Article

Characterization of a New DGKE Intronic Mutation inGenetically Unsolved Cases of Familial AtypicalHemolytic 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 Neunhauserer, Matteo Breno,Veronique Fremeaux-Bacchi, Richard Lifton, Giuseppe Remuzzi, and Marina Noris

AbstractBackground and objectives Genetic and acquired abnormalities causing dysregulation of the complementalternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized bythrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in asubstantial proportion of patients the disease-associated alterations are still unknown.

Design, setting, participants, & measurementsWhole-exome and whole-genome sequencing were performed intwo unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individualswas used totest for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE)protein was evaluated with western blotting.

Results Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal anyobvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encodingDGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing,allowing identification of a novel intronic DGKE mutation (c.888+40A.G) that segregated with disease. Se-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 incompound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelatedfamily. Homozygous and compound heterozygous patients presented similar clinical features, including aHUSpresentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical ofDGKE-associated aHUS.

Conclusions This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronicmutations 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 andclinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled withmRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.

Clin J Am Soc Nephrol 10: ccc–ccc, 2015. doi: 10.2215/CJN.08520814

IntroductionAtypical hemolytic uremic syndrome (aHUS) is a raredisorder resulting in thrombocytopenia, nonimmunemicroangiopathic hemolytic anemia, and acute kidneyfailure (1). It has a poor prognosis with approxi-mately 60% of patients progressing to ESRD and amortality rate between 4% and 25% (1,2). Extensivestudies showed that hyperactivation of the comple-ment alternative pathway is the main pathogeneticeffector mechanism leading to endothelial damageand microvascular thrombosis in most patients withaHUS (1,2). Genetic and autoimmune abnormalitiesaffecting complement proteins (complement factorH [FH], factor H–related proteins [FHRs], factor I[FI], factor B [FB], complement component 3 [C3],

membrane cofactor protein [MCP], and thrombo-modulin [THBD]) have been documented in nearly60% of patients (1–10). These findings paved the wayfor complement-tailored treatments (1,11) that have ledto impressive improvements in short- and long-termprognosis (12). However, the underlying cause remainselusive for a substantial proportion of patients. The ad-vent of next-generation sequencing has resulted inprogress toward filling these knowledge gaps, allow-ing for a rapid exome-/genome-wide search for path-ogenic mutations (13). Recently, using whole-exomesequencing (WES), Lemaire and colleagues success-fully identified recessive mutations in DGKE, encod-ing diacylglycerol kinase epsilon (DGKE), as a novelcause of aHUS (14). Patients showed a specific clinical

Due to the number ofcontributing authors,the affiliations areprovided in theSupplementalMaterial.

Correspondence: Dr.Giuseppe Remuzzi,IRCCS - Istituto diRicercheFarmacologiche MarioNegri, Centro AnnaMaria Astori, Scienceand Technology ParkKilometro Rosso, ViaStezzano 87, 24126Bergamo, Italy. E-mail:giuseppe.remuzzi@marionegri.it

www.cjasn.org Vol 10 June, 2015 Copyright © 2015 by the American Society of Nephrology 1

. Published on April 8, 2015 as doi: 10.2215/CJN.08520814CJASN ePress

phenotype characterized by onset in infancy, multiple re-lapsing episodes, and nephrotic-range proteinuria (14). Be-sides cobalamin C deficiency–associated aHUS (15), DGKEis the only other gene implicated in aHUS that does notencode a complement component (16).Patients carrying DGKE mutations did not show con-

sumption of serum complement components, suggestingthe existence of a novel pathogenetic mechanism for throm-botic microangiopathy (14). However, this assumption hasbeen challenged by the report of a DGKE-truncating muta-tion in two patients with moderate C3 consumption (17).The recent finding of combined DGKE and complementgene mutations in three patients suggests that complementdysregulation may have a role in modulating diseaseseverity in DGKE mutation carriers (18). Identification ofadditional patients with DGKE mutations will be helpfulto better characterize the phenotypic and prognostic het-erogeneity.Here, we report two families with recessive aHUS where

examination of DGKE variants obtained from WES andwhole-genome sequencing (WGS) allowed identificationof a novel, intronic DGKE mutation that segregates withdisease in both families and causes aberrant splicing ofDGKE transcripts.

Materials and MethodsaHUS was diagnosed on the basis of microangiopathic

hemolytic anemia and thrombocytopenia defined by he-matocrit ,30%, hemoglobin level ,100 g/L, serum lactatedehydrogenase level .460 U/L, undetectable haptoglobin,fragmented erythrocytes in peripheral blood smear, andplatelet count ,1503109/L, associated with acute kidneyfailure.Family 1 and 30 unrelated pediatric patients with aHUS

were recruited from the International Registry of HUS/Thrombotic Thrombocytopenic Purpura. Patient II-1 fromfamily 2 underwent clinical WGS as part of the GenomicsMedicine Clinic, in collaboration with Children’s Hospitalof Wisconsin and Froedtert Hospital (19). Twenty unrelatedFrench patients with pediatric aHUS undergoing geneticscreening in Paris were included in this study.Peripheral blood samples were collected for DNA, RNA,

and protein isolation. Samples from 89 Italian healthypersons were analyzed as controls.The study was approved by the Ethics Committee of the

Azienda Sanitaria Locale, Bergamo, Italy, and the Institu-tional Review Boards at the Medical College of Wisconsinand Yale University School of Medicine. Informed consentwas obtained from participants or by their parents accord-ing to the Declaration of Helsinki.Detailed description ofmaterials andmethods are reported

in the Supplemental Material.

ResultsIdentification of a Novel Homozygous Intronic DGKEMutation in Family 1We studied a consanguineous family (family 1, Figure

1A) from the North of Italy (South Tyrol) with two affectedsiblings (including a 13-year-old girl and 10-year-old boyat present) whose parents are healthy second cousins.

aHUS was diagnosed at 10 months in the girl (no. 452)and at 5 months in the boy (no. 1200). Both siblings hadthrombocytopenia, hemolytic anemia with schistocytes onblood smear, and renal impairment (Table 1). Mild pro-teinuria and hypertension were also documented. C3 lev-els were lower than normal, and C4 was normal. Completeremission was achieved for both patients with supportivetherapy, which only included correction of anemia withpacked erythrocytes and antihypertensive therapy. Bothsiblings had relapsing disease with one to three bouts ayear, often in concomitance with viral or bacterial infec-tions (Supplemental Figure 1), without evidence of C3 con-sumption. During relapses, they manifested renalimpairment (serum creatinine, no. 452: 1.4–1.7 mg/dl;no. 1200: 0.7–0.85 mg/dl), with hematuria and high-degreeproteinuria (.2 g/24 h). After every relapse, renal and he-matologic parameters returned to baseline with supportivetherapy alone or with plasma infusion/exchange. From theage of 8 (no. 452) and 5 (no. 1200) years, the siblings receivedtwo plasma infusions a year as prophylaxis. Patient 1200had a relapse at 7 years and 3 weeks after plasma infusion.The relapse was treated with plasma infusion with promptrecovery. Thereafter, no further relapse occurred in both chil-dren. At the last follow-up (age of no. 452, 13 years; age ofno. 1200, 10 years), the siblings had mild persistent protein-uria with normal renal function (Table 1).Sanger sequencing did not reveal any mutation in known

aHUS-associated genes (CFH, MCP, CFI, CFB, C3, andTHBD) or the CFH-H3 and MCPggaac risk haplotypes.Multiplex ligation-dependent probe amplification analysisshowed the presence of two copies of CFHR1 and CFHR3and excluded genomic CFH-CFHRs rearrangements.ELISA for anti-FH autoantibodies was negative. To iden-tify the genetic basis of aHUS in this family, we performedWES coupled with homozygosity mapping (20).WES performed on patient 452 showed a 72X mean cov-

erage over the targeted exons, with 95.1% of targets coveredat an average depth of 4X or higher. Variant detection iden-tified 30,267 single nucleotide variants (SNVs) and 1137insertions/deletions. After excluding variants with minorallele frequency (MAF) .1%, 4507 candidates remained.Among those, 143 (131 SNVs and 12 insertions/deletions)were homozygous protein-altering variants (nonsense, mis-sense, or affecting canonical splice sites). We focused onidentifying autozygous mutations because the affected sib-lings are from a consanguineous family and are likely tohave two recessive disease alleles inherited from a commonancestor. Homozygosity mapping on the basis of WES datawere used to identify all autozygous blocks, defined asruns of homozygosity of at least 1.5 Mb (20). This analysisyielded seven uninterrupted homozygous regions acrossthe genome (Supplemental Table 1), which collectivelyspanned 77.6 Mb (approximately 2.7% of the genome),therefore confirming parental consanguinity at the levelof second cousins (inbreeding coefficient, F=1/64). Withinthe homozygous blocks, we detected three missense var-iants in WDR6, UQCRC1, and ZMYND10, whose segrega-tion patterns were consistent with recessive inheritance(Supplemental Table 2). Only the WDR6 variant was notpresent in public databases (dbSNP137, 1000 Genomes Pro-ject, and National Heart, Lung, and Blood Institute ExomeVariant Server). However, none of the three segregating

2 Clinical Journal of the American Society of Nephrology

variants appeared to be convincingly causative for aHUS,and none were predicted to be damaging.Meanwhile, the article by Lemaire and colleagues (14), doc-

umenting the association of DGKEmutations and aHUS, waspublished. Similarities concerning disease inheritance mode(recessive), age of onset (,1 year), and clinical phenotype(recurrent disease) among our patients and the ones de-scribed by Lemaire et al. (14) led us to reanalyze in detailWES data around the DGKE locus. We found a homozygousstretch, including eight SNVs spanning theDGKE locus (Sup-plemental Table 3), whose length (approximately 1.4 Mb),however, was just under the 1.5 Mb cutoff used. Amongthose SNVs, seven had MAF.1%; the eighth was a novelhomozygous intronic variant (NM_003647.2: c.888+40A.G,Figure 2A), which had been filtered out by computationalpipeline parameters. This variant is located in intron 5 and isabsent from public databases. Sanger sequencing confirmedthat the c.888+40A.G variant segregates with disease, witha recessive pattern: both affected siblings are homozygous,and both healthy parents are heterozygous (Figure 2A). Inaddition, the c.888+40A.G mutation was not found in 178chromosomes from adult Italian controls.Analyses with GenScan software (21) predicted that in the

mutant sequence the probability of the correct splicing ofexon 5 decreases from 0.999 to 0.118 versus the wild-typesequence (Figure 2B). The presence of a longer exon 5 (exon5*, Figure 2B) was predicted with a probability of 0.882.Additional analyses with Human Splicing Finder software(22) predict that this mutation creates a gt cryptic splicingdonor site within intron 5, with a score higher than the ca-nonical donor site (Figure 2B). This change introduces newexonic splicing enhancer motifs for SF2/ASF, SRp40, andSF2/ASF (IgM-BRCA1) that are predicted to alter the nor-mal behavior of the splicing regulatory proteins that processDGKE pre-mRNA molecules (Supplemental Figure 2).

Characterization of the Effects of c.888+40A>G on DGKETranscript and ProteinDGKE is expressed in peripheral blood leukocytes (23). To

assess the effects of the c.888+40A.G mutation at a tran-scriptional level, we obtained peripheral blood samples fromthe siblings and parents from family 1 and from a healthyunrelated Italian control. RNA analysis was performed byRT-PCR using primers spanning exons 4–6 of the DGKEtranscript. Electrophoresis of the amplification productsshowed that the wild-type amplicon (273bp) found in thecontrol was absent in both affected children (Figure 3A). In-stead, their cDNA exhibited three additional bands with mo-lecular weights higher than that of the wild-type. The bandcorresponding to an approximately 300 bp amplicon (mu-tant isoform 1) was more prominent than the other two,measuring approximately 400 and 650 bp, respectively (mu-tant isoforms 2 and 3, Figure 3A). Parental samples revealedthe wild-type and mutant amplicons.The nucleotide sequences of each isoform were obtained

after extraction of the bands from the agarose gel. Mutantisoform 1 (312 bp) results from the retention of 39 intronicnucleotides that immediately follow exon 5 (c.888_889ins39,Figure 3B). This confirms in silico prediction that the neo-splicedonor site created by the mutation is used instead of the weakcanonical splice donor site of exon 5. Isoform 1 transcript ispredicted to yield a DGKE protein 13 amino acids longer thanthe wild-type (p.Lys296_Gly297ins13, Figure 4). Isoform 2 (407bp) differs from isoform 1 because it also includes a 95-bp-long pseudoexon (c.888+278–372) within intron 5 (Figure 3B)because of the recognition of cryptic splice acceptor and donorsites at positions 276–277 and 373–374 bp, respectively, down-stream of exon 5 (Supplemental Table 4). The resultant aber-rant mRNA (c.888_889ins134) is predicted to yield a truncatedDGKE protein because of a frameshift (p.Gly297Valfs*88, Fig-ure 4). This transcript is, however, likely to be degraded by

Figure 1. | Family trees. Pedigrees of family 1 (A) and family 2 (B) carryingDGKEmutations. 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 trianglesrepresent the DGKE c.888+40A.G mutation. Empty triangles represent the DGKE c.966G.A mutation. wt, wild-type allele.

Clin J Am Soc Nephrol 10: ccc–ccc, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 3

Tab

le1.

Clinical

charac

teristicsofpatients

withDGKEmutations

Cha

racteristic

Family

1Fa

mily

2

Patien

t452

Patien

t120

0Pa

tien

tII-1

Patien

tII-2

Patien

tII-4

Ons

etLast

Follo

w-U

pOns

etLast

Follo

w-U

pOns

etLast

Follo

w-U

pOns

etLast

Follo

w-U

pOns

etLast

Follo

w-U

p

Age

(y)

0.8

130.4

100.75

130.6

100.4

4SC

r(m

g/dl)

4.17

0.58

0.7

0.48

0.7

0.68

7.3

0.45

5.3

0.29

Platelet

coun

t(310

9 /L)

6333

996

310

85—

34—

122

—Hem

oglobin(g/dl)

10.7

15.2

10.9

14.1

9.8

—8

—8.3

—LDH

(IU/L)

4000

—90

8—

3396

—10

,181

——

—Total

bilirub

in(m

g/dl)

1.15

—1.4

——

——

——

—Schistocytes

Positive

—Po

sitive

—Po

sitive

—Po

sitive

—Po

sitive

—C3(m

g/dl)

7014

781

151

——

——

Normal

—C4(m

g/dl)

2033

2128

——

——

Normal

—Proteinu

ria(g/24

h)0.41

0.57

0.57

0.21

+++a,b

++a

—+++a,b

—+a

Prot/Creat

ratio

—1.53

—0.44

——

——

——

Hem

aturia

+a

Neg

ative

+a

Neg

ative

+++a,b

+++a,b

—+++a,b

—Traces

Normal

values

areas

follo

ws:serum

creatinine

:children,1–

5ye

ars,0.3–

0.5mg/

dl;ch

ildren5–

10ye

ars,0.5–

0.8mg/

dl,ch

ildren.10

years,0.5–

1.2mg/

dl.Platelet

coun

t:15

0–40

0310

9 /L.

Hem

oglobin:

child

ren5mon

thsto

1ye

ar,10.8–

12.5g/

dl;ch

ildren1–

5ye

ars,11

.7–13

.7g/

dl;ch

ildren5–

10ye

ars,12

–14

.4g/

dl;female.10

years,12

–16

g/dl;male.10

years14

–18

g/dl.LDH:

child

ren,1–

5ye

ars,12

5–20

6IU

/L;children5–

10ye

ars,10

4–20

1IU

/L;children10

–15

years,90

–19

9IU

/L.C

3:90

–18

0mg/

dl.C4:10

–40

mg/

dl.Proteinu

riain

child

ren,

0.2mg/

mgof

protein/

creatinine

ratioin

spot

urineor

,0.2g/

24hproteinu

riaor

nega

tive

dipstick.

Nep

hroticrang

eproteinu

riaisdefi

nedas

protein/

creatinine

ratio.2mg/

mgor

proteinu

ria.3.5g/

24h.

SCr,

serum

creatinine

;LDH,lactate

deh

ydroge

nase;C

3,serum

complem

entC

3leve

ls;C

4,serum

complem

entC

4leve

ls;P

rot,proteinu

ria;Creat,creatinine;+,m

ildleve

l;++,m

oderateleve

l;+++,

seve

releve

l.a D

ipstickwas

used

forthisev

alua

tion

.bStrong

lypo

sitive

resu

lt.

4 Clinical Journal of the American Society of Nephrology

nonsense-mediated mRNA decay, therefore explaining thelower intensity of the corresponding band versus the isoform1 band. Isoform 3 (645 bp) results from skipping both thecanonical and neo-splice donor sites: the splicing machineryonly recognizes the cryptic splice donor site that defined thepseudoexon of isoform 2 (Figure 3B) (24). Notably, this sitehas the highest consensus value for all splice donor siteswithin intron 5 predicted by the Human Splicing Finder soft-ware (Supplemental Table 4). Isoform 3 is characterized by in-frame retention of 372 intronic nucleotides (c.888_889ins372,Figure 3B), which would result in a protein with 124 addi-tional amino acids (p.Lys296_Gly297ins124, Figure 4A). Theseresults suggest that in the presence of the c.888+40A.G mu-tation, the weak canonical splice donor site of exon 5 is ig-nored by the spliceosome, resulting in the production of atleast three aberrant transcripts.Western blot of blood leukocyte lysates documented that

isoform 1 is expressed at the protein level, whereas nosignal was found for isoform 3 (Figure 4B).Exon 5 encodes part of the catalytic domain of DGKE

(Figure 4A) (23). Structural tridimensional modeling sug-gests that the 13 amino acid insertion in isoform 1 shouldresult in structural changes that may affect DGKE kinaseactivity (Supplemental Figure 3 and Supplemental Material).

Identification of a Second Family with the DGKE c.888+40A>G MutationA second unrelated kindred (family 2, Figure 1B) with

the same intronic mutation was identified independently

on the basis of WGS performed on patient II-1. Despiteexcellent coverage (96% of the reference genome at an av-erage depth of 33X), the initial data assessment did notreveal a clear molecular diagnosis (19). A previously de-scribed (14) heterozygous DGKE nonsense mutation,c.966G.A (p.Trp322*), was found in patient II-1 by WGSand was subsequently confirmed by Sanger in all relativesexcept the mother (Supplemental Figure 4A). A more in-depth analysis of all potential maternal-in-origin DGKEvariants was therefore performed. No other coding, nonsyn-onymous variants were identified, and there was no struc-tural variation within or near DGKE. Out of 52 intronic DGKEvariants, only eight were rare (MAF,1%, SupplementalFigure 4B). All were sequenced in the parents to find thoseoriginating from the maternal lineage. Genotyping of thethree affected (nos. II-1, II-2, and II-4, Figure 1B) and oneunaffected sibling (no. II-3) for the two maternal-specificvariants (Supplemental Table 5) revealed that only the af-fected individuals harbored both noncoding variants incompound heterozygosity with the c.966G.A mutation(Supplemental Figure 4C). The first variant (c.888+40A.G)appeared more promising and was the same found in homo-zygosity in family 1. The other (rs145743671) has an MAF of0.55% in control individuals and is .700 bp away fromexons 9 and 10.The three affected siblings, born to an outbred union, were

diagnosed with aHUS in early infancy. In individual II-1,aHUSwas diagnosed at 9 months. He presented with throm-bocytopenia, Coombs’ negative hemolytic anemia with

Figure 2. | Identification of the DGKE c.888+40A>G mutation in family 1. (A) Representative Sanger sequencing electropherograms ofDGKEc.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 theA→Gsubstitution (arrow), and their unaffectedparents (nos. 1417and1421) areheterozygous. The lowermost electropherogram isfrom a homozygouswild-type (wt) healthy control. (B) wt andmutant (mut) genomic nucleotide sequence ofDGKE around intron 5. The position ofthe 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 thecorresponding probabilities predictedby theGenScan software. The orange line traces the canonical splice donor site in thewild-type sequence andthe predicted new splice donor site in the mutant sequence, with the corresponding scores calculated by the Human Splicing Finder software.

Clin J Am Soc Nephrol 10: ccc–ccc, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 5

schistocytes, and mild renal impairment (Table 1). Markedhematuria and proteinuria were also documented. He hadfour relapses in the following year and a fifth relapse at 4 years(Supplemental Figure 4D, Supplemental Table 6). During thesecond and third relapses he had urine protein/creatinineratios of 46 and 19 mg/mg, respectively (normal ,0.2 mg/mg).Treatment was supportive for all episodes, and peritonealdialysis was only prescribed for the last one. At last follow-up (age 13 years), renal function, BP, and complement ac-tivity (CH50 and AH50) were normal, but hematuria andproteinuria were persistent (Table 1). Individuals II-2 andII-4 had aHUS onset at 7 and 5 months, respectively (Table 1),associated with acute renal failure. C3 and C4 levels, mea-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 ofage (Supplemental Figure 4D, Supplemental Table 6). Bothpatients required peritoneal dialysis only during the firstepisode. Nephrotic-range proteinuria was documented dur-ing relapses in individual II-4 (urine protein/creatinine ratio21.2–34.4) and at remission in individual II-2 (urine protein/creatinine ratio 2.6–8.8). Renal function, BP, CH50, andAH50 were normal for both patients at last follow-up, atages 10 and 4 years, respectively (Table 1). Both parentsand a 6-year-old sister are unaffected. The father is of NorthernEuropean descent, and the mother is half-German, half–NativeAmerican (Cherokee).

DGKE c.888+40A>G is a Founder Mutation that is Rare inaHUSBecause two unrelated families of European descent have

the exact same novel intronic variant, we reasoned that thismay be caused by a rare foundermutation.We cross-referencedthe list of the homozygous variants around DGKE locus inthe WES data for individual 452 (Supplemental Table 3)and found that all were heterozygous in the WGS data forpatient II-1. This concordance suggests a common origin forthe c.888+40A.G mutation in the two families. Sequencingdone to assess the frequency of the c.888+40A.G allele in 50patients with pediatric-onset aHUS without a molecular di-agnosis did not identify a single carrier.

DiscussionWe report on the identification of a novel intronic DGKE

mutation, c.888+40A.G, that interferes with normal mRNAsplicing in two unrelated multiplex kindreds with pheno-types consistent with DGKE-associated aHUS. The pathoge-nicity of c.888+40A.G is supported by several lines of evidence.First, this mutation cosegregates with disease following arecessive pattern in both families: it is homozygous forfamily 1 and compound heterozygous for family 2 in con-cert with a known nonsense DGKE mutation (14). Second,the probability of having by chance two unrelated kindredswith phenotype concordant with that of DGKE-associated

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 fromperipheral 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 redarrows in panel B) spanningDGKE exons 4–6. Only one band of 273 bp (wt isoform) was obtained from the healthy control individual, whereasa 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 healthyparents showed both the wt and the aberrant longer amplicons. (B) Diagrams of the normal (wt) and aberrant (mutant isoform 1 [Iso#1], mutantisoform 2 [Iso#2], mutant isoform 3 [Iso#3]) splicing of exon 5. Themutant Iso#1 is characterized by the retention of 39 nucleotides of intron 5.Themutant Iso#2 differs from Iso#1 for the further inclusion of a 95-bp-long pseudoexon from intron 5. Themutant Iso#3 is characterized by theretention of 372 nucleotides. The position of the c.888+40A.G is marked by a broken vertical red line.

6 Clinical Journal of the American Society of Nephrology

aHUS (14,17) and the same novel intronic mutation is verylow. Third, we demonstrate that this intronic mutation actsas a neo-splice site that not only results in the transcriptionof mutant mRNA isoforms predicted to be deleterious toDGKE catalytic activity, but also completely abrogateswild-type DGKE mRNA transcription.Finding multiple patients from unrelated kindreds to

have the exact same mutation is an unusual situation that isexplained either by a mutational hotspot or by remote com-mon ancestry. Our data support the notion that the patternobserved is caused by shared ancestry because all homo-zygous SNVs surroundingDGKE in family 1 are heterozygousin family 2.Serum C3 levels of the two affected siblings from family 1

were slightly depressed at disease onset, but they werealways normal when measured during disease relapse. Theydo not carry mutations in any of the known complement

aHUS–associated genes. It remains unclear whether aHUS-associated DGKE deficiency may directly initiate comple-ment activation or whether these patients carry anothergenetic abnormality in a modifier gene that affects com-plement biology.Despite the advances in understanding aHUS genetic

causes, approximately 40% of patients still do not carrymutations in CFH, MCP, CFI, CFB, C3, THBD, and DGKE ordeletions/rearrangements in CFH-CFHRs region (2,3,5,7,25).To our knowledge, this is the first report describing an in-tronic mutation located beyond exon/intron boundaries inDGKE and in general in aHUS-associated genes. Known in-tronic mutations far from exon/intron boundaries and caus-ing human disorders are rare (26–28). However, thesemutations may be underreported because they are not rou-tinely investigated by standard analysis with Sanger sequenc-ing, which is usually restricted to exons and canonical splice

Figure 4. | Sequence changes identified in the three mutant DGKE isoforms. (A) Schematic representation of the domains identified in DGKEprotein (from SMART, http://smart.embl-heidelberg.de) is illustrated (upper part of panel A) to show the relative position of the changes. In the lowerpart of panel Awe present the sequences of bases and translated amino acids of wt DGKE protein to allow for comparison with those of the threemutantDGKE forms caused by the c.888+40A.G.The subscripts below the amino acids located at the beginning and the endof each exonare usedto reveal their position relative to thewt protein. The sequence of exons 4, 5, and 6 are labeled in orange, green, and gray fonts, respectively. The redfont is used to show the sequences that areunique to the threemutantDGKEproteins. (B)Proteinblot of total proteins extracted fromperipheralbloodleukocytes. Lane 2: leukocytes from a healthy control; lane 3: leukocytes from patient 1200; lane 4: leukocytes from his mother (no. 1421) probedwith an anti-DGKE antibody against the 2–51 N-terminal amino acids. Lane 1 contains total proteins extracted from healthy human platelets asadditional control. Expected band size: DGKE wt, 64 kD; DGKE isoform 1, 65.5 kD; DGKE isoform 3, 78 kD (http://www.expasy.org/). A bandcorresponding toDGKE Iso#1 is present in the sample frompatient 1200, indicating that thismutant is expressed. Themutant Iso#1 andwtDGKE (ofcontrol samples) had a very similar migration pattern when subjected to gel electrophoresis because of the very small difference in the predictedmolecular weights. No band corresponding to Iso#3 was detected, indicating that this isoform is produced in a very small amount, this isoform isunstable and rapidly degraded in the cells, or its conformation negatively affects the integrity of the targeted DGKE epitope. The blot were probedwith 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.

Clin J Am Soc Nephrol 10: ccc–ccc, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 7

sites. Also, in WES, common criteria for selection of var-iants restrict the analysis to coding regions, including the2–4 bp immediately adjacent to the exons. The two familiesreported here suggest that a number of unexplained patientswith aHUS may be accounted for by intronic mutations thatcause aberrant splicing of known aHUS-associated genetranscripts.In recent years, WES and WGS have allowed the

discovery of a growing numbers of disease-associatedgenes (29); however, most variants in noncoding regionswill be missed by WES. This shortcoming could be over-come by WGS; however, because of the large number ofvariants encountered, the choice regarding the ones de-serving follow-up is a very challenging task. This is dem-onstrated by the fact that WGS failed to reveal a clearmolecular diagnosis in family 2 because intronic mutationswere initially filtered out.Another striking feature is the diverse transcriptional

effects of the mutant neo-splice site when it occurs in thecontext of a weak canonical splice donor and many strongcryptic donor and acceptor sites. The same phenomenonmay occur in other DGKE introns, and perhaps in intronsof other known aHUS-associated genes. It would be veryinteresting to determine if one could effectively prioritizeat-risk introns via genome-wide identification of all weakdonor and/or acceptor splice sites.Because the DGKE c.888+40A.G mutation was shown

to be recurrent among patients with early-onset aHUS, di-agnostic and research laboratories should consider the in-clusion of intron 5 in the routine genetic screening ofDGKE. Sequencing of intronic regions of aHUS-associatedgenes followed by analysis of mRNAs sequence couldprovide a valuable, still underexploited tool to resolve pa-tients with unknown genetic defects. Transcriptome se-quencing (30) could soon represent an alternative valuablestrategy to directly detect mutations, including variants thataffect splicing.

AcknowledgmentsWe thank Annalisa Sorosina, RamonaMaranta, Marta Alberti for

Sanger sequencing, Elena Brini, and Alessandro Guffanti fromGenomnia forWESanddataanalysis.Wealso thankErinLoringandCarol Nelson-Williams for help with family 2 sample handlingand genomic DNA preparation, respectively. We thank the patientsand their relatives; without their contribution this work could nothave been done.

Supported by Fondazione ART per la Ricerca sui Trapianti ARTONLUS (Milano, Italy), Fondazione Aiuti per la Ricerca sulle Ma-lattie Rare ARMR ONLUS (Bergamo, Italy), grants from the Euro-pean Union Seventh Framework Programme FP7-EURenOmics(project no. 305608), and The Genomnia Minigrant (Lainate, Milan,Italy) in collaboration with Life Technologies. Mathieu Lemaire isthe recipient of a Kidney Research Scientist Core Education andNational Training Program Post-Doctoral Fellowship Award fromthe Kidney Foundation of Canada and is a member of the In-vestigative PhD Medicine program at Yale University School ofMedicine. Rossella Piras is a recipient of a research contract fromProgetto DDD Onlus, Associazione per la lotta alla DDD.

DisclosuresMarina Noris and Véronique Frémeaux-Bacchi have received

honoraria from Alexion Pharmaceuticals for giving lectures and

participating in advisory boards. Giuseppe Remuzzi has consul-tancy agreements with AbbVie, Alexion Pharmaceuticals, BayerHealthcare, Reata Pharmaceuticals, Novartis Pharma,AstraZeneca,Otsuka Pharmaceutical Europe, Concert Pharmaceuticals: no per-sonal remuneration is accepted, and compensations are paid to hisinstitution for research and educational activities. None of theseactivities has had any influence on the results or interpretation inthis article. The other authors declare no conflicts of interest.

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Received: August 27, 2014 Accepted: February 9, 2015

C.M. and M.L. contributed equally to this work.

Published online ahead of print. Publication date available at www.cjasn.org.

This article contains supplemental material online at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN.08520814/-/DCSupplemental.

Clin J Am Soc Nephrol 10: ccc–ccc, June, 2015 A DGKE Intronic Mutation in Familial HUS, Mele et al. 9