- siblings had relapsing disease with one to three bouts a tion in two patients with moderate C3 consumption (17). year, often in concomitance with viral or bacterial infec- The recent finding of combined DGKE and complement tions (Supplemental Figure 1), without evidence of C3 con- gene mutations in three patients suggests that complement sumption. During relapses, they manifested renal dysregulation may have a role in modulating disease impairment (serum creatinine, no. 452: 1.4–1.7 mg/dl; severity in DGKE mutation carriers (18). Identification of no. 1200: 0.7–0.85 mg/dl), with hematuria and high-degree additional patients with DGKE mutations will be helpful proteinuria (.2 g/24 h). After every relapse, renal and he- to better characterize the phenotypic and prognostic het- matologic parameters returned to baseline with supportive erogeneity. therapy alone or with plasma infusion/exchange. From the Here, we report two families with recessive aHUS where age of 8 (no. 452) and 5 (no. 1200) years, the siblings received examination of DGKE variants obtained from WES and two plasma infusions a year as prophylaxis. Patient 1200 whole-genome sequencing (WGS) allowed identification had a relapse at 7 years and 3 weeks after plasma infusion. of a novel, intronic DGKE mutation that segregates with The relapse was treated with plasma infusion with prompt disease in both families and causes aberrant splicing of recovery. Thereafter, no further relapse occurred in both chil- DGKE transcripts. dren. At the last follow-up (age of no. 452, 13 years; age of no. 1200, 10 years), the siblings had mild persistent protein- uria with normal renal function (Table 1). Materials and Methods Sanger sequencing did not reveal any mutation in known aHUS was diagnosed on the basis of microangiopathic aHUS-associated genes (CFH, MCP, CFI, CFB, C3,and hemolytic anemia and thrombocytopenia defined by he- THBD)ortheCFH-H3 and MCPggaac risk haplotypes. matocrit ,30%, hemoglobin level ,100 g/L, serum lactate Multiplex ligation-dependent probe amplification analysis dehydrogenase level .460 U/L, undetectable haptoglobin, showed the presence of two copies of CFHR1 and CFHR3 fragmented erythrocytes in peripheral blood smear, and andexcludedgenomicCFH-CFHRs rearrangements. platelet count ,1503109/L, associated with acute kidney ELISA for anti-FH autoantibodies was negative. To iden- failure. tify the genetic basis of aHUS in this family, we performed Family 1 and 30 unrelated pediatric patients with aHUS WES coupled with homozygosity mapping (20). were recruited from the International Registry of HUS/ WES performed on patient 452 showed a 72X mean cov- Thrombotic Thrombocytopenic Purpura. Patient II-1 from erage over the targeted exons, with 95.1% of targets covered family 2 underwent clinical WGS as part of the Genomics at an average depth of 4X or higher. Variant detection iden- Medicine Clinic, in collaboration with Children’s Hospital tified 30,267 single nucleotide variants (SNVs) and 1137 of Wisconsin and Froedtert Hospital (19). Twenty unrelated insertions/deletions. After excluding variants with minor French patients with pediatric aHUS undergoing genetic allele frequency (MAF) .1%, 4507 candidates remained. screening in Paris were included in this study. Among those, 143 (131 SNVs and 12 insertions/deletions) Peripheral blood samples were collected for DNA, RNA, were homozygous protein-altering variants (nonsense, mis- and protein isolation. Samples from 89 Italian healthy sense, or affecting canonical splice sites). We focused on persons were analyzed as controls. identifying autozygous mutations because the affected sib- The study was approved by the Ethics Committee of the lings are from a consanguineous family and are likely to Azienda Sanitaria