ADCK4-Associated Glomerulopathy Causes Adolescence-Onset FSGS

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†‡ | | Emine Korkmaz,* Beata S. Lipska-Zie˛ tkiewicz, Olivia Boyer,§ ¶ Olivier Gribouval,§ † †† ‡‡ Cecile Fourrage,** Mansoureh Tabatabaei, Sven Schnaidt, Safak Gucer, Figen Kaymaz,§§ || ††† ‡‡‡ Mustafa Arici, Ayhan Dinckan,¶¶ Sevgi Mir,*** Aysun K. Bayazit, Sevinc Emre, ||| ||| Ayse Balat,§§§ Lesley Rees, Rukshana Shroff, Carsten Bergmann,¶¶¶ Chebl Mourani,**** |†††† ‡‡‡‡ † Corinne Antignac,§ Fatih Ozaltin,* §§§§ Franz Schaefer, and the PodoNet Consortium BRIEF COMMUNICATION *Nephrogenetics Laboratory, Department of Pediatric Nephrology, Hacettepe University Faculty of Medicine, Ankara, Turkey; †Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine, Heidelberg, Germany; ‡Department of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland; §Institut National de la Santé et de la Recherche Médicale (INSERM) Unité mixte de recherche (UMR) U1163, Laboratory of Hereditary Kidney Diseases, Paris, France; |Université Paris Descartes, Sorbonne Paris Cité, Imagine Institute, Paris, France; ¶Department of Pediatric Nephrology, Necker Hospital, Assistance Publique– Hôpitaux de Paris, Paris, France; **Paris Descartes University Bioinformatics Platform, Imagine Institute, Paris, France; ††Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany; Departments of ‡‡Pediatric Pathology, §§Histology and Embryology, and ||Nephrology, Hacettepe University Faculty of Medicine, Ankara, Turkey; ¶¶Department of Surgery, Akdeniz University Faculty of Medicine, Antalya, Turkey; ***Department of Pediatric Nephrology, Ege University Medical Faculty, Bornova, Izmir, Turkey; †††Department of Pediatric Nephrology, Cukurova University, Adana, Turkey; ‡‡‡Department of Pediatric Nephrology, Istanbul Medical Faculty, University of Istanbul, Capa, Istanbul, Turkey; §§§Department of Pediatric Nephrology, Gaziantep University Medical Faculty, Gaziantep, Turkey; |||Renal Unit, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom; ¶¶¶Bioscientia Institute for Medical Diagnostics GmbH, Center for Human Genetics, Ingelheim, Germany; ****Hotel Dieu de France, Department of Pediatrics and Pediatric Nephrology, Beirut, Lebanon; ††††Department of Genetics, Necker Hospital, Assistance Publique–Hôpitaux de Paris, Paris, France; ‡‡‡‡Department of Pediatric Nephrology, Hacettepe University Faculty of Medicine, Ankara, Turkey; and §§§§Hacettepe University Center for Biobanking and Genomics, Ankara, Turkey

ABSTRACT 1 Hereditary defects of coenzyme Q10 biosynthesis cause steroid-resistant nephrotic exhibit organ-selective phenotypes. In syndrome (SRNS) as part of multiorgan involvement but may also contribute to isolated the kidney, mitochondriopathies typi- SRNS. Here, we report 26 patients from 12 families with recessive mutations in ADCK4. cally cause proximal tubulopathy2; how- Mutation detection rate was 1.9% among 534 consecutively screened cases. Patients ever, glomerular dysfunction has been with ADCK4 mutations showed a largely renal-limited phenotype, with three subjects reported with mitochondrial DNA exhibiting occasional seizures, one subject exhibiting mild mental retardation, and one mutations in the tRNALEU gene and subject exhibiting retinitis pigmentosa. ADCK4 nephropathy presented during adoles- coenzyme Q biosynthesis defects.2–5 cence (median age, 14.1 years) with nephrotic-range proteinuria in 44% of patients and advanced CKD in 46% of patients at time of diagnosis. Renal biopsy specimens uni- formly showed FSGS. Whereas 47% and 36% of patients with mutations in WT1 and NPHS2 Received December 20, 2014. Accepted March 13, , respectively, progressed to ESRD before 10 years of age, ESRD occurred 2015. almost exclusively in the second decade of life in ADCK4 nephropathy. However, E.K. and B.S.L.-Z. contributed equally to this work. CKD progressed much faster during adolescence in ADCK4 than in WT1 and NPHS2 nephropathy, resulting in similar cumulative ESRD rates (.85% for each disorder) in the Published online ahead of print. Publication date third decade of life. In conclusion, ADCK4-related glomerulopathy is an important novel available at www.jasn.org. differential diagnosis in adolescents with SRNS/FSGS and/or CKD of unknown origin. Correspondence: Dr. Beata S. Lipska-Zie˛ tkiewicz, Department of Biology and Genetics, Medical Uni- J Am Soc Nephrol 27: 63–68, 2016. doi: 10.1681/ASN.2014121240 versity of Gdansk, Debinki 1str, 80211 Gdansk, Po- land, or Dr. Fatih Ozaltin, Department of Pediatric Nephrology, Hacettepe University Faculty of Medi- cine, 06100, Sihiye, Ankara, Turkey. Email: b.lipska@ Mitochondrial cytopathies are clinically involve multiple organ systems and often gumed.edu.pl or [email protected] and genetically heterogeneous disor- present with prominent neurologic and Copyright © 2016 by the American Society of ders. Althoughmostmitochondriopathies myopathic features in childhood, a few Nephrology

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Coenzyme Q (ubiquinone; CoQ10)isa component of the mitochondrial respi- ratory chain,4 a potent lipophilic anti- taster

oxidant, and a cofactor for mitochondrial Mutation prediction dehydrogenases and in pyrimidine nucleoside biosynthesis. CoQ10 is synthe- sized ubiquitously through a multien- SIFT

zyme complex at the inner mitochondrial score 0 damaging disease causing membrane. Mutations in several genes 0 damaging disease causing encoding enzymes of the CoQ10 biosyn- thetic pathway (COQ2, COQ6,and PDSS2) are associated with a glomerular score damaging damaging phenotype. These have been collectively PolyPhen 2 termed CoQ10 glomerulopathies. Re- cently, recessive mutations in ADCK4 93 0.173 benign 0 damaging disease causing 98 1.0 probably

(AarF Domain Containing Kinase-4) NA NA NA disease causing 102 1.0 probably have been added to this list as a novel score Grantham difference cause of steroid-resistant nephrotic syndrome (SRNS).6 ADCK4 interacts with components of the CoQ10 biosynthesis pathway, and patients with ADCK4 mutations have reduced cellular CoQ10 6,7 content. The selective glomerular Finder 3.0 phenotype of patients with ADCK4 mu- Human Splicing exonic ESE site positions potentially breaking ESE site positions potentially breaking ESE site tations may be the result of relative exonic cryptic acceptor site, or alteration of exonic ESE site, or creation of exonic ESS site mutation in early exonic enrichment of ADCK4 and lacking ex- pression of the related protein ADCK3 in podocytes, whereas ADCK3 expres- sion exceeds that of ADCK4 in most other body tissues.6 Conservation We identiﬁed a new patient cohort with within conserved region ADCK4 glomerulopathy among 534 con- highly conserved, highly conserved potential activation of b secutive SRNS cases. ADCK4 mutations

were found in ten patients (1.9%) from a

mostly consanguineous families. In ﬁve b families the mutation was found in further 0.3% 1:10,000 0.3% 0 (not reported) low conservation potential creation of , 0 (not reported) highly conserved mutation in late exonic affected siblings (Supplemental Figure 1: 0 (not reported) Families III–VII). Two additional families comprising nine affected subjects in ADCK4 ﬁ – whom mutations were identi ed – by genome-wide linkage analysis or exome subdomain) sequencing (Supplemental Figure 1: Fami- (helical) lies I, II), yielding a total cohort of 26 patients. Mutational analysis revealed four novel sequence variants and three previously reported homozygous mutations, namely c.645delT, c.1199_1200dupA, and c.532C.T; p.(Arg178Trp) substitution.6 p.(Pro310Leu) kinase (ABC1 p.(Leu98Arg) transmembrane The novel c.1339dupG variant was found in four apparently unrelated Kurdish AA p.(Ala498Glu) families originating from a region in southeast Turkey, suggesting a founder Summary of bioinformatic analyses of the detected novel sequence variants T G . effect. Bioinformatic information on . thenovelvariantsisgiveninTable1 Novel variant Residue change Protein domain MAF In-house allele frequency database representative for Turkish population (collection of 373 individual genomes; accessed October 22, 2014). MAF, minor allele frequency; estimation based on data of 2577 individual genomes cataloged by the 1000 Genomes Project; 6503 samples collected at NHLBI Exome Sequencing Project and data from 60,706 c.1493_1494CC . c.1339dupG p.(Glu447Glyfs10) c.929C Table 1. ESE, exonic splicinga enhancer; ESS, exonic splicing silencer; NA, not applicable. b individuals aggregated by the Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org); (accessed January 31, 2015). and the online supplement. c.293T

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The phenotypic profile is summarized in posterior encephalopathy whereas the than FSGS were commonly observed at Table 2. The disease first manifested in other two require continued anticonvul- time of diagnosis in NPHS2 (Mesangio- adolescence, typically with mild to mod- sive therapy. One patient presented with proliferative GN, Minimal change GN) erate proteinuria with no or mild edema. retinitis pigmentosa. No histories of and WT1 nephropathy (diffuse mesa- However, advanced CKD was present in hearing problems, cardiomyopathy, ngial sclerosis). In ADCK4 patients, ad- almost half of patients at time of diagno- muscle weakness, optical nerve atrophy, vanced CKD at time of diagnosis was sis and progression to ESRD occurred in or hematologic or endocrinologic abnor- more prevalent than in NPHS2.Ofpa- 22 of the 26 patients within a median of 9 malities were reported in any patient. tients with ADCK4 disease, 38.5% pre- (interquartile range 0–44) months from Serum lactate was episodically elevated sented with CKD5, compared with diagnosis. Hematuria was present at in 4 of 11 patients tested, and transient 15.6% of WT1 and 2.9% of NPHS2 cases time of diagnosis in 25% of patients, in- creatine kinase elevation was noted in (P,0.001). Whereas 47% of WT1 and cluding one case with a chief complaint two patients during episodes of AKI. 36% of NPHS2 patients progressed to of macroscopic hematuria accompanied The clinical phenotype of ADCK4- ESRD before reaching 10 years of age, by only trace proteinuria. FSGS was di- related glomerulopathy was compared ESRD occurred almost exclusively in agnosed in all biopsies, including two with the phenotypes of the two most the second decade of life in ADCK4 differentiated as collapsing and tip lesion common genetic podocytopathies, i.e., nephropathy (Figure 1). However, CKD subtypes. NPHS2-andWT1-associated nephrop- progression was much faster during ad- Signs and symptoms compatible with athies (Figure 1, Table 2). Patients with olescence in ADCK4 than in WT1 and neurologic dysfunction were reported ADCK4-related glomerulopathy were NPHS2 nephropathy, resulting in similar in six patients (Supplemental Table 1). significantlyolderattimeofdiagnosis, cumulative ESRD rates (.85%) for the Mild mental retardation and agora- with no cases manifesting before 5 years three genetic forms of SRNS in the third phobia were each present in one case of age, and they presented with less se- decade of life. Neurologic deficits were and two siblings had primary nocturnal vere proteinuria and less edema than more frequent in ADCK4 disease. Renal enuresis. Three patients developed WT1-orNPHS2-associated disease. Hy- and urinary tract malformations oc- electroencephalogram-confirmed sei- pertension was less common than in curred almost exclusively in WT1 . Other zures, including two while on dialysis. WT1 nephropathy. FSGS was the histo- congenital anomalies, mostly heart One subject was eventually diagnosed pathologic diagnosis in all biopsied structural defects, were anecdotally re- with hypertension-related reversible ADCK4 cases, whereas diagnoses other ported in all groups.

Table 2. Comparison of clinical characteristics at time of diagnosis and prospective kidney survival of patients with ADCK4- related SRNS versus patients with NPHS2- and WT1-related glomerulopathy from the PodoNet Registry8 ADCK4 SRNS NPHS2 SRNS WT1 SRNSd n 26 140 66 Age at ﬁrst reported manifestation, years 14.1 (10.8–17.0) 3.4 (1.1–6.6)d 2.0 (0.7–5.4)d Asymptomatic, incidental diagnosis 26.9% 22.9% 28.1% Edema (none/mild/moderate/severe) 54/42/4/0% 48/17/16/19% 47/19/24/10% Proteinuria (subnephrotic/nephrotic range) 57.1/43.9% 14.9/85.1%d 19.4/80.6%c Hematuria 25.0% 44.0% 26.3% Hypertension 30.8% 15.7% 41.5%b CKD stage 3–5 46.1%a 13.6%a,d 23.4%a,b including RTT: 26.9%a 2.9%a,d 15.6%a Age at start of RRT, years 16.1 (13.7–18.0)a 12.9 (7.6–19.4)a,d 10.9 (2.3–17.0)a,d Histopathological diagnosis FSGS/global glomerulosclerosis 61.5% 49.3% 45.0% Diffuse mesangial sclerosis 0 0.7% 33.3%d Mesangioproliferative GN 0 12.9% 4.5% Minimal change GN 0 10.7% 1.5% Other 0 5.0% 3.0% No data/ not performed 38.5% 21.4% 13.7% Neurologic abnormalities (seizures, NI, behavioral problems) 24.0% 5.0%c 6.1%bb Congenital organ abnormalities CAKUT 4.0% CAKUT 0% CAKUT 42.2%d Other 8.0% Other 5.7% Other 4.5% Data are given as median (interquartile range) or percentage. Sixty-one of the patients with WT1 mutations were previously described by Lipska et al. (2014)9 aPercentages given are relative to all observation with information on a speciﬁc variable. bP,0.05. cP,0.01. dP,0.001.

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dysfunction in particular. In patients creatine kinase during AKI are of ques- with mutations in PDSS2, COQ2,and tionable specificity and relevance. COQ6, the other mitochondriopathy Hence, within the wide spectrum of genes associated with SRNS, renal symp- mitochondrial disorders ADCK4 muta- toms usually occur as part of a multisys- tions lead to the most selective glomer- temic disease complex encompassing ular involvement, possibly related to progressive encephalopathy, ataxia, sei- preferential enrichment of ADCK4 in zures, mental retardation, deafness, reti- podocytes. The cytosolic as well as mito- nopathy, hypertrophic cardiomyopathy, chondrial localization of ADCK4 pro- and generalized myopathy.1–5,10 By con- tein in podocytes has led to speculation trast, ADCK4 disease typically manifests that ADCK4 may exert additional func- 6 as an isolated nephropathy with only tions other than CoQ10 biosynthesis. occasional extrarenal symptomatology. Notwithstanding the preferential renal Combining our cohort with five previ- phenotype, patients diagnosed with ously published cases with available ADCK4 nephropathy should undergo data on extrarenal involvement,6 we systematic and repeated screening for oversee 30 patients from 17 families subclinical extrarenal symptoms. with detailed phenotypic information. Our systematic screening of more Figure 1. Age at attainment of ESRD by Among these, 15 patients (50%) never than 500 prospective SRNS cases suggests genetic cause of glomerulopathy. Thirty- showed any extrarenal system involve- that ADCK4 nephropathy may be the five patients with ADCK4 glomerulo- ment. Three patients presented seizures third most common hereditary cause of pathy (current series plus nine previously (thereof two while on dialysis), and two SRNS, with a detection rate of one in 50 6 published cases ), 140 cases of NPHS2- patients each had mild mental retarda- patients compared with one in eight for associated SRNS and 66 cases of WT1- tion and behavioral problems. Two cases NPHS28 and one in 18 for WT1.9 The associated SRNS from PodoNet Registry.8,9 of goiter and single cases of retinitis pig- renal phenotype of ADCK4 disease is mentosa and a lupus-like syndrome were characterized by an insidious onset at Immunosuppressive therapy was ap- reported. Occasionally observed tran- adolescence with mild to moderate pro- plied in 11 cases, of which only in two sient mild elevations of lactate and teinuria and absence of relevant edema siblingsanapparentpartialinitialresponse in the majority of cases. As a conse- to oral steroids was reported. One patient quence of the oligosymptomatic early progressed to ESRD at age 14 years,where- course, advanced CKD is often present as the other is still on conservative therapy at the time of diagnosis. The compari- 8 years after diagnosis of SRNS and is son of renal survival of patients with currently on cyclosporin and angiotensin- ADCK4, WT1,andNPHS2 glomeru- converting enzyme inhibition, with lopathies respectively demonstrates the persistent proteinuria of 2.5 g/m2 per unusual evolution of renal function in day. Two patients were diagnosed while the mitochondriopathy, with almost all still being asymptomatic with only mini- patients progressing to ESRD between mal proteinuria and normal kidney func- 12 and 23 years of age. Hence, at the tion, thanks to the identification of current stage of knowledge SRNS pro- ADCK4 mutations in older siblings with gressing toward ESRD in the first de- established CKD. These subjects were cade of life almost rules out ADCK4 started on CoQ10 supplementation soon disease, whereas ADCK4 nephropathy after diagnosis and demonstrated a de- is an important differential diagnosis crease of proteinuria by 50% and 80%, to consider in cases of adolescent-onset respectively, within 6 weeks of treatment multidrug-resistant proteinuria with (Figure 2, Supplemental Materials). FSGS on biopsy. In this group, where Our assembly and detailed pheno- genetic causes are found in less than typic characterization of the largest 10% of cases by conventional screen- Figure 2. Changes in albuminuria after ADCK4 nephropathy cohort to date al- ing,11,12 mutations in ADCK4 may be starting CoQ10 supplementation in patients NPHS2 lowed us to demonstrate or corroborate with early-stage ADCK4 glomerulopathy. as common as those in and ADCK4 WT1. several features that make Changes shown for two subjects detected Of course, the experience derived disease unique among the hereditary in the asymptomatic early stage of the dis- from the first two disease cohorts com- glomerulopathies in general and ease as a result of our study (further pre- prising patients from 20 families with among those related to mitochondrial sented in Supplemental Material). 15 different mutations is still limited.

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It remains to be seen whether the sug- CONCISE METHODS no. 2012-305608 (EURenOmics), from gested detection rate will be confirmed Bundesministerium für Bildung und For- and whether the uniform disease pattern ADCK4 screening was performed in 534 schung (BMBF) through the e-Rare initiative will diversify with the identification of consecutive SRNS patients from the Podo- (PodoNet), the Polish Ministry of Science more patients and mutations. Net Registry and in-house biobanks at Nec- and Education (grant N402631840). F.O. fi The CoQ10 glomerulopathies repre- kerHospitalinParis,France,theHacettepe was supported by the Scienti c and Techno- sent the first hereditary forms of SRNS University Nephrogenetics Laboratory, logical Research Council of Turkey (grant for which a causative molecular therapy Ankara, Turkey, and the Molecular Genetics 108S417) and by the Hacettepe University is potentially available. Oral CoQ10 sup- Unit at Bioscientia, Ingelheim, Germany. Infrastructure Projects (grant 06A101008 and plementation may reverse proteinuria Clinical information was available for 349 011A101003). and stabilize kidney function if applied patients, including 233 unrelated patients early in the disease course.5,6,10,13 The negative for mutations in the first-line DISCLOSURES commonly late diagnosis of ADCK4 SRNS-associated genes (NPHS2, exons 8– disease so far has precluded efficient 9ofWT1) and 116 not previously tested None. therapy in most affected patients. This individuals. situation may change in the near future The PodoNet, Necker, and Bioscientia co- REFERENCES with earlier diagnosis thanks to in- horts underwent high-throughput sequencing usingcustom-designed multi-gene NGS panels creased awareness of the disease entity, 1. Chinnery PF: Mitochondrial Disorders Over- inclusion of the mitochondrial genes for FSGS and related glomerulopathies. Se- view. In: GeneReviewsÒ, edited by Pagon in routinely performed next generation quencing was performed using the MiSeq/ RA, Adam MP, Ardinger HH, et al, Seattle, sequencing (NGS) panel screening, and HiSeq platform (Illumina, San Diego, CA). All WA, University of Washington, Seattle, pp – proteinuria screening of asymptomatic findings were verified by Sanger sequencing, 1993 2015, [Internet], Available from http:// www.ncbi.nlm.nih.gov/books/NBK1224/ which was also used to test eligible family siblings of affected patients as accom- 2. Doimo M, Desbats MA, Cerqua C, Cassina M, plished in two children in this report. members. Trevisson E, Salviati L: Genetics of coenzyme Both subjects indeed demonstrated a Comparator cohorts with SRNS related to q10 deficiency. Mol Syndromol 5: 156–162, significant decrease of proteinuria on mutations in NPHS2 (n=140) or WT1 (n=66) 2014 8,9 3. Guéry B, Choukroun G, Noël LH, Clavel P, CoQ10 supplementation, raising hopes were extracted from the PodoNet Registry. Rötig A, Lebon S, Rustin P, Bellané-Chantelot Whole-genome linkage analysis using that timely treatment may preserve po- C, Mougenot B, Grünfeld JP, Chauveau D: docyte and kidney function in children 250K single nucleotide polymorphism array The spectrum of systemic involvement in with ADCK4 nephropathy. (Affymetrix, Santa Clara, CA) followed by adults presenting with renal lesion and mi- Based on the preliminary evidence homozygosity mapping using VIGENOS tochondrial tRNA(Leu) gene mutation. JAm – presented here, we propose to perform software was performed in two index families Soc Nephrol 14: 2099 2108, 2003 4. Emma F, Bertini E, Salviati L, Montini G: Renal ADCK4 (Supplemental Figure 1: Families I and II). sequencing, ideally as part of involvement in mitochondrial cytopathies. NGS panel screening, in all patients Illumina TruSeq Exome Enrichment Kit Pediatr Nephrol 27: 539–550, 2012 with adolescent-onset proteinuric kid- was used for paired-end whole exome se- 5. Ozaltin F: Primary coenzyme Q10 (CoQ 10) ney disease in whom autoimmune eti- quencing performed on an Illumina HiSeq deficiencies and related nephropathies. Pe- – ologies have been ruled out on clinical 2000 sequencing system (Illumina, San diatr Nephrol 29: 961 969, 2014 6. Ashraf S, Gee HY, Woerner S, Xie LX, Vega- Diego, CA). and biochemical grounds. Genetic Warner V, Lovric S, Fang H, Song X, Cattran screening should be prioritized over Detailed clinical information on renal and DC, Avila-Casado C, Paterson AD, Nitschké kidney biopsy, particularly in cases of extrarenal symptoms was obtained on all P, Bole-Feysot C, Cochat P, Esteve-Rudd familial disease occurrence or parental ADCK4 patients by way of a standardized J, Haberberger B, Allen SJ, Zhou W, consanguinity. questionnaire. The patient-level data are Airik R, Otto EA, Barua M, Al-Hamed MH, Kari JA, Evans J, Bierzynska A, Saleem ADCK4 giveninSupplementalTable1.Statistical In conclusion, glomerulo- MA, Böckenhauer D, Kleta R, El Desoky S, pathy is a novel cause of adolescent- analyses were performed using the STATIS- Hacihamdioglu DO, Gok F, Washburn J, onset SRNS caused by defective CoQ10 TICA 9.1 (StatSoft; Tulsa, OK) data analysis Wiggins RC, Choi M, Lifton RP, Levy S, Han Z, biosynthesis in podocytes. This recessive software system. Salviati L, Prokisch H, Williams DS, Pollak M, Mendelian disease may present with Clarke CF, Pei Y, Antignac C, Hildebrandt F: ADCK4 mutations promote steroid-resistant signs and symptoms of systemic mito- nephrotic syndrome through CoQ10 bio- chondrial dysfunction, but more often synthesis disruption. JClinInvest123: 5179– manifests as isolated FSGS. Despite the ACKNOWLEDGMENTS 5189, 2013 late clinical manifestation, rapid progres- 7. Desbats MA, Lunardi G, Doimo M, Trevisson sion to end-stage renal disease is com- The research leading to these results has E, Salviati L: Genetic bases and clinical mani- festations of coenzyme Q10 (CoQ 10) de- mon. Early diagnosis will help to identify received funding from the European Com- ficiency. JInheritMetabDis38: 145–156, 2015 ’ children at early disease stages who are munity s Seventh Framework Programme 8. Trautmann A, Bodria M, Ozaltin F, Gheisari eligible for oral CoQ10 supplementation. (FP7/2007-2013) under grant agreement A, Melk A, Azocar M, Anarat A, Caliskan S,

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Emma F, Gellermann J, Oh J, Baskin E, Ksiazek S, Ovunc B, Schoeb DS, McLaughlin HM, T, Firszt-Adamczyk A, Dusek J, Simonetti GD, J, Remuzzi G, Erdogan O, Akman S, Dusek Airik R, Vlangos CN, Gbadegesin R, Hinkes B, Gok F, Gheissari A, Emma F, Krmar RT, J, Davitaia T, Özkaya O, Fotios P, Firszt- Saisawat P, Trevisson E, Doimo M, Casarin A, Fischbach M, Printza N, Simkova E, Mele C, Adamczyk A, Urasinski T, Testa S, Krmar RT, Pertegato V, Giorgi G, Prokisch H, Rötig A, Ghiggeri GM, Schaefer F; PodoNet Consor- Hyla-Klekot L, Pasini A, Özcakar ZB, Sallay P, Nürnberg G, Becker C, Wang S, Ozaltin F, tium: Genetic screening in adolescents with Cakar N, Galanti M, Terzic J, Aoun B, Caldas Topaloglu R, Bakkaloglu A, Bakkaloglu SA, steroid-resistant nephrotic syndrome. Kid- Afonso A, Szymanik-Grzelak H, Lipska BS, Müller D, Beissert A, Mir S, Berdeli A, ney Int 84: 206–213, 2013 Schnaidt S, Schaefer F; for the PodoNet Con- Varpizen S, Zenker M, Matejas V, Santos- 12. Boyer O, Benoit G, Gribouval O, Nevo F, sortium Spectrum of Steroid Resistant and Ocaña C, Navas P, Kusakabe T, Kispert A, Tête MJ, Dantal J, Gilbert-Dussardier B, Congenital Nephrotic Syndrome in Children: Akman S, Soliman NA, Krick S, Mundel P, Touchard G, Karras A, Presne C, Grunfeld JP, The PodoNet Registry Cohort. Clin J Am Soc Reiser J, Nürnberg P, Clarke CF, Wiggins RC, Legendre C, Joly D, Rieu P, Mohsin N, Nephrol 10: 592–600, 2015 Faul C, Hildebrandt F: COQ6 mutations Hannedouche T, Moal V, Gubler MC, Broutin 9. Lipska BS, Ranchin B, Iatropoulos P, in human patients produce nephrotic syn- I, Mollet G, Antignac C: Mutations in INF2 Gellermann J, Melk A, Ozaltin F, Caridi G, drome with sensorineural deafness. JClin are a major cause of autosomal dominant Seeman T, Tory K, Jankauskiene A, Zurowska Invest 121: 2013–2024, 2011 focal segmental glomerulosclerosis. JAm A, Szczepanska M, Wasilewska A, Harambat 11. Lipska BS, Iatropoulos P, Maranta R, Caridi Soc Nephrol 22: 239–245, 2011 J, Trautmann A, Peco-Antic A, Borzecka G, Ozaltin F, Anarat A, Balat A, Gellermann 13. Montini G, Malaventura C, Salviati L: Early H, Moczulska A, Saeed B, Bogdanovic R, J, Trautmann A, Erdogan O, Saeed B, coenzyme Q10 supplementation in primary Kalyoncu M, Simkova E, Erdogan O, Vrljicak Emre S, Bogdanovic R, Azocar M, Balasz- coenzyme Q10 deﬁciency. NEnglJMed K, Teixeira A, Azocar M, Schaefer F; PodoNet Chmielewska I, Benetti E, Caliskan S, Mir S, 358: 2849–2850, 2008 Consortium: Genotype–phenotype associa- Melk A, Ertan P, Baskin E, Jardim H, Davitaia tions in WT1 glomerulopathy. Kidney Int 85: T, Wasilewska A, Drozdz D, Szczepanska 1169–1178, 2014 M, Jankauskiene A, Higuita LM, Ardissino 10. Heeringa SF, Chernin G, Chaki M, Zhou W, G, Ozkaya O, Kuzma-Mroczkowska E, This article contains supplemental material online at Sloan AJ, Ji Z, Xie LX, Salviati L, Hurd TW, Soylemezoglu O, Ranchin B, Medynska A, http://jasn.asnjournals.org/ lookup/suppl/doi:10.1681/ Vega-Warner V, Killen PD, Raphael Y, Ashraf Tkaczyk M, Peco-Antic A, Akil I, Jarmolinski ASN.2014121240/-/DCSupplemental.

68 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 63–68, 2016 SUPPLEMENTAL INFORMATION

TABLE S-1. Clinical and demographic characteristics of the patients with mutations in the ADCK4 gene.

ID Mutation Gender Age at 1st Symptoms Initial Initial Initial Hematuria Histopathology Age at Response to Neurological signs Other extra- Lactate CK manifestation at onset creatinine proteinuri GFR ESRD immunosuppresive and symptomes renal signs (years) [mg/dL] a ml/min/1.73m2 (years) treatment (mmol/L) (U/L) I-1 c.1339dupG M 14 mild edema 0.9 2.1 g/d 73 yes FSGS 17.7 no response to — — 1.2 (2011) 153 (p.Glu447Glyfs*10) steroids, CsA hom. 1.9 (2014)

I-2 c.1339dupG F 7.3 mild edema 0.6 0.78 g/d >75 no FSGS 12.6 no response to epilepsy: generalised Large ASD, 1.6 163 (p.Glu447Glyfs*10) steroids; bioccipital spike and intermediate hom. wave pattern; normal ASVD, no CsA/other brain MRI surgically treatment corrected I-3 c.1339dupG F 17 mild edema NA NA NA no not performed 18 not treated with — — NA CK-MB (p.Glu447Glyfs*10) steroids/CsA/other hom. 16 U/L I-4 c.1339dupG F 27 mild edema, NA NA NA no not performed 31 not treated with — — NA CK-MB (p.Glu447Glyfs*10) hypertension steroids/CsA/other hom. 23 U/L I-5 c.1339dupG F 7 incidental NA 5-6mg/m2/d NA macroscopic not performed — not treated with — — 2.5 90 (p.Glu447Glyfs*10) (5.3 yrs obs) steroids/CsA/other hom. II-1 c.1339dupG F 25.7 edema, vague loin 0.98 3.6 g/d 63 no FSGS 35.4 no response to — — 1.39 NA (p.Glu447Glyfs*10) pain, headaches, steroids; hom. medullary no CsA/other nephrocalcinosis, treatment hypertension II-2 c.1339dupG M 16.7 incidental 14.2 300 mg/dL 4.9 no not performed 16.7 not treated with — trace MI, TI NA 101 (p.Glu447Glyfs*10) in spot urine steroids/CsA/other hom. II-3 c.1339dupG M 13.5 incidental 0.99 6.76 g/d 120 no FSGS/GGS 16.6 no response to mild mental — 1.9 NA (p.Glu447Glyfs*10) steroids; retardation hom. no CsA/other treatment; II-4 not tested M 22 headaches NA NA <15 NA not performed 22 not treated with NA NA NA NA steroids/CsA/other

III-1 c.293T>G F 5.9 incidental 0.4 1.39 g/m2/d >75 no FSGS -- partial response to — primary 0.9 99 (p.Leu98Arg) hom. (8.4 yrs obs) steroids; partial response nocturnal to CsA enuresis

III-2 c.293T>G M 13.3 mild edema 0.6 2 g/m2/d >75 no FSGS 14 partial response to — primary 0.7 144 (p.Leu98Arg) hom. steroids nocturnal enuresis IV-1 c.532C>T M 14.3 hypertension, 14.6 1.5 g/d 7.5 no FSGS 14.3 not treated with — hypermetro NA NA (p.Arg178Trp) hom. polyuria, steroids/CsA/other pia, polidypsja astigmatism Pre-term (born at 34wks) IV-2 c.532C>T M 9.8 mild edema, 13.4 2.5 g/d 2.5 no FSGS 9.8 not treated with — hypermetro NA NA (p.Arg178Trp) hom. hypertension steroids/CsA/other pia, astigmatism V-1 c.293T>G F 13.5 moderate 1.5 +++ (dipstick) 64 no FSGS/GGS 16.1 not treated with — lupus-like 0.5-3.3 NA (p.Leu98Arg) hom. edema steroids/CsA/other symptoms

V-2 c.293T>G F 27 incidental 0.51 0.38 g/d 136 no not performed — not treated with — — NA normal (p.Leu98Arg) hom. (0.4 yrs obs) steroids/CsA/other CoQ10 supplementation VI-1 c.1339dupG M 14.9 chronic renal 15 1.3 g/m2/d 3.5 yes not performed 14.9 not treated with — — NA 2098 at acute (p.Glu447Glyfs*10) failure steroids/CsA/other renal hom. decompensation 69 (2014) VI-2 c.1339dupG F 13.2 hypertension 5.8 0.36 g/mmol 12 yes not performed 13.2 not treated with Epilepsy (grand mal) — 0.9 88 (p.Glu447Glyfs*10) uAlbCr (+++) steroids/CsA/other diagnosed at 10yrs hom.

VI-3 c.1339dupG M 18 chronic renal NA NA NA NA not performed 18 not treated with — — NA NA (p.Glu447Glyfs*10) failure steroids/CsA/other hom.

VI-4 c.1339dupG M 9 incidental, 0.53 0.044 142 no not performed — not treated with — — 1.1 78 (p.Glu447Glyfs*10) asymptomatic g/mmol (0.3 yrs steroids/CsA/other hom. uAlbCr obs) CoQ10 supplementation

VII-1 c.748G>A M 16.9 hypertension, 2.5 2.64g/m2/d 28 yes FSGS 17.4 no response to — — NA NA (p.Asp250Asn) hom. headaches steroids; no CsA/other treatment VII-2 c.748G>A F 13.4 mild edema, 4.0 0.13g/m2/d 15 no FSGS 13.7 no response to — — NA NA (p.Asp250Asn) hom. hypertension, steroids; headaches no CsA/other treatment VIII-1 c.645delT M 15.1 mild edema, 7.4 0.18g/m2/d 9.5 no FSGS 15.8 no response to — — NA NA (p.Phe215Leufs*14) hypertension steroids; hom. no CsA/other treatment IX-1 c.1199_1200dupA M 10.8 edema NA NA NA no FSGS – tip 15.9 no response to — — NA NA (p.His400Asnfs*11) lesion steroids; hom. no CsA/other treatment X-1 c.929C>T F 5.1 incidental 0.83 0.3 g/d 57 no FSGS NOS 13.6 not treated with Generalized seizures — 0.81 38 (p.Pro310Leu) htz; steroids/CsA/other 2° to hypertension c.1493_1494CC>AA while on PD; brain (p.Ala498Glu) htz. MRI: PRES; inconstant defects of visual fields; agoraphobia XI-1 c.645delT M 14.2 edema, 18.4 0.56 g/mmol 4 no FSGS NOS 15.2 not treated with — retinitis NA 1179 at acute renal (p.Phe215Leufs*14) fatigue, uPCr steroids/CsA/other pigmentosa; decompensation and hom. chronic renal hypospadias 2° heart failure failure and 2° heart failure XII-1 c.1339dupG M 17.6 chronic NA ‘nephrotic NA yes cFSGS 18.0 no response to — — NA NA (p.Glu447Glyfs*10) renal failure -range’ steroids, MMF hom. Legend: NA – not available; hom – homozygous; htz – heterozygous; M – male; F – female; uPCr – urinary protein/creatinine ratio; uAlbCr – urinary albumin/creatinine ratio; FSGS – focal segmental glomerulosclerosis; cFSGS – collapsing FSGS; NOS – non otherwise specified; GGS – global glomerulosclerosis; ESRD – end-stage renal disease; CsA – ciclosporin A; MMF – Mycophenolate mofetil; PD – peritoneal dialysis; US – ultrasound; MRI – magnetic resonance imaging; EEG – electroencephalography; PRES – posterior reversible encephalopathy syndrome; MI – mitral insufficiency; TI – triscuspid insufficiency; ASD – atrial septum defect; ASVD – atrioventricular septal defect; CK – creatine kinase; CK-MB – creatine kinase MB isoenzyme;

S-2 Detailed clinical information on the subjects placed on CoQ10 supplementation at early stage of ADCK4 glomerulopathy.

Patient VI-4 was diagnosed with mild albuminuria at still normal GFR at 9 years of age. After mutation confirmation he received CoQ10 supplementation at 30mg/kg per day. Six weeks later albuminuria had dropped by 80%. The patient complained of mild side effects of treatment including loss of appetite and abdominal pain.

Patient V-2 was diagnosed with mild proteinuria at age 27 years. After confirmation of the mutation she received CoQ10 at a starting dose of 10mg/kg per day. Albuminuria decreased by approximately 50% within 6 weeks. Moreover, she subjectively reported better physical fitness and reduced fatigue and weakness. The patient refused to increase the COD10 dose to the recommended 30 mg/kg/d due to financial constraints.

Figure S-3. Family Pedigrees

Family pedigrees showing affected members with confirmed ADCK4-related glomerulopathy. Due to limited space and/or scarce family information, some family members are shown jointly (i.e. the number of siblings is indicated by the corresponding digit within the ideogram).

S-4 EVALUATION OF THE PATHOGENIC CHARACTER OF THE DETECTED NOVEL SEQUENCE VARIANTS

A. In-silico analyses of the effect on protein structure and function

The absence of structural information for ADCK4 precludes detailed assessment of mutation effects on protein structure and function (Protein Data Bank; PDB last accessed 12 Oct, 2014). Nevertheless, the variants are highly likely to be pathogenic in view of the results obtained from a number of in- silico prediction tools (as summarized in Table 1):

1. Three databases, namely ExAC, NHLBI ESP and 1000Genomes, comprising jointly sequencing information of ~ 70,000 individuals were searched to estimate minor allele frequency of the detected variants. The three missense variants were found to be novel, while c.1339dupG allele has been reported at a frequency of 3:100,000. The latter is in line with our observation that this variant is present in general Turkish population with an estimated frequency of ~ 3:1,000 (Table 1). It most likely points towards a putative founder effect in the local population. Indeed, this ADCK4 mutation was found in homozygous state in four apparently unrelated Kurdish families originating from a region in South Eastern Turkey.

2. Multiple-alignment analysis of orthologs of ADCK4 from different species was performed using ClustalW algorithm in order to identify conserved amino acid residues. c.293T>C is predicted to produce a residue change from hydrophobic leucine to hydrophilic arginine in a highly conserved lipophilic helical transmembrane domain. The predicted change most likely disturbs the location of helical structure as it changes its hydrophobicity profile significantly, probably causing its misplacement in the membrane. c.929C>T is predicted to affect another highly conserved residue, Pro310 lying in the critical ABC1 sub-domain of the protein kinase. The exceptional conformational rigidity of proline affects the secondary structure of protein near a proline residue. Its replacement by a hydrophobic branched-chain leucine possibly would result in disruption of the domain structure and hence its proper functioning. The third missense variant c.1493_1494CC>AA is predicted to affect an evolutionarily not conserved alanine residue at the C-terminal part of the protein. Nevertheless, the residue lies adjacent to a region of high homology and the steric hindrance caused by significantly bigger side chain of glutamine may influence protein folding in that region. The forth variant c.1339duG results in a frameshift and leads to premature truncation of the predicted protein.

3. The three most commonly used bioinformatical predictors of variants' pathogenecity, namely PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/) and Mutation Taster (http://www.mutationtaster.org/) predicted all novel variants to be highly deleterious (Table 1). The only exception regarded p.Ala498Glu substitution, that was classified as pathogenic by SIFT and Mutation Taster but tolerable by PolyPhen-2.

B. Verification of splicing signals

The effect of the detected mutations on pre-mRNA splicing was performed using web in silico predictor Human Splicing Finder 3.0 (http://www.umd.be/HSF3/) to estimate possible alterations in either exonic splicing enhancer or the splice site acceptor/donor motifs. All variants were identified as having potential effect on splicing. The results of the analyses are summarized in the Table 1.

S-5 DETAILED METHODS DESCRIPTION

Sanger sequencing

DNA was extracted from peripheral blood following the standard phenol-chloroform protocol or using commercially available kits (Qiagen; Hilden, Germany). The exons of ADCK4 gene with mutations identified by high-throughput sequencing together with their adjacent intronic junctions were analyzed by direct sequencing using ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA). The NCBI 544aa isoform A of ADCK4 (NM_024876.3), corresponding to ENSEMBL transcript ENST00000324464 (ENSG00000123815) was used as reference sequence.

Homozygosity mapping

In Family I, the index patient (I-3) and her relatives (cases: I-1, I-2 and five non-affected members of the family) were genotyped using 250K SNP array (Affymetrix, Santa Clara, CA) according to the manufacturer’s protocol. Genotype files (CHP files) were generated with Affymetrix GTYPE software and were transferred to the VIGENOS (Visual Genome Studio) program (Hemosoft, Ankara, Turkey), which facilitates visualization of a large quantity of genomic data in comprehensible visual screens. We identified only one uninterrupted homozygous region across the genome, which was located on chromosome 19.

Exome Capture and Sequencing

Illumina system (Illumina, San Diego, CA) was used for whole-exome sequencing experiments. Genomic DNA samples were prepared for sequencing by using the Illumina TruSeq Sample Preparation kit. Exonic regions of the samples were captured by Illumina TruSeq Exome Enrichment Kit. Illumina TruSeq PE Cluster Kit v3-cBot-HS was used for paired-end cluster generation and TruSeq SBS Kit v3-HS reagent kit used for sequencing the post-capture libraries. Initial clustering was performed on an Illumina cBot machine. Paired-end sequencing was done on an Illumina HiSeq 2000 system with read length of 104. All procedures were carried out according to the manufacturer’s instructions. Base calling and image analysis was done using Illumina's Real Time Analysis software version 1.13 with default parameters.

High-throughput Sequencing

Different approaches were performed. In the first one, all exons and adjacent intronic boundaries of ADCK4 were targeted by a custom SeqCap EZ choice sequence capture library (NimbleGen, Madison, Wisconsin, USA) as part of a large NGS multi-gene panel for FSGS and related glomerulopathies and subsequently sequenced on an Illumina MiSeq or HiSeq platform according to the manufacturer´s protocol and as described previously. Patients described in this manuscript were analyzed with an

average coverage of more than 200-fold. Bioinformatic analysis was performed using SeqPilot SeqNext moduleTM (v3.5.2, JSI medical systems, Kippenheim, Germany) and in-house bioinformatic pipelines established at the Center for Human Genetics at Bioscientia. All mutations were confirmed by Sanger sequencing.

The second approach comprised a custom-designed multiplex PCR kit (MASTR FSGS, Multiplicom, Niel, Belgium) was used to amplify 546 coding exons of 31 glomerulopathy-related genes, ADCK4 included. PCR performed on 8 to 16 different DNA samples were therefore barcoded, pooled and sequenced paired end (2x300 bp) with MiSeq® Reagent Kit v3 on the MiSeq System (Illumina, San Diego, CA) generating a maximum of 25 million reads and 15 Gb of sequence. The average coverage was more than 500-fold. The pilot study used the beta (evaluation) version of the kit, hence further technical description of the methodology remains confidential at this point.

Alignment, post-processing, calling and annotation

Aligment was made by BWA. Downstream processing was carried out with the Genome Analysis Toolkit (GATK), SAMtools, and Picard, following documented best practices (www.broadinstitute.org/ gatk/guide/topic?name=best-practices). Variant calls were made both by the GATK Unified Genotyper and SAM-tools. The two calling sets were merged using GATK combined variant.

The variants were evaluated using a software system developed by the Paris Descartes University Bioinformatics platform. All the annotation processes were based on the Ensembl database (ver. 74), dbSNPs (ver. 135), EVS (ESP6500SI-V2), 1000 Genomes Project (release date: 21.05.2011), and local SNPs databases (>3000 full exomes).

Statistics

STATISTICA 9.1 (StatSoft; Tulsa, OK, USA) was the data analysis software system used for statistical analyses. Frequencies were compared using chi-squared tests with continuity corrections or Fisher exact test with Freeman-Halton extension for 2x3 and 2x4 tables when applicable. For continuous variables, differences between groups were evaluated using Kruskal-Wallis test for global and Mann- Whitney U tests for pairwise comparisons. Kaplan-Meier survival probability estimates and log-rank tests were performed to estimate the effect of mutations on long-time kidney survival rates. Due to the exploratory character of the analysis, no adjustment for multiple comparisons was done.

S -6 PODONET COLLABORATORS

Chile: Marta Azocar, Santiago; Lily Quiroz, Santiago; Colombia: Lina Maria Serna Higuita, Medellín; Czech Republic: Jiří Dušek, Prague; France: Bruno Ranchin, Lyon; Michel Fischbach, Strasbourg; Georgia: Tinatin Davitaia, Tbilisi; Germany: Jutta Gellermann, Berlin; Jun Oh, Hamburg; Anette Melk, Hannover; Franz Schaefer, Heidelberg; Marianne Wigger, Rostock; Greece: Papachristou Fotios, Thessaloniki; Hungary: Peter Sallay, Budapest; Iran: Alaleh Gheissari, Isfahan; Italy: Guiseppe Remuzzi, Bergamo; Andrea Pasini, Bologna; Gian Marco Ghiggeri, Genova; Gianluigi Ardissino, Milano; Elisa Benetti, Padova; Francesco Emma, Rome; Lebanon: Bilal Aoun, Beirut; Pauline Abou- Jaoudé, Byblos; Lithuania: Augustina Jankauskiene, Vilnius; Poland: Anna Wasilewska, Bialystok; Lidia Hyla-Klekot, Chorzow; Aleksandra Zurowska, Gdansk; Dorota Drozdz, Krakow; Marcin Tkaczyk, Lodz; Halina Borzecka, Lublin; Magdalena Silska, Poznan; Tomasz Jarmolinski, Szczecin; Agnieszka Firszt-Adamczyk, Torun; Joanna Ksiazek, Warsaw; Elzbieta Kuzma-Mroczkowska, Warsaw; Anna Medynska, Wroclaw; Maria Szczepanska, Zabrze; Portugal: Alberto Caldas Afonso, Porto; Helena Jardim, Porto; Serbia: Amira Peco-Antic, Belgrade; Radovan Bogdanovic, Belgrade; Sweden: Rafael T. Krmar, Stockholm; Switzerland: Giacomo D. Simonetti, Bern; Syria: Bassam Saeed, Damascus; Turkey: Ali Anarat, Adana; Aysin Bakkaloglu, Ankara; Ayse Balat, Gaziantep; Z. Esra Baskin, Ankara; Nilgun Cakar, Ankara; Ozlem Erdogan, Ankara; Birsin Özcakar, Ankara; Fatih Ozaltin, Ankara; Onur Sakallioglu, Ankara; Oguz Soylemezoglu, Ankara; Sema Akman, Antalya; Faysal Gok, Gulhane; Salim Caliskan, Istanbul; Cengiz Candan, Istanbul; Sevinc Emre, Istanbul; Sevgi Mir, Izmir; Ipek Akil, Manisa; Pelin Ertan, Manisa; Ozan Özkaya, Samsun; Mukaddes Kalyoncu, Trabzon; United Arab Emirates: Eva Simkova, Dubai; Entesar Alhammadi, Sharjah; Ukraine: Roman Sobko, Lviv.