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 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 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 (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 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 Locale, Bergamo, Italy, and the Institu- have two recessive disease alleles inherited from a common tional Review Boards at the Medical College of Wisconsin ancestor. Homozygosity mapping on the basis of WES data and Yale University School of Medicine. Informed consent were used to identify all autozygous blocks, defined as was obtained from participants or by their parents accord- runs of homozygosity of at least 1.5 Mb (20). This analysis ing to the Declaration of Helsinki. yielded seven uninterrupted homozygous regions across Detailed description of materials and methods are reported the genome (Supplemental Table 1), which collectively in the Supplemental Material. spanned 77.6 Mb (approximately 2.7% of the genome), therefore confirming parental consanguinity at the level of second cousins (inbreeding coefficient, F=1/64). Within Results the homozygous blocks, we detected three missense var- Identification of a Novel Homozygous Intronic DGKE iants in WDR6, UQCRC1, and ZMYND10, whose segrega- Mutation in Family 1 tion patterns were consistent with recessive inheritance We studied a consanguineous family (family 1, Figure (Supplemental Table 2). Only the WDR6 variant was not 1A) from the North of Italy (South Tyrol) with two affected present in public databases (dbSNP137, 1000 Genomes Pro- siblings (including a 13-year-old girl and 10-year-old boy ject, and National Heart, Lung, and Blood Institute Exome at present) whose parents are healthy second cousins. Variant Server). However, none of the three segregating Clin J Am Soc Nephrol 10: 1011–1019, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 1013

Figure 1. | Family trees. Pedigrees of family 1 (A) and family 2 (B) carrying DGKE mutations. Square symbols represent male family members, and circles represent female family members. Consanguineous unions are represented by double horizontal lines. Black-filled symbols rep- resent affected individuals, and white symbols represent unaffected individuals. Slashes represent deceased individuals. Black-filled triangles represent the DGKE c.888+40A.G mutation. Empty triangles represent the DGKE c.966G.A mutation. wt, wild-type allele. variants appeared to be convincingly causative for aHUS, Characterization of the Effects of c.888+40A>G on DGKE and none were predicted to be damaging. Transcript and Protein Meanwhile, the article by Lemaire and colleagues (14), doc- DGKE is expressed in peripheral blood leukocytes (23). To umenting the association of DGKE mutations and aHUS, was assess the effects of the c.888+40A.G mutation at a tran- published. Similarities concerning disease inheritance mode scriptional level, we obtained peripheral blood samples from (recessive), age of onset (,1 year), and clinical phenotype the siblings and parents from family 1 and from a healthy (recurrent disease) among our patients and the ones de- unrelated Italian control. RNA analysis was performed by scribed by Lemaire et al. (14) led us to reanalyze in detail RT-PCR using primers spanning exons 4–6 of the DGKE WES data around the DGKE . We found a homozygous transcript. Electrophoresis of the amplification products stretch, including eight SNVs spanning the DGKE locus (Sup- showed that the wild-type amplicon (273bp) found in the plemental Table 3), whose length (approximately 1.4 Mb), control was absent in both affected children (Figure 3A). In- however, was just under the 1.5 Mb cutoff used. Among stead, their cDNA exhibited three additional bands with mo- those SNVs, seven had MAF.1%; the eighth was a novel lecular weights higher than that of the wild-type. The band homozygous intronic variant (NM_003647.2: c.888+40A.G, corresponding to an approximately 300 bp amplicon (mu- Figure 2A), which had been filtered out by computational tant isoform 1) was more prominent than the other two, pipeline parameters. This variant is located in intron 5 and is measuring approximately 400 and 650 bp, respectively (mu- absent from public databases. Sanger sequencing confirmed tant isoforms 2 and 3, Figure 3A). Parental samples revealed that the c.888+40A.G variant segregates with disease, with the wild-type and mutant amplicons. a recessive pattern: both affected siblings are homozygous, The nucleotide sequences of each isoform were obtained and both healthy parents are heterozygous (Figure 2A). In after extraction of the bands from the agarose gel. Mutant addition, the c.888+40A.G mutation was not found in 178 isoform 1 (312 bp) results from the retention of 39 intronic from adult Italian controls. nucleotides that immediately follow exon 5 (c.888_889ins39, Analyses with GenScan software (21) predicted that in the Figure 3B). This confirms in silico prediction that the neo-splice mutant sequence the probability of the correct splicing of donorsitecreatedbythemutationisusedinsteadoftheweak exon 5 decreases from 0.999 to 0.118 versus the wild-type canonical splice donor site of exon 5. Isoform 1 transcript is sequence (Figure 2B). The presence of a longer exon 5 (exon predicted to yield a DGKE protein 13 amino acids longer than 5*, Figure 2B) was predicted with a probability of 0.882. the wild-type (p.Lys296_Gly297ins13, Figure 4). Isoform 2 (407 Additional analyses with Human Splicing Finder software bp) differs from isoform 1 because it also includes a 95-bp- (22) predict that this mutation creates a gt cryptic splicing long pseudoexon (c.888+278–372) within intron 5 (Figure 3B) donor site within intron 5, with a score higher than the ca- because of the recognition of cryptic splice acceptor and donor nonical donor site (Figure 2B). This change introduces new sites at positions 276–277 and 373–374 bp, respectively, down- exonic splicing enhancer motifs for SF2/ASF, SRp40, and stream of exon 5 (Supplemental Table 4). The resultant aber- SF2/ASF (IgM-BRCA1) that are predicted to alter the nor- rant mRNA (c.888_889ins134) is predicted to yield a truncated mal behavior of the splicing regulatory proteins that process DGKE protein because of a frameshift (p.Gly297Valfs*88, Fig- DGKE pre-mRNA molecules (Supplemental Figure 2). ure 4). This transcript is, however, likely to be degraded by 04Ciia ora fteAeia oit fNephrology of Society American the of Journal Clinical 1014

Table 1. Clinical characteristics of patients with DGKE mutations

Family 1 Family 2

Patient 452 Patient 1200 Patient II-1 Patient II-2 Patient II-4 Characteristic Last Last Last Last Last Onset Onset Onset Onset Onset Follow-Up Follow-Up Follow-Up Follow-Up Follow-Up

Age (y) 0.8 13 0.4 10 0.75 13 0.6 10 0.4 4 SCr (mg/dl) 4.17 0.58 0.7 0.48 0.7 0.68 7.3 0.45 5.3 0.29 Platelet count (3109/L) 63 339 96 310 85 — 34 — 122 — Hemoglobin (g/dl) 10.7 15.2 10.9 14.1 9.8 — 8 — 8.3 — LDH (IU/L) 4000 — 908 — 3396 — 10,181 ——— Total bilirubin (mg/dl) 1.15 — 1.4 ——————— Schistocytes Positive — Positive — Positive — Positive — Positive — C3 (mg/dl) 70 147 81 151 ————Normal — C4 (mg/dl) 20 33 21 28 ————Normal — Proteinuria (g/24 h) 0.41 0.57 0.57 0.21 +++a,b ++a — +++a,b — +a Prot/Creat ratio — 1.53 — 0.44 —————— Hematuria +a Negative +a Negative +++a,b +++a,b — +++a,b — Traces

Normal values are as follows: serum creatinine: children ,1–5 years, 0.3–0.5 mg/dl; children 5–10 years, 0.5–0.8 mg/dl, children .10 years, 0.5–1.2 mg/dl. Platelet count: 150–400 3109/L. Hemoglobin: children 5 months to 1 year, 10.8–12.5 g/dl; children 1–5 years, 11.7–13.7 g/dl; children 5–10 years, 12–14.4 g/dl; female .10 years, 12–16 g/dl; male .10 years 14–18 g/dl. LDH: children ,1–5 years, 125–206 IU/L; children 5–10 years, 104–201 IU/L; children 10–15 years, 90–199 IU/L. C3: 90–180 mg/dl. C4: 10–40 mg/dl. Proteinuria in children , 0.2 mg/mg of protein/ creatinine ratio in spot urine or , 0.2 g/24 h proteinuria or negative dipstick. Nephrotic range proteinuria is defined as protein/creatinine ratio .2 mg/mg or proteinuria .3.5 g/24 h. SCr, serum creatinine; LDH, lactate dehydrogenase; C3, serum complement C3 levels; C4, serum complement C4 levels; Prot, proteinuria; Creat, creatinine; +, mild level; ++, moderate level; +++, severe level. aDipstick was used for this evaluation. bStrongly positive result. Clin J Am Soc Nephrol 10: 1011–1019, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 1015

Figure 2. | Identification of the DGKE c.888+40A>G mutation in family 1. (A) Representative Sanger sequencing electropherograms of DGKE c.888+40A.G mutation in individuals from family 1 and healthy controls. The affected children from family 1 (patients 452 and 1200) are ho- mozygous for the A→G substitution (arrow), and their unaffected parents (nos. 1417 and 1421) are heterozygous. The lowermost electropherogram is from a homozygous wild-type (wt) healthy control. (B) wt and mutant (mut) genomic nucleotide sequence of DGKE around intron 5. The position of the c.888+40A.G mutation is marked in red. The green lines trace the sequences of exon 5 and 6 in the wild-type and mutant DNA, with the corresponding probabilities predicted by the GenScan software. The orange line traces the canonical splice donor site in the wild-type sequence and the predicted new splice donor site in the mutant sequence, with the corresponding scores calculated by the Human Splicing Finder software. nonsense-mediated mRNA decay, therefore explaining the on the basis of WGS performed on patient II-1. Despite lower intensity of the corresponding band versus the isoform excellent coverage (96% of the reference genome at an av- 1 band. Isoform 3 (645 bp) results from skipping both the erage depth of 33X), the initial data assessment did not canonical and neo-splice donor sites: the splicing machinery reveal a clear molecular diagnosis (19). A previously de- only recognizes the cryptic splice donor site that defined the scribed (14) heterozygous DGKE nonsense mutation, pseudoexon of isoform 2 (Figure 3B) (24). Notably, this site c.966G.A (p.Trp322*), was found in patient II-1 by WGS has the highest consensus value for all splice donor sites and was subsequently confirmed by Sanger in all relatives within intron 5 predicted by the Human Splicing Finder soft- except the mother (Supplemental Figure 4A). A more in- ware (Supplemental Table 4). Isoform 3 is characterized by in- depth analysis of all potential maternal-in-origin DGKE frame retention of 372 intronic nucleotides (c.888_889ins372, variants was therefore performed. No other coding, nonsyn- Figure 3B), which would result in a protein with 124 addi- onymous variants were identified, and there was no struc- tional amino acids (p.Lys296_Gly297ins124, Figure 4A). These tural variation within or near DGKE. Out of 52 intronic DGKE results suggest that in the presence of the c.888+40A.Gmu- variants, only eight were rare (MAF,1%, Supplemental tation, the weak canonical splice donor site of exon 5 is ig- Figure 4B). All were sequenced in the parents to find those nored by the spliceosome, resulting in the production of at originating from the maternal lineage. Genotyping of the least three aberrant transcripts. three affected (nos. II-1, II-2, and II-4, Figure 1B) and one Western blot of blood leukocyte lysates documented that unaffected sibling (no. II-3) for the two maternal-specific isoform 1 is expressed at the protein level, whereas no variants (Supplemental Table 5) revealed that only the af- signal was found for isoform 3 (Figure 4B). fected individuals harbored both noncoding variants in Exon 5 encodes part of the catalytic domain of DGKE compound heterozygosity with the c.966G.Amutation (Figure 4A) (23). Structural tridimensional modeling sug- (Supplemental Figure 4C). The first variant (c.888+40A.G) gests that the 13 amino acid insertion in isoform 1 should appeared more promising and was the same found in homo- result in structural changes that may affect DGKE kinase zygosity in family 1. The other (rs145743671) has an MAF of activity (Supplemental Figure 3 and Supplemental Material). 0.55% in control individuals and is .700 bp away from exons 9 and 10. Identification of a Second Family with the DGKE c.888 The three affected siblings, born to an outbred union, were +40A>G Mutation diagnosed with aHUS in early infancy. In individual II-1, A second unrelated kindred (family 2, Figure 1B) with aHUS was diagnosed at 9 months. He presented with throm- the same intronic mutation was identified independently bocytopenia, Coombs’ negative hemolytic anemia with 1016 Clinical Journal of the American Society of Nephrology

Figure 3. | DGKE c.888+40A>G mutation causes an aberrant splicing of exon 5. (A) Agarose gel of the RT-PCR on cDNA obtained from peripheral blood leukocytes of homozygous mutated patients from family 1 (patients 452 and 1200), their heterozygous healthy parents (nos. 1417 and 1421), and a homozygous wild-type (wt) healthy control (CTR). Amplification products were obtained using primers (shown as red arrows in panel B) spanning DGKE exons 4–6. Only one band of 273 bp (wt isoform) was obtained from the healthy control individual, whereas a strong band (mutant isoform 1, approximately 310 bp long) and 2 weak bands of higher molecular weight (mutant isoforms 2 and 3, ap- proximately 400 and 650 bp long, respectively) than the wt amplicon were present in the cDNA of both patients. The heterozygous healthy parents showed both the wt and the aberrant longer amplicons. (B) Diagrams of the normal (wt) and aberrant (mutant isoform 1 [Iso#1], mutant isoform 2 [Iso#2], mutant isoform 3 [Iso#3]) splicing of exon 5. The mutant Iso#1 is characterized by the retention of 39 nucleotides of intron 5. The mutant Iso#2 differs from Iso#1 for the further inclusion of a 95-bp-long pseudoexon from intron 5. The mutant Iso#3 is characterized by the retention of 372 nucleotides. The position of the c.888+40A.G is marked by a broken vertical red line. schistocytes, and mild renal impairment (Table 1). Marked DGKE c.888+40A>G is a Founder Mutation that is Rare in hematuria and proteinuria were also documented. He had aHUS four relapses in the following year and a fifth relapse at 4 years Because two unrelated families of European descent have (Supplemental Figure 4D, Supplemental Table 6). During the the exact same novel intronic variant, we reasoned that this second and third relapses he had urine protein/creatinine may be caused by a rare founder mutation. We cross-referenced ratios of 46 and 19 mg/mg, respectively (normal ,0.2 mg/mg). the list of the homozygous variants around DGKE locus in Treatment was supportive for all episodes, and peritoneal the WES data for individual 452 (Supplemental Table 3) dialysis was only prescribed for the last one. At last follow- and found that all were heterozygous in the WGS data for up (age 13 years), renal function, BP, and complement ac- patient II-1. This concordance suggests a common origin for tivity (CH50 and AH50) were normal, but hematuria and the c.888+40A.G mutation in the two families. Sequencing proteinuria were persistent (Table 1). Individuals II-2 and done to assess the frequency of the c.888+40A.G allele in 50 II-4hadaHUSonsetat7and5months,respectively(Table1), patients with pediatric-onset aHUS without a molecular di- associated with acute renal failure. C3 and C4 levels, mea- agnosis did not identify a single carrier. sured in individual II-4 during the acute phase, were normal. Individual II-2 had two relapses both within 1 year of di- agnosis, whereas II-4 had three relapses, all before 4 years of Discussion age (Supplemental Figure 4D, Supplemental Table 6). Both We report on the identification of a novel intronic DGKE patients required peritoneal dialysis only during the first mutation, c.888+40A.G, that interferes with normal mRNA episode. Nephrotic-range proteinuria was documented dur- splicing in two unrelated multiplex kindreds with pheno- ing relapses in individual II-4 (urine protein/creatinine ratio types consistent with DGKE-associated aHUS. The pathoge- 21.2–34.4) and at remission in individual II-2 (urine protein/ nicity of c.888+40A.G is supported by several lines of evidence. creatinine ratio 2.6–8.8). Renal function, BP, CH50, and First, this mutation cosegregates with disease following a AH50 were normal for both patients at last follow-up, at recessive pattern in both families: it is homozygous for ages 10 and 4 years, respectively (Table 1). Both parents family 1 and compound heterozygous for family 2 in con- and a 6-year-old sister are unaffected. The father is of Northern cert with a known nonsense DGKE mutation (14). Second, European descent, and the mother is half-German, half–Native the probability of having by chance two unrelated kindreds American (Cherokee). with phenotype concordant with that of DGKE-associated Clin J Am Soc Nephrol 10: 1011–1019, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 1017

Figure 4. | Sequence changes identified in the three mutant DGKE isoforms. (A) Schematic representation of the domains identified in DGKE protein (from SMART, http://smart.embl-heidelberg.de) is illustrated (upper part of panel A) to show the relative position of the changes. In the lower part of panel A we present the sequences of bases and translated amino acids of wt DGKE protein to allow for comparison with those of the three mutant DGKE forms caused by the c.888+40A.G. The subscripts below the amino acids located at the beginning and the end of each exon are used to reveal their position relative to the wt protein. The sequence of exons 4, 5, and 6 are labeled in orange, green, and gray fonts, respectively. The red font is used to show the sequences that are unique to the three mutant DGKE proteins. (B) Protein blot of total proteins extracted from peripheral blood leukocytes. Lane 2: leukocytes from a healthy control; lane 3: leukocytes from patient 1200; lane 4: leukocytes from his mother (no. 1421) probed with an anti-DGKE antibody against the 2–51 N-terminal amino acids. Lane 1 contains total proteins extracted from healthy human platelets as additional control. Expected band size: DGKE wt, 64 kD; DGKE isoform 1, 65.5 kD; DGKE isoform 3, 78 kD (http://www.expasy.org/). A band corresponding to DGKE Iso#1 is present in the sample from patient 1200, indicating that this mutant is expressed. The mutant Iso#1 and wt DGKE (of control samples) had a very similar migration pattern when subjected to gel electrophoresis because of the very small difference in the predicted molecular weights. No band corresponding to Iso#3 was detected, indicating that this isoform is produced in a very small amount, this isoform is unstable and rapidly degraded in the cells, or its conformation negatively affects the integrity of the targeted DGKE epitope. The blot were probed with an antiactin antibody as control for loading. DGKE, diacylglycerol kinase epsilon; wt, wild-type; C1, DGKE kinase C conserved region 1 (C1) domains; DAGKc, diacylglycerol kinase catalytic domain; DAGKa, diacylglycerol kinase accessory domain; LC, low-complexity region; TR, transmembrane region; Iso#1, isoform 1; Iso#2, isoform 2; Iso#3, isoform 3. aHUS (14,17) and the same novel intronic mutation is very aHUS–associated genes. It remains unclear whether aHUS- low. Third, we demonstrate that this intronic mutation acts associated DGKE deficiency may directly initiate comple- as a neo-splice site that not only results in the transcription ment activation or whether these patients carry another of mutant mRNA isoforms predicted to be deleterious to genetic abnormality in a modifier gene that affects com- DGKE catalytic activity, but also completely abrogates plement biology. wild-type DGKE mRNA transcription. Despite the advances in understanding aHUS genetic Finding multiple patients from unrelated kindreds to causes, approximately 40% of patients still do not carry have the exact same mutation is an unusual situation that is mutations in CFH, MCP, CFI, CFB, C3, THBD,andDGKE or explained either by a mutational hotspot or by remote com- deletions/rearrangements in CFH-CFHRs region (2,3,5,7,25). mon ancestry. Our data support the notion that the pattern To our knowledge, this is the first report describing an in- observed is caused by shared ancestry because all homo- tronic mutation located beyond exon/intron boundaries in zygous SNVs surrounding DGKE in family 1 are heterozygous DGKE and in general in aHUS-associated genes. Known in- in family 2. tronic mutations far from exon/intron boundaries and caus- Serum C3 levels of the two affected siblings from family 1 ing human disorders are rare (26–28). However, these were slightly depressed at disease onset, but they were mutations may be underreported because they are not rou- always normal when measured during disease relapse. They tinely investigated by standard analysis with Sanger sequenc- do not carry mutations in any of the known complement ing, which is usually restricted to exons and canonical splice 1018 Clinical Journal of the American Society of Nephrology

sites. Also, in WES, common criteria for selection of var- participating in advisory boards. Giuseppe Remuzzi has consul- iants restrict the analysis to coding regions, including the tancy agreements with AbbVie, Alexion Pharmaceuticals, Bayer 2–4 bp immediately adjacent to the exons. The two families Healthcare, Reata Pharmaceuticals, Novartis Pharma, AstraZeneca, reported here suggest that a number of unexplained patients Otsuka Pharmaceutical Europe, Concert Pharmaceuticals: no per- with aHUS may be accounted for by intronic mutations that sonal remuneration is accepted, and compensations are paid to his cause aberrant splicing of known aHUS-associated gene institution for research and educational activities. None of these transcripts. activities has had any influence on the results or interpretation in Inrecentyears,WESandWGShaveallowedthe this article. The other authors declare no conflicts of interest. discovery of a growing numbers of disease-associated genes (29); however, most variants in noncoding regions References will be missed by WES. This shortcoming could be over- 1. Noris M, Remuzzi G: Atypical hemolytic-uremic syndrome. N come by WGS; however, because of the large number of Engl J Med 361: 1676–1687, 2009 variants encountered, the choice regarding the ones de- 2. Mele C, Remuzzi G, Noris M: Hemolytic uremic syndrome. serving follow-up is a very challenging task. This is dem- Semin Immunopathol 36: 399–420, 2014 3. Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, onstrated by the fact that WGS failed to reveal a clear Daina E, Fenili C, Castelletti F, Sorosina A, Piras R, Donadelli R, molecular diagnosis in family 2 because intronic mutations Maranta R, van der Meer I, Conway EM, Zipfel PF, Goodship TH, were initially filtered out. Remuzzi G: Relative role of genetic complement abnormalities Another striking feature is the diverse transcriptional in sporadic and familial aHUS and their impact on clinical effects of the mutant neo-splice site when it occurs in the phenotype. Clin J Am Soc Nephrol 5: 1844–1859, 2010 4. Fremeaux-Bacchi V, Fakhouri F, Garnier A, Bienaime´ F, Dragon- context of a weak canonical splice donor and many strong Durey MA, Ngo S, Moulin B, Servais A, Provot F, Rostaing L, cryptic donor and acceptor sites. The same phenomenon Burtey S, Niaudet P,Descheˆnes G, Lebranchu Y,Zuber J, Loirat C: may occur in other DGKE introns, and perhaps in introns Genetics and outcome of atypical hemolytic uremic syndrome: A of other known aHUS-associated genes. It would be very nationwide French series comparing children and adults. Clin J Am Soc Nephrol 8: 554–562, 2013 interesting to determine if one could effectively prioritize fi 5. Bresin E, Rurali E, Caprioli J, Sanchez-Corral P, Fremeaux-Bacchi at-risk introns via genome-wide identi cation of all weak V, Rodriguez de Cordoba S, Pinto S, Goodship TH, Alberti M, donor and/or acceptor splice sites. Ribes D, Valoti E, Remuzzi G, Noris M; European Working Party Because the DGKE c.888+40A.Gmutationwasshown on Complement Genetics in Renal Diseases: Combined com- to be recurrent among patients with early-onset aHUS, di- plement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype. J Am Soc Nephrol 24: 475–486, agnostic and research laboratories should consider the in- 2013 clusion of intron 5 in the routine genetic screening of 6. Kavanagh D, Richards A, Fremeaux-Bacchi V,Noris M, Goodship DGKE. Sequencing of intronic regions of aHUS-associated T, Remuzzi G, Atkinson JP: Screening for complement system genes followed by analysis of mRNAs sequence could abnormalities in patients with atypical hemolytic uremic syn- drome. Clin J Am Soc Nephrol 2: 591–596, 2007 provide a valuable, still underexploited tool to resolve pa- 7. Maga TK, Nishimura CJ, Weaver AE, Frees KL, Smith RJ: Muta- tients with unknown genetic defects. Transcriptome se- tions in alternative pathway complement proteins in American quencing (30) could soon represent an alternative valuable patients with atypical hemolytic uremic syndrome. Hum Mutat strategy to directly detect mutations, including variants that 31: E1445–E1460, 2010 affect splicing. 8. Esparza-Gordillo J, Goicoechea de Jorge E, Buil A, Carreras Berges L, Lo´pez-Trascasa M, Sa´nchez-Corral P, Rodrı´guez de Co´rdoba S: Predisposition to atypical hemolytic uremic syn- Acknowledgments drome involves the concurrence of different susceptibility alleles We thank Annalisa Sorosina, Ramona Maranta, Marta Alberti for in the regulators of complement activation gene cluster in 1q32. Sanger sequencing, Elena Brini, and Alessandro Guffanti from Hum Mol Genet 14: 703–712, 2005 9. Venables JP, Strain L, Routledge D, Bourn D, Powell HM, Genomnia for WES and data analysis. We also thank Erin Loring and Warwicker P, Diaz-Torres ML, Sampson A, Mead P, Webb M, Carol Nelson-Williams for help with family 2 sample handling Pirson Y, Jackson MS, Hughes A, Wood KM, Goodship JA, and genomic DNA preparation, respectively. We thank the patients Goodship TH: Atypical haemolytic uraemic syndrome associ- and their relatives; without their contribution this work could not ated with a hybrid complement gene. PLoS Med 3: e431, 2006 have been done. 10. Dragon-Durey MA, Blanc C, Garnier A, Hofer J, Sethi SK, Zimmerhackl LB: Anti-factor H autoantibody-associated hemo- Supported by Fondazione ART per la Ricerca sui Trapianti ART lytic uremic syndrome: Review of literature of the autoimmune ONLUS (Milano, Italy), Fondazione Aiuti per la Ricerca sulle Ma- form of HUS. Semin Thromb Hemost 36: 633–640, 2010 lattie Rare ARMR ONLUS (Bergamo, Italy), grants from the Euro- 11. Legendre CM, Licht C, Muus P, Greenbaum LA, Babu S, pean Union Seventh Framework Programme FP7-EURenOmics Bedrosian C, Bingham C, Cohen DJ, Delmas Y, Douglas K, Eitner F, Feldkamp T, Fouque D, Furman RR, Gaber O, Herthelius M, (project no. 305608), and The Genomnia Minigrant (Lainate, Milan, Hourmant M, Karpman D, Lebranchu Y, Mariat C, Menne J, Italy) in collaboration with Life Technologies. Mathieu Lemaire is Moulin B, Nu¨rnberger J, Ogawa M, Remuzzi G, Richard T, the recipient of a Kidney Research Scientist Core Education and Sberro-Soussan R, Severino B, Sheerin NS, Trivelli A, National Training Program Post-Doctoral Fellowship Award from Zimmerhackl LB, Goodship T, Loirat C: Terminal complement the Kidney Foundation of Canada and is a member of the In- inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med 368: 2169–2181, 2013 vestigative PhD Medicine program at Yale University School of 12. Rathbone J, Kaltenthaler E, Richards A, Tappenden P, Bessey A, Medicine. Rossella Piras is a recipient of a research contract from Cantrell A: A systematic review of eculizumab for atypical Progetto DDD Onlus, Associazione per la lotta alla DDD. haemolytic uraemic syndrome (aHUS). BMJ Open 3: e003573, 2013 Disclosures 13. Biesecker LG, Green RC: Diagnostic clinical genome and exome sequencing. N Engl J Med 370: 2418–2425, 2014 Marina Noris and Véronique Frémeaux-Bacchi have received 14. Lemaire M, Fre´meaux-Bacchi V,Schaefer F, Choi M, Tang WH, Le honoraria from Alexion Pharmaceuticals for giving lectures and Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji W, Clin J Am Soc Nephrol 10: 1011–1019, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 1019

Overton JD, Mane SM, Nu¨rnberg G, Altmu¨ller J, Thiele H, Morin variants cause a glomerular microangiopathy that mimics D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, membranoproliferative GN. J Am Soc Nephrol 24: 377–384, Simkova E, Nu¨rnberg P, Rioux-Leclerc N, Moeckel GW, Gubler 2013 MC, Hwa J, Loirat C, Lifton RP: Recessive mutations in DGKE 24. Homolova K, Zavadakova P, Doktor TK, Schroeder LD, Kozich V, cause atypical hemolytic-uremic syndrome. Nat Genet 45: 531– Andresen BS: The deep intronic c.903+469T.C mutation in the 536, 2013 MTRR gene creates an SF2/ASF binding exonic splicing en- 15. Sharma AP, Greenberg CR, Prasad AN, Prasad C: Hemolytic hancer, which leads to pseudoexon activation and causes the uremic syndrome (HUS) secondary to cobalamin C (cblC) dis- cblE type of homocystinuria. Hum Mutat 31: 437–444, 2010 order. Pediatr Nephrol 22: 2097–2103, 2007 25. Loirat C, Noris M, Fremeaux-Bacchi V: Complement and the 16. Lung M, Shulga YV, Ivanova PT, Myers DS, Milne SB, Brown HA, atypical hemolytic uremic syndrome in children. Pediatr Topham MK, Epand RM: Diacylglycerol kinase epsilon is selec- Nephrol 23: 1957–1972, 2008 tive for both acyl chains of phosphatidic acid or diacylglycerol. 26. Flanagan SE, Xie W, Caswell R, Damhuis A, Vianey-Saban C, J Biol Chem 284: 31062–31073, 2009 Akcay T, Darendeliler F, Bas F, Guven A, Siklar Z, Ocal G, 17. Westland R, Bodria M, Carrea A, Lata S, Scolari F, Fremeaux- Berberoglu M, Murphy N, O’Sullivan M, Green A, Clayton PE, Bacchi V, D’Agati VD, Lifton RP, Gharavi AG, Ghiggeri GM, Banerjee I, Clayton PT, Hussain K, Weedon MN, Ellard S: Next- Sanna-Cherchi S: Phenotypic expansion of DGKE-associated generation sequencing reveals deep intronic cryptic ABCC8 and diseases. J Am Soc Nephrol 25: 1408–1414, 2014 HADH splicing founder mutations causing hyperinsulinism by 18. Sa´nchez Chinchilla D, Pinto S, Hoppe B, Adragna M, Lopez L, pseudoexon activation. Am J Hum Genet 92: 131–136, 2013 Justa Roldan ML, Pena~ A, Lopez Trascasa M, Sa´nchez-Corral P, 27. Costantino L, Claut L, Paracchini V, Coviello DA, Colombo C, Rodrı´guez de Cordoba S: Complement mutations in diac- Porcaro L, Capasso P, Zanardelli M, Pizzamiglio G, Degiorgio D, ylglycerol kinase-«-associated atypical hemolytic uremic syn- Seia M: A novel donor splice site characterized by CFTR mRNA drome. Clin J Am Soc Nephrol 9: 1611–1619, 2014 analysis induces a new pseudo-exon in CF patients. J Cyst Fibros 19. Jacob HJ, Abrams K, Bick DP, Brodie K, Dimmock DP, Farrell M, 9: 411–418, 2010 Geurts J, Harris J, Helbling D, Joers BJ, Kliegman R, Kowalski G, 28. Aten E, Sun Y,Almomani R, Santen GW, Messemaker T,Maas SM, Lazar J, Margolis DA, North P, Northup J, Roquemore-Goins A, Breuning MH, den Dunnen JT: Exome sequencing identifies a Scharer G, Shimoyama M, Strong K, Taylor B, Tsaih SW, branch point variant in Aarskog-Scott syndrome. Hum Mutat 34: Tschannen MR, Veith RL, Wendt-Andrae J, Wilk B, Worthey EA: 430–434, 2013 Genomics in clinical practice: lessons from the front lines. Sci 29. Rabbani B, Tekin M, Mahdieh N: The promise of whole-exome Transl Med 5: cm5, 2013 sequencing in medical genetics. J Hum Genet 59: 5–15, 2014 20. Seelow D, Schuelke M: HomozygosityMapper2012–bridging the 30. Ozsolak F, Milos PM: RNA sequencing: Advances, challenges gap between homozygosity mapping and deep sequencing. and opportunities. Nat Rev Genet 12: 87–98, 2011 Nucleic Acids Res 40: W516–520, 2012 21. Burge C, Karlin S: Prediction of complete gene structures in hu- Received: August 27, 2014 Accepted: February 9, 2015 man genomic DNA. J Mol Biol 268: 78–94, 1997 22. Desmet FO, Hamroun D, Lalande M, Collod-Be´roud G, Claustres C.M. and M.L. contributed equally to this work. M, Be´roud C: Human Splicing Finder: An online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37: e67, 2009 23. Ozaltin F, Li B, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Published online ahead of print. Publication date available at www. Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen cjasn.org. S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang C, Chen P, Lu D, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, This article contains supplemental material online at http://cjasn. Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, asnjournals.org/lookup/suppl/doi:10.2215/CJN.08520814/-/ Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M: DGKE DCSupplemental. Supplemental Information

CHARACTERIZATION OF A NEW DGKE INTRONIC MUTATION IN GENETICALLY

UNSOLVED CASES OF FAMILIAL ATYPICAL HEMOLYTIC UREMIC SYNDROME

Caterina Mele1* PhD, Mathieu Lemaire2,3* MD, Paraskevas Iatropoulos1 MD, Rossella Piras1

ChemPharmD, Elena Bresin1 MD, Serena Bettoni1 BiotechD, David Bick4,5,6 MD, Daniel Helbling4

MD, Regan Veith5 MD, Elisabetta Valoti1 BiolSciD, Roberta Donadelli1 BiolSciD, Luisa Murer7

MD, Maria Neunhäuserer8 MD, Matteo Breno1 PhD, Véronique Frémeaux-Bacchi9 PhD, Richard

Lifton2,3,10 MD, Giuseppe Remuzzi1,11 MD, and Marina Noris1 PhD

1IRCCS - Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare

Diseases “Aldo e Cele Daccò”, Ranica, Bergamo, Italy

2Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

3Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut,

USA

4Human and Molecular Genetic Center, Medical College of Wisconsin, Milwaukee, Wisconsin,

USA

5Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

6Genomics Medicine Clinic, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

7Unit of Pediatric Nephrology, Azienda Ospedaliera di Padova, Padua, Italy

8Unit of Pediatry, Südtiroler Sanitätsbetrieb, Bruneck, Italy

1

9Department of Immunology, Assistance Publique-Hopitaux de Paris, Hopital Europeen George-

Pompidou and INSERM UMRS 1138, Cordelier Research Center, Team “Complement and

Diseases”, Paris, France

10Yale Center for Mendelian Genomics, New Haven, Connecticut, USA

11Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

*Equally contributed

2

SUPPLEMENTAL METHODS

Diagnosis

Atypical HUS (aHUS) was diagnosed in the patients included in this study based on microangiopathic hemolytic anemia and thrombocytopenia defined on the bases of hematocrit less than 0.3 (30%), hemoglobin (Hb) level less than 100 g/L (10 g/dL), serum lactate dehydrogenase

(LDH) level higher than 460U/L, undetectable haptoglobin level, fragmented erythrocytes in peripheral blood smear, and platelet count less than 150X109/L (150,000/µl) associated with acute kidney failure. Familial aHUS was diagnosed when two or more members of the same family were affected by the disease at least 6 months apart and exposure to a common trigger infectious agent was excluded. Sporadic aHUS was diagnosed when one or more episodes of the disease manifested in an individual with no familial history of the disease.

Complement profile assessment

Serum concentrations of complement components C3 and C4 were evaluated by kinetic nephelometry. For Family#2, CH50 and AH50 were performed in a CLIA-certified clinical laboratory, following standard protocols.

Complement gene alteration and anti-FH antibody screening

Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures.

PCR products of the coding sequence and the intronic flanking regions of complement factor H

(CFH), membrane cofactor protein (MCP), complement factor I (CFI), complement component C3

(C3), complement factor B (CFB), and thrombomodulin (THBD) genes were analyzed by Sanger sequencing on the 3730 DNA Analyzer (Applied Biosystem).

3

Screening for genomic disorders affecting CFH, complement factor H-related 1 (CFHR1), CFHR2,

CFHR3, and CFHR5 was undertaken using multiplex ligation-dependent probe amplification using a kit from MRC Holland (SALSA MLPA kit P236-A3 ARMD) and homemade probes for CFH exon 19, intron 20, intron 21, intron 22 and exon 23.1

Screening for anti-FH autoantibodies was undertaken using an ELISA assay.2 The normal ranges were set as mean ±2 standard deviation of the values recorded in healthy individuals. Value up the higher limit of the normal ranges were considered as high, whereas values below the lower limit of the normal ranges were considered as low.

Whole-exome sequencing, homozygosity mapping and analysis of variants

Whole-exome sequencing (WES) of patient#452 and bioinformatics analysis of the raw sequence datasets (up to single nucleotide variant and insertion/deletion call evaluation and preliminary annotation) were undertaken at Genomnia srl (Lainate, Milano, Italy). Targeted enrichment and sequencing were performed by using an ABI SOLiD optimized SureSelect Human All Exon 50Mb

Kit (Agilent, Santa Clara, CA, USA) and the SOLiDTM 4 System (Life Technologies, Carlsband,

CA, USA) with 50bp fragment reads, respectively. Accuracy enhancement of the raw datasets was performed using the SAET program (SOLiD Accuracy Enhancement Tool, http://solidsoftwaretools.com/gf/project/saet/). Mapping was performed using the mapping module of the LifeTech LifeScope ver2.5.1 analysis pipeline against the reference

GRCh37/hg19. Single nucleotide variants (SNVs) were called by the DiBayes algorithm using parameters optimized for rare variant detection. Small insertions and deletions (indels) were identified with the LifeScope Small Indels Analysis tool. Sequence coverage of exons was evaluated with ad hoc developed Perl scripts and information from the pipeline output. Primary variant annotation was performed similarly with ad hoc developed Perl scripts. Called SNVs and indels were annotated using ANNOVAR software.3

4

Homozygosity mapping was performed in patient #452 byHomozygosityMapper4, 5 using genotypes inferred from WES data. Genotypes were called using SNVs with a coverage of at least 10 reads.

To minimize the number of runs of homozygosity that occurred by chance, search parameters were set as follows: minimum detected length > 1.5Mb; number of consecutive SNVs required >40.

Sanger sequencing was performed to validate WES results and to assess segregation pattern in

Family#1. In silico analyses for the DGKE c.888+40A>G mutation were performed using GenScan

(http://genes.mit.edu/GENSCAN.html)6 and Human Splicing Finder v3

(http://www.umd.be/HSF/).7 Primers used for PCR and Sanger sequencing targeting the DGKE c.888+40A>G were: forward 5’-CAGATGAAGGAAAATGTTGGA-3’; reverse 5’-

GGATGGCTTGAACCTGAGAA-3’. DNA samples from 89 healthy Italian individuals were used as controls.

RNA analysis

RNA was extracted from fresh whole blood samples using the NucleoSpin® RNA Blood kit

(MACHEREY-NAGEL) following manufacturer’s instructions. Extracted RNA was reverse transcribed into cDNA by using hexanucleotides and the Superscript II RT enzyme kit (Invitrogen). cDNA was amplified by RT-PCR using a forward primer designed on exon 4 (5′-

ATCCTGGCCAACTCTCGTAG-3′) and a reverse primer on exon 6 (5′-

TGTTCCCAGAGGCAAAACTG-3′) of DGKE. The amplification products were run on a 1% agarose gel and visualized in a UV transilluminator. The single bands were excised from gel, purified with the QIAquick Gel Extraction Kit (Qiagen) according to manufacturer’s protocol, and directly sequenced using the same primers as for PCR.

Protein expression analysis

Peripheral blood leukocytes were isolated from EDTA-collected blood by centrifugation at 1400g for 15 min. Lysis of contaminating red blood cells was performed with ACK1x lysis buffer 5

(Ammonium-Chloride-Potassium, 150mM NH4Cl, 1000mM KHCO3, 100mM EDTA•Na2•2H2O).

Platelets (as positive control) were prepared from normal whole blood collected on sodium citrate by differential centrifugation as previously described.8 Cell lysates were prepared using a lysis buffer containing 0.1% Triton-X100 in 50mM Tris-HCl pH7.4, 150mM NaCl, 0.1%SDS, 1mM

EDTA, with protease inhibitors (Complete Protease Inhibitor cocktail Tablets, Roche).

Fifty micrograms of proteins were subjected to SDS-PAGE (Sodium Dodecyl Sulphate -

PolyAcrylamide Gel Electrophoresis) and analyzed by protein blotting. After blocking, membrane was probed with rabbit polyclonal anti-DGKE (1:750, abcam, ab84331, against the 2-51 N-terminal amino-acids) and rabbit polyclonal anti-actin (1:30,000, Sigma-Aldrich, A5060). Membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:30,000, Vector

Laboratories, PI-1000).

Whole-genome sequencing and analysis of variants

All testing was carried out in CLIA certified / College of American Pathologist (CAP)-accredited laboratories. Blood was drawn and genomic DNA extracted following standard protocols. Whole- genome sequencing (WGS) (paired-end, 100 base pairs reads) of patient #II-1 was done on Illumina

HiSeq2000 genome analyzer (Illumina Clinical Services Laboratory, San Diego California). Base calling, read mapping (against the reference human genome NCBI36/hg18) and variant calling were done locally by Illumina Clinical Services Laboratory using standard softwares. Variant annotation was done with CarpeNovo, a software developed and implemented at Medical College of

Wisconsin, Milwaukee, Wisconsin, USA.9, 10

DGKE structural modeling

Protein structure predictions of DGKE for the wild-type molecule, isoform #1 and isoform#3 were carried out in the online platform I-TASSER.11 Due to insertions/alternative splicing, submitted amino acids ranged from 215 to 360 (wild-type), 215 to 373 (isoform#1), and 215 to 484 6

(isoform#3), respectively, corresponding to the DGKE kinase domain. 3D reconstruction of the amino acid chains were visualized with Jmol (Jmol: an open-source Java viewer for chemical structures in 3D, http://www.jmol.org/).

7

SUPPLEMENTAL RESULTS

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Birth HUS

R1 R2 R3 #452 R4 R5 R6 R7 R8 R9 R10 R11

Birth

HUS R1 R2 #1200 R3 R4 R5 R6

R7 R8

Supplemental Figure 1. Clinical course for the two affected siblings from Family#1.

This timeframe indicates the relative timing of major clinical events for the two affected siblings from this kindred, labeled #452 (proband) and #1200, including births, aHUS diagnoses and relapses. Abbreviations: HUS, first episode of aHUS; Rx, relapse of aHUS.

8

EXON 5 (144bp) Intron 5 (630bp) EXON 6 (158bp)

wt …TAGGTTTTTG…GATTAAGGTATTAGTCTTTAAGAACTACTACAGAAGGACTTCTAAGATAAATATACTATACATATA…ATTCTAGGGACAAG...TAGATCGGTA... AACTACTA (SC35) TACATA (SC35) CTACTAC (SRp40) CTACAGA (SRp40) TACAGA (SRp55) ACAGAAG (SRp40) CAGAAGG (SF2/ASF-IgM/BRCA1) CAGAAGG (SF2/ASF) GACTTCTA (SC35)

EXON 5 (144bp) Intron 5 (630bp) EXON 6 (158bp)

mut …TAGGTTTTTG…GATTAAGGTATTAGTCTTTAAGAACTACTACAGAAGGACTTCTAAGGTAAATATACTATACATATA…ATTCTAGGGACAAG...TAGATCGGTA... AACTACTA (SC35) TCTAAGG (SRp40) TACATA (SC35) CTACTAC (SRp40) CTAAGGT (SF2/ASF) CTACAGA (SRp40) CTAAGGT (SF2/ASF-IgM/BRCA1) TACAGA (SRp55) ACAGAAG (SRp40) CAGAAGG (SF2/ASF-IgM/BRCA1) CAGAAGG (SF2/ASF) GACTTCTA (SC35)

Supplemental Figure 2. The DGKE c.888+40A>G mutation is predicted to create new exonic splicing enhancer (ESE) motifs in intron 5.

This figure illustrates how the non-coding DGKE mutation c.888+40A>G is predicted by Human

Splicing Finder v3 software to result in the formation of new ESEs that would allow for the aberrant binding of various mRNA regulatory proteins, such as serine/arginine protein 40 (SRp40) or serine/arginine-rich splicing factor 1 (SF2/ASF). This change is predicted to alter the correct assembly of the splicing regulatory proteins on DGKE pre-mRNA. The diagram shows the normal

(wt) and the mutant (mut) sequences of DGKE intron 5. The position of the c.888+40A>G is marked in red. Wild-type ESE motifs are highlighted in green. The new ESE motifs in the mutant sequence are highlighted in red.

9

DGKE structural modeling

The best model obtained by the online platform I-TASSER11 for the DGKE wild-type kinase domain showed an overall good prediction (C-score 0.10, expected TM-score 0.73±11, indicating a correct folding). The top structurally similar proteins, selected from a database of known structures, were a putative diacylglycerol kinase from Bacillus antracis (PDB#3T5P) or various homologous proteins of bacterial origin (details not shown). The best functional predictions identified the protein as a diacylglycerol kinase based on the Enzyme Commission (EC) number (2.7.1.107; C-score =

0.317, TM-score = 755) and on the top hits on (GO) terms (GO:0004143; C-score =

0.59, TM-score = 0.73, GO-score for GO:0004143= 0.94). Other GO terms included ATP binding, metal ion binding and lipid kinase.

Isoform #1 best model displayed a C-score of -0.81 with an expected TM-score of 0.61±0.14 indicating a reduced confidence in the prediction compared to the wild-type domain. Structural and functional matches were comparable to those predicted for the wild-type sequence. The EC number and GO terms associated with the best hits were highly similar to wild-type, but with reduced accuracy (C-score= 0.244, TM-score= 0.699 and C-score= 0.48, TM-score = 0.66 and GO-score of

GO:0004143 = 0.83 for EC number and GO respectively). Finally, the best model for isoform#3 had a low C-score (-1.89 with an expected TM-score of 0.49±0.15) indicating a bad quality of the model with a likely incorrect topology, making it unreliable for predictions (details not shown).

10

Supplemental Figure 3. Graphical representation of predicted 3D models for the isolated kinase domain of wild-type and mutant DGKE proteins.

The impact of the c.888+40A>G on the structure of DGKE protein was assessed using the online prediction software I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). Panels A and B illustrate the predicted structure of wild-type DGKE kinase domain. Panels C and D show the impact of the c.888+40A>G on the structure of the kinase domain of one of the mutant DGKE proteins, predicted from mRNA sequence of isoform#1. The panels on the left contain the predicted secondary structures, while the panels on the right show the reconstruction of the molecular surface.

Wild-type residues are depicted in white, while the in-frame insertion is depicted in green.

11

Supplemental Figure 4. Sequencing and clinical data for patients from Family#2.

(A) Electropherograms of DGKE c.966G>A (p.Trp322*) in individuals from Family#2. (B)

Strategy used to filter out all of the DGKE variants found by WGS in patient #II-1. Both coding and non-coding variants were considered. The only non-synonymous mutation was the paternal allele c.966G>A(p.Trp322*), which is known to be pathological in many cases of DGKE nephropathy.

After filtering out variants based on minor allele frequency data (<1%), a total of 0 non-coding variants remained for analysis. The parental origin of each allele was determined by Sanger

12 sequencing. For details, please see Supplemental Table 5. (C) Electropherograms of DGKE c.888+40A>G mutation in individuals from Family#2. The affected children (#II-1, #II-2 and #II-4) are compound heterozygous for the DGKEc.888+40A>G and c.966G>A. The healthy father (#I-1) and child (#II-2) are heterozygous carriers of the c.966G>A, while the healthy mother (#I-2) is heterozygous carriers of the c.888+40A>G. (D) Clinical course for the three affected siblings with compound heterozygous mutations in DGKE. This timeframe indicates the relative timing of major events for the three affected siblings from this kindred, including births, aHUS diagnoses and relapses. The timeline was generated using the software Aeon Timeline (version 1.2.4).

Abbreviations: HUS, first episode of aHUS; Rx, relapse of aHUS.

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Supplemental Table 1. Genomic regions of homozygosity in patient#452

N° of Protein- SNVs and Rare* Chr Start End Lenght (bp) annotated altering indels homozygous genes variants#

3 10,088,343 13,679,203 3,590,860 27 48 0 0 3 46,753,976 54,596,815 7,842,839 150 163 9 3 8 69,002,965 121,210,069 52,207,104 181 185 3 0 12 53,227,803 54,974,803 1,747,000 51 50 0 0 16 66,886,777 68,712,730 1,825,953 65 63 1 0 19 13,445,208 16,197,377 2,752,169 61 96 1 1 19 40,901,496 48,517,387 7,615,891 180 233 2 0 Total 77,581,816 715 838 16 4 GRCh37/hg19 coordinates. *MAF<=1%. #Nonsense, missense, frameshift indels or affecting canonical splice sites. Abbreviations: chr = ; SNV = single nucleotide variant; indel = insertion/deletion.

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Supplemental Table 2. Homozygous rare variants found within the homozygous regions of patients #452 (ANNOVAR output) and segregation analysis in Family#1 Nucleotide Aminoacidic Mutation Mutation Segregating with Chr Position Ref Alt Gene RefSeq mRNA ESP6500 1,000G TSI dbSNP137 SIFT PP2 LRT GERP change change Taster Assessor the disease? 3 48,637,143 C T UQCRC1 NM_003365 c.G1303A p.E435K 0.0018 0.0014 0.0102 rs139999010 0.06 0.003 0.00003 0.1221 0.345 4.84 Yes 3 49,050,826 G A WDR6 NM_018031 c.G1949A p.R650H NA NA NA NA 0.56 0.003 0.13329 0.4727 1.39 4.47 Yes 3 50,379,343 C T ZMYND10 NM_015896 c.G1019A p.R340Q 0.0032 0.0014 0.005 rs148328402 0.32 0.008 0.00322 0.1372 1.155 4.12 Yes 19 14,736,326 C T EMR3 NM_032571 c.G1898A p.G633D 0.0088 0.01 0.0102 rs117374816 0.25 0.725 . 0.0305 0.345 0.674 No* GRCh37/hg19 coordinates. *Patient #1200 was heterozygous.

Abbreviations: chr = chromosome; Ref = reference allele; Alt = alternate allele; ESP6500 = National Heart, Lung, and Blood Institute Exome Variant Server database; 1,000G = 1,000 Genomes Project database; TSI = Italian population from Tuscany (1,000 Genomes subpopulation); SIFT = sorting intolerant from tolerant algorithm (possible values: "deleterious" <=0.05, "tolerated" >0.5); PP2 = Polyphen 2 algorithm (scores built on HumanVar database; possible values: "probably damaging" 1-0.909, "possibly damaging" 0.908-0.447, "benign" 0.446-0); LRT = likelihood ratio test (LRT and Mutation Taster scores range from 0 "neutral" to 1 "damaging"); GERP = Genomic Evolutionary Rate Profiling (GERP score ranges from -12.3 for less conserved to 6.17 for more conserved residues). Mutation Assessor possible values: "non-functional" < 1.938, "functional" > 1.938.

Notes: the UQCRC1 variant (rs139999010) has a MAF>1% in the Italian population from Tuscany, thus it was filtered out. The ZMYND10 variant (rs148328402) is reported in OMIM (Online Mendelian Inheritance in Man) database in association with Primary Ciliary Dyskenysia type 22, thus it was excluded as possible cause of aHUS in Family#1. The WDR6 variant was excluded since it is predicted not to be damaging by in silico analysis. In addition, WDR6 protein is a negative regulator of cell proliferation, and somatic mutations in this gene have been found in cancer (COSM1045996).

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Supplemental Table 3. SNVs around DGKE locus #452 #II-1 Chr Position Reference Variant Gene dbSNP137 ESP6500 1,000G Zygosity Zygosity 17 het het 54,534,634 G A ANKFN1 rs10852985 0.69597 0.79 17 hom het 54,872,439 T C C17orf67 rs72837329 0.141335 0.13 17 hom het 54,872,555 C A C17orf67 rs72837330 0.138067 0.13 17 hom het 54,892,348 G C C17orf67 rs72837358 0.126227 0.11 17 hom het 54,912,339 G A DGKE rs1048159 0.127576 0.11 17 hom het 54,925,262 G T DGKE rs6503775 0.862448 0.89 17 hom het 54,925,466 A G DGKE NA NA NA 17 hom het 54,978,794 G A TRIM25 rs205498 0.762494 0.78 17 hom het 55,027,850 T C COIL rs8071691 0.866677 0.92 17 het het 55,950,064 C T CUEDC1 rs35704289 0.315162 0.22 GRCh37/hg19 coordinates.

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Supplemental Table 4. Potential DGKE intron 5 splice sites Position Splice site type Motif New potential splice site Consensus value* c.888+14 Acceptor ttagtctttaagaa ttagtctttaagAA 78.49 c.888+25 Acceptor gaactactacagaa gaactactacagAA 80.9 c.888+28 Acceptor ctactacagaagga ctactacagaagGA 76.84 c.888+38 Acceptor aggacttctaagat aggacttctaagAT 71.55 c.888+112 Acceptor ttttttcttgagat ttttttcttgagAT 84.89 c.888+118 Acceptor cttgagatacagtc cttgagatacagTC 81.31 c.888+136 Acceptor actgtcacccaggc actgtcacccagGC 85.94 c.888+143 Acceptor cccaggctggagtg cccaggctggagTG 72 c.888+148 Acceptor gctggagtgcagcg gctggagtgcagCG 77.91 c.888+156 Acceptor gcagcggcgcagac gcagcggcgcagAC 77.1 c.888+162 Acceptor gcgcagacacagct gcgcagacacagCT 76.48 c.888+186 Acceptor tccatttctcaggt tccatttctcagGT 90.99 c.888+193 Acceptor ctcaggttcaagcc ctcaggttcaagCC 74.61 c.888+210 Acceptor ctcccacctcagcc ctcccacctcagCC 87.72 c.888+219 Acceptor cagcctacaaagtt cagcctacaaagTT 71.6 c.888+227 Acceptor aaagttgctgagac aaagttgctgagAC 66.1 c.888+234 Acceptor ctgagactataggt ctgagactatagGT 79.1 c.888+276 Acceptor tattttttgtagag tattttttgtagAG 82.37 c.888+278 Acceptor ttttttgtagagac ttttttgtagagAC 80.66 c.888+282 Acceptor ttgtagagacaggg ttgtagagacagGG 83.5 c.888+328 Acceptor cctgggctcaagcg cctgggctcaagCG 74.49 c.888+355 Acceptor ggcctcccaaagtg ggcctcccaaagTG 74.94 c.888+376 Acceptor tacatgggtgagcc tacatgggtgagCC 65.86 c.888+399 Acceptor aacctaatatagaa aacctaatatagAA 72.1 c.888+431 Acceptor tttattatgcagat tttattatgcagAT 88.58 c.888+442 Acceptor gatgaatattagac gatgaatattagAC 68.12 c.888+452 Acceptor agacattgccagct agacattgccagCT 77.15 c.888+462 Acceptor agcttcaaatagtt agcttcaaatagTT 72.16 c.888+473 Acceptor gttaaaaatgagat gttaaaaatgagAT 67.38 c.888+487 Acceptor gttttgattaagca gttttgattaagCA 75.21 c.888+494 Acceptor ttaagcaattagca ttaagcaattagCA 70.8 c.888+500 Acceptor aattagcacaagct aattagcacaagCT 70.4 c.888+506 Acceptor cacaagctttagca cacaagctttagCA 72.41 c.888+521 Acceptor aacaacatacagtt aacaacatacagTT 77.94 c.888+583 Acceptor attattacccagca attattacccagCA 83.16 c.888+590 Acceptor cccagcaaatagtc cccagcaaatagTC 70.08 c.888+596 Acceptor aaatagtctgagag aaatagtctgagAG 68.08 c.888+598 Acceptor atagtctgagagaa atagtctgagagAA 70.72 c.888+629 Acceptor tccatattctagGG tccatattctagGG 82.11 (WT) c.888+1 Donor AAGgtatta AAGgtatta 75.59 (WT) c.888+73 Donor tgggttttg TGGgttttg 65.08 c.888+188 Donor caggttcaa CAGgttcaa 71.67 c.888+236 Donor taggtgtgc TAGgtgtgc 85.88 c.888+238 Donor ggtgtgcgc GGTgtgcgc 70.38 c.888+285 Donor agggtttca AGGgtttca 66.01 c.888+373 Donor tgggtgagc TGGgtgagc 89.83 c.888+591 Donor atagtctga ATAgtctga 67.55 *Consensus values (calculated by Human Splicing Finder) ranges from 0 to 100. Only splice sites with consensus values above 65 were considered. Splice sites with consensus values higher than 80 are strong splice sites; less strong sites have consensus values that range from 70 to 80. Splice sites with consensus values of 65 to 70 are weak. The two cryptic splice acceptor and donor sites leading to mutant isoforms #2 and #3 are highlighted in yellow. Wild-type donor splice site of exon 5 and wild-type acceptor splice site of exon 6 are highlighted in green.

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Supplemental Table 5. Details for the parental origin for the 8 rare and non-coding variants extracted from WGS data performed on patient #II-1. Position on chromosome 17 Ref/Var Genotypes Origin of Variant# dbSNP id MAF hg18 hg19 nucleotides Father Mother mutant allele 1 52273472 54918473 G/A rs144054661 0.78% G/A G/G PATERNAL 2 52274278 54919279 C/T rs114187815 0.91% C/T C/C PATERNAL 3 52274354 54919355 G/A rs115382235 1.00% G/A G/G PATERNAL 4 52277530 54922531 A/G rs77950042 0.91% G/A A/A PATERNAL 5 52280465 54925466 A/G Not Found novel A/A A/G MATERNAL 6 52287719 54932720 G/T rs74963470 0.87% G/T G/G PATERNAL 7 52290158 54935159 C/T Not Found novel C/T C/C PATERNAL 8 52291937 54936938 G/A rs145743671 0.55% G/G G/A MATERNAL

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Supplemental Table 6. Quantitative documentation of the evolution of proteinuria over time for the three affected siblings from Family#2. Urine solutes concentrations (mg/dL) Prot:creat Patients Date of sampling Protein Creatinine ratioA

#II-1 Jan 2002B 1084 23.6 45.9

March 2002B 634 33.9 18.7

May 2008 49 82.5 0.6

#II-2 May 2008 366 139.9 2.6

Nov 2009 1016 115.92 8.8

#II-4 March 2011B 2589 75.25 34.4

Jan 2013B 1204 56.9 21.2 Notes: please refer to supplemental Figure 4D for details about the time course of clinical events over time. ANormal urine protein:creatinine ratio in children < 0.2. B Indicates admission for aHUS relapse the same month.

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