A Kidney-Disease Gene Panel Allows a Comprehensive

www.kidney-international.org clinical investigation

A kidney-disease gene panel allows a comprehensive genetic diagnosis of cystic and glomerular inherited kidney diseases Gemma Bullich1,2, Andrea Domingo-Gallego1,2, Ivań Vargas3, Patricia Ruiz1, Laura Lorente-Grandoso1, Mońica Furlano2, Gloria Fraga4,A´ lvaro Madrid5, Gema Ariceta5, Mar Borregań6, Juan Alberto Pin˜ero-Fernańdez7, Lidia Rodrı´guez-Penã8, Maria Juliana Ballesta-Martıńez8, Isabel Llano-Rivas9, Mireia Aguirre Men˜ica10, Jose´ Ballarıń2, David Torrents11,12, Roser Torra2,13 and Elisabet Ars1,2,13

1Molecular Biology Laboratory, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau, Universitat Autònoma de Barcelona, Red de Investigación Renal (REDINREN), Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain; 2Nephrology Department, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau, Universitat Autònoma de Barcelona, REDinREN, Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain; 3Informatics Department, Institut de diagnòstic per la imatge, Barcelona, Catalonia, Spain; 4Pediatric Nephrology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Catalonia, Spain; 5Pediatric Nephrology Department, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Catalonia, Spain; 6Genetics Department, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Catalonia, Spain; 7Nephrology Department, Pediatrics Service, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 8Clinical Genetics Department, Pediatrics Service, Hospital Universitario Virgen de la Arrixaca, Centre for Biomedical Research on Rare Diseases, Murcia, Spain; 9Genetics Department, Hospital Universitario Cruces, Biocruces Health Research Institute, Centre for Biomedical Research on Rare Diseases, Barakaldo-Bizkaia, Spain; 10Department of Pediatrics, Nephrology Unit, Hospital Universitario Cruces, Barakaldo-Bizkaia, Spain; 11Barcelona Supercomputing Center, Joint Barcelona Supercomputing Center–Centre for Genomic Regulation (CRG)- Institute for Research in Biomedicine (IRB) Research Program in Computational Biology, Barcelona, Spain; and 12Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

Molecular diagnosis of inherited kidney diseases remains a Therefore, in 17% of cases our genetic analysis was crucial challenge due to their expanding phenotypic spectra as to establish the correct diagnosis. Complex inheritance well as the constantly growing list of disease-causing patterns in autosomal dominant polycystic kidney disease genes. Here we develop a comprehensive approach for and Alport syndrome were suspected in seven and six genetic diagnosis of inherited cystic and glomerular patients, respectively. Thus, our kidney-disease gene panel nephropathies. Targeted next generation sequencing of is a comprehensive, noninvasive, and cost-effective tool for 140 genes causative of or associated with cystic or genetic diagnosis of cystic and glomerular inherited kidney glomerular nephropathies was performed in 421 patients, a diseases. This allows etiologic diagnosis in three-quarters validation cohort of 116 patients with previously known of patients and is especially valuable in patients with mutations, and a diagnostic cohort of 207 patients with unspeciﬁc or atypical phenotypes. suspected inherited cystic disease and 98 patients with Kidney International (2018) -, -–-; https://doi.org/10.1016/ glomerular disease. In the validation cohort, a sensitivity of j.kint.2018.02.027 99% was achieved. In the diagnostic cohort, causative KEYWORDS: autosomal dominant polycystic kidney disease; ciliopathies; mutations were found in 78% of patients with cystic genetic testing; glomerulopathies; inherited kidney diseases; targeted next- disease and 62% of patients with glomerular disease, generation sequencing ª mostly familial cases, including copy number variants. Copyright 2018, International Society of Nephrology. Published by Elsevier Inc. All rights reserved. Results depict the distribution of different cystic and glomerular inherited diseases showing the most likely diagnosis according to perinatal, pediatric and adult disease onset. Of all the genetically diagnosed patients, nherited kidney diseases (IKDs) are the leading cause of chronic kidney disease (CKD) in children and account for 15% were referred with an unspeciﬁed clinical diagnosis I and in 2% genetic testing changed the clinical diagnosis. at least 10% of cases of end-stage renal disease (ESRD) in Europe.1 The most common IKDs are cystic and glomerular nephropathies.2,3 Correspondence: Elisabet Ars, Molecular Biology Laboratory, Fundació Cystic IKDs encompass different diseases and syndromes Puigvert, Cartagena 340-350, Barcelona, Catalonia 08025, Spain. E-mail: characterized by the formation of renal cysts that disrupt the [email protected] structure of the nephron. To date, about 100 genes causing 13RT and EA contributed equally. cystic IKDs have been described; most are expressed in the Received 13 September 2017; revised 9 February 2018; accepted 15 primary cilium of renal tubular cells, so these diseases are February 2018 globally called ciliopathies.4 Cystic IKDs manifest with a

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broad range of phenotypes ranging from adult-onset mild RESULTS disease to perinatal lethal disease. Autosomal dominant Validation cohort polycystic kidney disease (ADPKD) is the most common IKD. Mutations were identified in 115 of the 116 patients in the It is typically an adult-onset disease caused by mutations in validation cohort. A total of 134 of the 135 previously known PKD1 or PKD2. Autosomal recessive polycystic kidney disease mutations were detected in their correct heterozygous or ho- (ARPKD) generally presents in the perinatal period and is mozygous state, for a sensitivity of 99%. The detected muta- caused by mutations in the PKHD1 gene. About 2% to 5% of tions included 76 single nucleotide variants (SNVs), 26 small ADPKD patients show an early and severe phenotype clini- (20 base pairs or less) deletions, 9 small insertions, 4 insertions cally indistinguishable from ARPKD.5 Some of these severely and deletions, 17 large deletions, and 2 large insertions. The affected patients carry >1 mutation in cystic IKD genes, only undetected mutation was a small insertion in PKD1 exon probably aggravating the phenotype.6 Clinical manifestations 1. No spurious pathogenic mutations were found in any of of cystic IKD can be mimicked by mutations in HNF1B, these samples. The results of the validation cohort have been 17,18 which can cause a spectrum of related diseases (HNF1B-RDs), partially published for ADPKD and SRNS. as well as by mutations in genes that typically cause nephronophthisis-related ciliopathies (NPHP-RCs), especially Diagnostic cohort in the perinatal period and early childhood.5 NPHP-RCs Molecular diagnosis of cystic IKD. We identified disease- include a broad range of pediatric AR diseases with high causing mutations in 78% (161 of 207) of patients with genetic heterogeneity.4 Renal cysts can also be present in suspected cystic IKD (Supplementary Table S1), 44% of several multisystemic diseases such as tuberous sclerosis whom had a positive family history of cystic IKDs. Com- complex (TSC), autosomal dominant polycystic liver disease, parison between the clinical suspicion and the definitive oral-facial-digital syndrome, and renal coloboma syndrome.7 molecular diagnosis is shown in Figure 1a. The clinical Glomerular IKDs manifest with proteinuria or micro- diagnosis was confirmed in 129 patients and changed in 3 hematuria or both caused by structural defects in the patients, and a molecular diagnosis was made in 29 patients glomerular basement membrane or the podocytes. Currently, referred with an unspecific cystic disease (Figure 1b). The >50 genes responsible for glomerular IKDs have been detected mutations included SNVs (n ¼ 131), small deletions described.8 Alport syndrome (AS) is the most common (n ¼ 46) and insertions (n ¼ 10), insertions and deletions glomerular IKD and can be caused by mutations in the (n ¼ 1), and large deletions (n ¼24). We designated as COL4A5 (X-linked AS), COL4A3,orCOL4A4 genes (ARAS or disease-causing mutations those variants predicted to be autosomal dominant AS [ADAS]).9 Phenotypic overlap with definitely pathogenic or likely pathogenic, 86 of whom were AS can be found in MYH9-RD and COL4A1-RD.10,11 Steroid- novel (nontruncating likely pathogenic variants are listed in resistant nephrotic syndrome (SRNS) has a high genetic Supplementary Table S2). heterogeneity, with >40 causative genes described to date. All patients with prenatal presentation of a suspected cystic The genetic diagnostic performance is inversely correlated IKD were referred with an unspecified clinical diagnosis, and with age of onset, ranging from almost 100% of patients with our genetic analysis allowed the etiologic diagnosis in 81% congenital NS to around 30% of families manifesting before (25 of 31) of them (Figure 2, Supplementary Table S1). The 25 years of age and much lower in sporadic cases. NPHS2, most prevalent cystic IKD with prenatal presentation was NPHS1, and WT1 are the most frequently mutated genes.12,13 ARPKD (42%, 13 of 31), followed by NPHP-RC (19%, 6 of Focal segmental glomerulosclerosis (FSGS) represents the 31), HNF1B-RD (16%, 5 of 31), and ADPKD (3%, 1 of 31). A most common biopsy finding in pediatric patients with genetic cause of the disease was identified in 79% (19 of 24) of SRNS.14 Adult familial FSGS has been found to be mostly fetuses with oligo- or anhydramnios that resulted in a legal caused by mutations in COL4A3, COL4A4, NPHS2 (p.R229Q termination of pregnancy. These patients carried mutations in variant in trans with a pathogenic mutation), INF2, and PKDH1 (n ¼ 13), NPHP3 (n ¼ 2), TMEM67 (n ¼ 2), NEK8 TRPC6.12,15,16 (n ¼ 1), or HNF1B (n ¼ 1). A disease-causing mutation was In this study, we used a genetic diagnosis approach based identified in 86% (6 of 7) of prenatally diagnosed patients on targeted next-generation sequencing of 140 genes causa- who survived, carrying mutations in HNF1B (n ¼ 4), PKD1 tive of cystic and glomerular IKDs in a total of 421 patients, (n ¼ 1), or CEP290 (n ¼ 1). including a validation cohort (n ¼ 116) with previously The diagnostic yield in patients with pediatric diagnosis known mutations and a diagnostic cohort (n ¼ 305) with (from birth to 18 years) of cystic IKDs was 72% (46 of 64) suspected inherited cystic (n ¼ 207) and glomerular (n ¼ 98) (Figure 2, Supplementary Table S1). ARPKD and ADPKD were diseases. We aimed to (i) develop a global tool for compre- the most frequent molecular diagnoses (both in 19%, 12 of 64), hensive and efficient diagnosis of cystic and glomerular IKDs but their frequency was only slightly higher than that of that is able to identify all types of genetic variants; (ii) NPHP-RC (in 16%, 10 of 64), followed by HNF1B-RD (9%, 6 determine the distribution of the different inherited cystic of 64). A genetic cause of the disease was identified in 30% (3 and glomerular diseases within our cohort depending on the of 10) of pediatric patients referred with an unspecificcystic age at disease onset; (iii) elucidate the genetic cause of disease IKD. Their molecular diagnoses were ARPKD (patient UPKD- in patients with atypical phenotypes. 014), NPHP-RC (patient UPKD-022), and HNF1B-RD

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a Clinical suspicion Molecular diagnosis

Prenatal 81% ARPKD NPHP-RC HNF1B-RD ADPKD n = 31 n = 25 n = 13 n = 6 n = 5 n = 1

ADPKD 88% ADPKD OFD ADPLD n = 101 n = 89 n = 87 n = 1 n = 1

ARPKD 85% ARPKD n = 13 n = 11 n = 11 = 207

n HNF1B-RD 70% HNF1B-RD n = 10 n = 7 n = 7

NPHP-RC 61% NPHP-RC PAX2-RD Cysc IKD Cysc n = 18 n = 11 n = 10 n = 1

TSC 74% TSC n = 19 n = 14 n = 14

Unspeciﬁed 27% ARPKD NPHP-RC HNF1B-RD n = 15 n = 4 n = 2 n = 1 n = 1

AS 82% AS COL4A1-RD n = 55 n = 45 n = 44 n = 1

SRNS/FSGS 30% SRNS/FSGS = 98 n = 27 n = 8 n = 8 n

COL4A1-RD 100% COL4A1-RD n = 2 n = 2 n = 2

MYH9-RD 100% MYH9-RD n n n Glomerular IKD = 1 = 1 = 1

Unspeciﬁed 39% SRNS/FSGS PAX2-RD AS n = 13 n = 5 n = 3 n = 1 n = 1

b Cysc IKD Glomerular IKD

22% 37% Diagnosis conﬁrmed 2% New diagnosis Diagnosis changed 14% 57% 62% No mutaon idenﬁed

5% 1%

Figure 1 | (a) Mutation detection rate and resulting molecular diagnosis for each cystic and glomerular inherited kidney diseases (IKDs). Patients with a prenatal presentation were referred with an unspeciﬁed clinical diagnosis. (b) Impact of the genetic testing on the clinical diagnosis of cystic and glomerular IKDs. ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; ARPKD, autosomal recessive polycystic kidney disease; AS, Alport syndrome; FSGS, focal segmental glomerulosclerosis; NPHP-RC, nephronophthisis-related ciliopathies; OFD, oral-facial-digital syndrome; RD, related disease; SRNS; steroid-resistant nephrotic syndrome; TSC, tuberous sclerosis complex.

(patient UPKD-028). A change in clinical diagnosis was made In patients with adult-onset cystic disease, a molecular in a patient with initial suspicion of NPHP-RC who presented diagnosis was achieved in 80% of patients (90 of 112) with microcysts and CKD at the age of 17 months. His mother (Figure 2, Supplementary Table S1). The most prevalent cystic had an unspeciﬁed glomerulopathy. At the age of 13 years, the IKD was ADPKD, accounting for 67% (75 of 112) of patients. proband presented CKD stage III with proteinuria. Ophthal- For the unspeciﬁed cystic IKD patients, we obtained a mo- mological revision was normal. Genetic testing detected a PAX2 lecular diagnosis in 20% (1 of 5), consisting of ARPKD (pa- mutation (patient PAX2-003). tient UPKD-015) but with extremely mild clinical

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100

70 Not idenﬁed OFD

60 ADPLD PAX2-RD

50 TSC HNF1B-RD Percentage 40 NPHP-RC ADPKD-CIP 30 ADPKD ARPKD 20

0 Prenatal (n = 31) Birth–18 yr (n = 64) >18 yr (n = 112) Figure 2 | Distribution of cystic inherited kidney diseases according to the age at diagnosis. ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; ARPKD, autosomal recessive polycystic kidney disease; CIP, complex inheritance patterns; NPHP-RC, nephronophthisis-related ciliopathies; OFD, oral-facial-digital syndrome; RD, related disease; TSC, tuberous sclerosis complex. presentation. A change in clinical diagnosis was made in 2 p.(Arg3348Gln) reported in the ADPKD mutation database as patients with initial suspicion of ADPKD. Patient OFD1-001, highly likely pathogenic and likely pathogenic, respectively, with oral-facial-digital syndrome, was a 35-year-old woman together with a previously reported likely hypomorphic presenting with ESRD and multiple bilateral renal cysts variant p.(Arg2765Cys) in cis with the p.(Arg3348Gln) without extrarenal manifestations who carried an OFD1 variant. The remaining 5 ADPKD patients had an earlier mutation. Patient ADPLD-001, with autosomal dominant presentation or a more severe phenotype than typical ADPKD polycystic liver disease, was a 40-year-old woman with bilat- or both, but had no available family members to analyze eral renal cysts, multiple hepatic cysts, and an intracranial whether the variants were inherited in cis, in trans, or pre- aneurysm, whose mother and maternal aunt presented with sented a de novo mutation (ADPKD-192, -245, -272, -282, multiple hepatic and renal cysts with normal renal function. -325). All 3 affected family members carried a truncating mutation Molecular diagnosis of glomerular IKD. We identified in the PRKCSH gene. disease-causing mutations in 62% (61 of 98) of patients with Mosaic mutations were identified in 3 patients with TSC. suspected glomerular IKDs (Supplementary Table S3), 81% of Mutant allele frequency was 14% (246 of 5024 reads) in pa- whom had a positive family history of glomerulopathy. The tient TSC-021, 15% (127 of 722 reads) in patient TSC-032, comparison between the clinical suspicion and the definitive and 9% (190 of 2109 reads) in patient TSC-040. Complex molecular diagnosis is shown in Figure 1a. The clinical sus- inheritance patterns in ADPKD genes were suspected in 7 picion was confirmed in 55 patients and changed in 1 patient, patients. Clinical evidence for a potential contribution of all and a diagnosis was made in 5 patients referred with an the variants to the severity of the disease could be assessed in unspecific glomerulopathy (Figure 1b). The detected muta- 2 of them. Patient ADPKD-216 reached ESRD at 51 years of tions included SNVs (n ¼ 61), small deletions (n ¼ 10) and age and carried the PKD2 truncating mutation p.(Ser74- insertions (n ¼ 3), insertions and deletions (n ¼ 1), and large Profs*43) together with the PKD1 missense variant deletions (n ¼ 2) and insertions (n ¼ 2), of whom 43 were p.(Glu1811Lys), classified as indeterminate clinical signifi- novel (nontruncating likely pathogenic variants are listed in cance in the ADPKD mutation database (http://pkdb.mayo. Supplementary Table S4). edu/). Patient ADPKD-184 reached ESRD at 36 years old All 5 patients with congenital-onset glomerular IKD (from and carried 2 PKD1 variants in trans, p.(Glu2771Lys) and birth to 3 months) were molecularly confirmed as congenital

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nephrotic syndrome (Figure 3, Supplementary Table S3). were found in 43% (3 of 7) of patients referred with an un- Disease-causing mutations were detected in the NPHS1 (n ¼ specific glomerular IKD, carrying compound heterozygous 4) and the WT1 (n ¼ 1) genes. NPHS1 mutations (patient UGLO-002) and heterozygous Among patients with pediatric onset of the disease (from 4 WT1 (UGLO-003) or COL4A4 (UGLO-004) mutations. months to 18 years) a molecular diagnosis was achieved in Digenic inheritance with mutations in 2 COL4A genes was 52% (26 of 50) (Figure 3, Supplementary Table S3), 77% of suspected in 6 patients, 2 with a pediatric onset of the disease whom had a positive family history of glomerulopathy. AS and 4 with adult-onset disease. Clinical evidence for a po- was the most frequent molecular diagnosis, accounting for tential contribution of both variants to the phenotype was 38% of patients (19 of 50), one-half of them with X-linked AS found in patient AS-057, who reached ESRD at 26 years and (9 of 19). Molecularly confirmed SRNS represented 8% (4 of had bilateral hearing loss. Molecular analysis revealed a 50) of patients in this cohort. A high genetic heterogeneity nonsense mutation in COL4A5 together with a missense was found in these patients, with mutations not only in the variant in COL4A4 predicted to be highly likely pathogenic. most frequently mutated genes but also in less-studied genes Her sister, who carried only the COL4A4 variant, presented such as CUBN and NUP293. A genetic cause of the glomer- with microhematuria, proteinuria, and bilateral hearing loss ular disease was found in 33% (2 of 6) of pediatric patients at 32 years with normal renal function at 47 years. In contrast, with an unspecified clinical diagnosis, who carried mutations intrafamilial phenotypic variability not attributable to the 2 in LMX1B (patient UGLO-001) and PAX2 (patient UGLO- COL4 variants was found in family AS-252. The proband of 005). A molecular diagnosis different from the clinical diag- this family had CKD stage IV at 54 years, whereas only nosis was found in patient COL4A1-RD-003 with clinical microhematuria with normal renal function was detected in suspicion of AS due to microhematuria, proteinuria, her 48- and 50-year-old sisters. However, molecular analysis congenital cataracts, and microcornea but carrying a de novo revealed that all of them carried heterozygous COL4A3 mutation in COL4A1. Of the 24 pediatric patients with (p.T781_G783del) and COL4A4 (p.Arg1682Gln) variants negative genetic testing, 79% (19 of 24) presented with predicted to be likely pathogenic. In the remaining patients nonfamilial SRNS, 17% (4 of 24) with unspecified glomer- (AS-269, AS-277, AS-287, and AS-288), no more family ulopathy, and 4% (1 of 24) with clinical suspicion of AS. members were available to assess the contribution of both In patients with adult-onset disease, we identified a variants to the phenotype. disease-causing mutation in 70% (30 of 43) of patients (Figure 3, Supplementary Table S3), 93% of whom had a DISCUSSION positive family history of glomerulopathy. The most frequent Here, we present our 3-year experience using an extensive molecular diagnosis was AS, accounting for 61 (26 of 43) of 140–kidney disease gene panel for the genetic diagnosis of a patients, mostly ADAS (19 of 26). Disease-causing mutations Spanish cohort of 421 patients with suspected cystic or

100

80 Not idenﬁed PAX2-RD 70 MYH9-RD 60 COL4A1-RD SRNS/FSGS 50 ADAS

Percentage 40 XLAS female COL4A digenic 30 ARAS XLAS male 20 CNS 10

0 Birth–12 mo (n = 5) 1–18 yr (n = 50) >18 yr (n = 43) Figure 3 | Distribution of glomerular inherited kidney diseases according to the age at disease onset. ADAS, autosomal dominant Alport syndrome; ARAS, autosomal recessive Alport syndrome; CNS, congenital nephrotic syndrome; FSGS, focal segmental glomerulosclerosis; RD, related disease; SRNS; steroid-resistant nephrotic syndrome; XLAS, X-linked Alport syndrome.

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glomerular IKDs. Our results depict the distribution of sequencing; and (iii) some complex genes such as PKD1 inherited cystic and glomerular diseases according to the age cannot be evaluated.28 at disease onset and show how genetic testing allows the In our cohort, 15% (34 of 222) of all the genetically achievement of a definitive diagnosis that in some patients diagnosed patients were referred with an unspecified clinical may even differ from the initial clinical suspicion. diagnosis and 2% (4 of 222) had an inaccurate clinical Clinicians are being confronted with an expanding diagnosis. Therefore, in 17% of cases, our genetic analysis was phenotypic spectra of IKDs as well as a constantly growing list crucial to establish the correct diagnosis. This is especially of disease-causing genes.19,20 Therefore, broader diagnostic relevant in distinguishing between different cystic IKDs in approaches are required. Whole exome sequencing has been severe fetal cases detected by prenatal ultrasound screening. used as a global diagnostic tool for IKD,21 but it is not an Some of these patients have a perinatal lethal disease, but the accurate approach to screen for mutations in the main identification of their pathogenic mutations will enable pre- causative gene of IKDs, PKD1, which has 6 almost identical cise genetic counseling of their parents, who may benefit from pseudogenes. PKD1 complexity also explains why it has been a future prenatal or preimplantational genetic diagnosis. Our excluded from several cystic kidney disease panels. Moreover, approach allowed a definitive diagnosis in approximately 80% some genes such as TTC21B and PAX2, initially involved in of the patients with a prenatal presentation of cystic IKDs, cystic IKD, have recently been associated with glomerular one-half of them affected by ARPKD. A renal gene panel has – IKDs,22 25 as illustrated by our 2 patients carrying PAX2 been used for the study of a highly consanguineous Saudi mutations, 1 of them with clinical suspicion of NPHP-RC Arabian cohort with antenatal cystic IKD. Genetic diagnosis (PAX2-003) and the other with unspecified glomerular IKD was achieved in 62% to 70% of patients, most of them having (UGLO-005). Because cystic and glomerular nephropathies NPHP-RC.29 are the most common IKDs, we have developed a genetic Cystic kidneys in children may develop as a result of diagnostic approach based on capture enrichment, which has different diseases and syndromes that vary with regard to proved suitable for PKD1,17,26 to simultaneously sequence their mode of inheritance, age of onset of disease, and severity 140 genes causative of these diseases. A high sensitivity and of renal and extrarenal phenotypes. Clinical approaches based specificity for detection of all types of genetic variants, from on the kidney size, the localization of cysts, and the presence SNVs to copy number variants (CNVs), was demonstrated in of extrarenal features have been described.30 However, there is a large validation cohort. clinical overlap among these diseases that hampers their In our diagnostic cohort, the genetic cause was identified correct diagnosis. In our patients with pediatric onset of cystic in 78% of patients with suspected cystic IKDs (44% familial IKDs, the main genetic diagnoses were ARPKD and ADPKD cases) and 62% of patients with suspected glomerular IKDs but 16% (10 of 64) were referred with an unspecified clinical (81% familial cases). These high diagnostic rates might be diagnosis. explained by our strict inclusion criteria for genetic testing, The most prevalent cystic IKD in adult-onset patients was especially in patients with suspected glomerular IKDs, most ADPKD, accounting for >60% of patients. In this context, it of whom had a positive family history. A further plus of our should be borne in mind that genetic diagnosis of ADPKD is approach is that it allows detection of CNVs, which accounted only indicated in specific situations.31 Complex inheritance for 10% of the genetically diagnosed patients (23 of 222), patterns in ADPKD patients may involve incompletely consisting of patients with HNF1B-RD (n ¼ 9), NPHP-RC penetrant or hypomorphic PKD1 alleles that cause mild cystic (n ¼ 5), ADPKD (n ¼ 3), TSC (n ¼ 2), X-linked AS (n ¼ disease when inherited alone or aggravate the severity of the 2), and ADAS (n ¼ 2). Thus, CNVs might account for a cystic disease in patients with another mutation inherited in nonnegligible proportion of patients with cystic and trans.32,33 Detailed phenotyping is essential to interpret the glomerular IKDs and should be assessed in the routine ge- pathogenicity of these combinations of variants, as illustrated netic diagnosis. Recently, a similar approach based on targeted by patient ADPKD-184. He was incidentally diagnosed at 33 next-generation sequencing of a 127-gene panel was applied years with CKD stage IV and reached ESRD at 36 years old. to a small cohort of 56 families, achieving a 59% diagnostic He carried 2 likely pathogenic PKD1 variants in trans and a yield without detection of any CNV.27 Another recent study hypomorphic variant (p.[Glu2771Lys];[Arg2765Cy- proposed targeted exome sequencing of >2000 Online s;Arg3348Gln]), according to the ADPKD mutation database Mendelian Inheritance in Man (OMIM) disease genes with classification. Two fully penetrant PKD1 mutations in trans subsequent phenotype-based analysis limited to 207 renal are predicted to result in embrionic lethal disease. Thus, the genes (subdivided into 10 multigene panels) for genetic patient’s phenotype indicates that only p.[Glu2771Lys] is a testing. A genetic diagnosis was identified in 43% of their 135 fully penetrant PKD1 pathogenic mutation and the other 2 studied families, consecutively referred to their diagnostic PKD1 variants in cis [Arg2765Cys;Arg3348Gln] may be 2 genetic service with no strict inclusion criteria. An advantage hypomorphic alleles. These alleles may contribute to his 30- of sequencing the broad range of OMIM genes is that addi- year earlier onset of ESRD, in comparison to the 67-year tional genes can be further analyzed. However, this approach median age at onset of ESRD for a carrier of a non- has several limitations: (i) it does not detect CNVs; (ii) some truncanting PKD1 mutation.34 Also, patient ADPKD-216 regions are incompletely covered, requiring Sanger with digenic inheritance, carrying a PKD2 truncating

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mutation and PKD1 missense variant, reached ESRD 28 years helps the diagnosis of patients with unspecific or atypical earlier than the median age for a carrier of a PKD2 muta- phenotypes. Finally, the strict clinical inclusion criteria tion.34 Our results support a relatively high frequency of established in this study are intended to favor genetic testing mosaic mutations in TSC patients, which may be undetected in patients with likely monogenic cause of nephropathy, being if insufficient depth of coverage is achieved. Mosaic TSC among the key points that explain our high diagnostic yield. patients have been reported in 6% to 58% of the 10% to 15% Consequently, the likelihood of causative mutation identifi- of TSC patients with no mutations identified after conven- cation cannot be extrapolated in patients not fulfilling our tional molecular diagnosis assessment.35,36 inclusion criteria. Genetic testing in glomerular nephropathies is mainly In conclusion, massive parallel sequencing of our kidney indicated in familial cases and also in patients with a very disease gene panel is a comprehensive, noninvasive, efficient, early onset glomerulopathy. We identified the disease-causing and cost-effective tool for genetic diagnosis of cystic and mutation in all congenital-onset patients, all of whom carried glomerular IKDs. Our approach allows the etiologic diagnosis podocyte gene mutations. In patients with an age at disease in three-quarters of affected individuals, enabling a more onset >1 year, our diagnostic yield was halved and mainly precise estimation of the risk of progression of renal disease limited to familial cases, AS (mostly X-linked AS) being the and risk of extrarenal manifestations. Furthermore, it allows most frequent diagnosis. In children, SRNS and AS are clin- the identification of at-risk relatives and enables genetic ically distinguishable entities but in adult-onset patients, the counseling. phenotypes of these diseases frequently overlap. Of note, we obtained a higher diagnostic rate for adults, compared with METHODS children, with suspected glomerular IKDs. This difference Patients might be explained by the fact that the main clinical diagnosis A total of 421 index patients with suspected IKDs were studied, in adults is AS, which has a genetic diagnostic yield of around including a validation cohort (n ¼ 116) with previously known 80%,28,37 whereas about one-half of our pediatric patients mutations and a diagnostic cohort (n ¼ 305). The patients in the presented with SRNS, which has a genetic diagnostic rate of validation cohort carried a total of 135 mutations in the following around 30%.13 Until the advent of next-generation genes: PKD1, PKD2, PKHD1, HNF1B, NPHP1, UMOD, TSC2, sequencing technologies, the only genes contemplated for COL4A5, COL4A3, NPHS1, NPHS2, WT1, INF2, TRPC6, and GLA. genetic diagnosis of adult-onset FSGS were NPHS2, INF2, The diagnostic cohort comprised 305 index patients referred to our n ¼ n ¼ TRPC6, ACTN4, and CD2AP.12,38 The identification of het- laboratory with suspected cystic ( 207) or glomerular ( 98) IKD from January 1, 2014 to December 31, 2016 that fulfilled spe- erozygous mutations in the COL4A3 and COL4A4 genes as cific inclusion criteria for genetic testing. Informed consent was causative of adult-onset FSGS has broadened the genetic fi 15,39 obtained for all patients. A speci c clinical diagnosis was suspected spectrum associated with this pathological entity. Our in 161 patients with cystic IKDs and 85 patients with glomerular results support a high frequency of ADAS, also named thin IKDs, whereas the remaining patients had an unspecified clinical basement membrane nephropathy or collagen IV nephropa- diagnosis. Patients of the diagnostic cohort were classified into 3 thy,40 in adult-onset patients, usually with FSGS as the pre- groups depending on the age at diagnosis. senting histological lesion and positive family history of Patients referred for genetic testing with suspected cystic IKDs glomerular disease.16,41 Digenic inheritance involving COL4A (n ¼ 207) were classified into prenatal, pediatric (from birth to 18 genes has been reported in patients with a more severe years), and adult (>18 years) onset. Prenatal (n ¼ 31) and pediatric n ¼ phenotype than those with only 1 mutated gene,42,43 as in our ( 64) onset groups included patients with any or a combination patient AS-057. In contrast, family AS-252, with digenic in- of the following: increased renal echogenicity, loss of cortico- medullary differentiation, renal cysts, or abnormal-sized kidneys for heritance, presented a high phenotypic variability, with severe the age on renal ultrasound. Adult-onset patients consisted of 112 disease in the proband but only microhematuria in her 2 patients with bilateral renal cysts and a family history of cystic IKDs sisters, suggesting that other genetic and environmental fac- or sporadic cases with a clear suspicion of a specific cystic IKDs. tors may explain the high phenotypic variability found in AS. Patients with suspected glomerular IKD (n ¼ 98) were grouped Our genetic testing approach is cost-effective for several into congenital (from birth to 3 months), pediatric (from 4 months reasons. First, the overall cost of sequencing our 140-gene to 18 years), and adult (>18 years) onset. Patients with congenital- panel is about 50% to 70% cheaper than the Sanger onset nephropathy included 5 patients with congenital nephrotic sequencing and multiplex ligation-dependent probe amplifi- syndrome. Patients with pediatric-onset disease (n ¼ 50) satisfied 1 cation analysis of the genes involved in ADPKD (PKD1/ of the following criteria: (i) family history of nephropathy or con- PKD2)orAS(COL4A3/COL4A4/COL4A5) and even cheaper sanguinity together with hematuria, proteinuria, CKD, renal biopsy for high genetic heterogeneity diseases such as NPHP-RC or showing FSGS, or ultrastructural anomalies in the glomerular basement membrane or a combination of these; (ii) hematuria and SRNS/FSGS. Second, a short turnaround time is achieved by proteinuria with ultrastructural anomalies in the glomerular base- processing 24 patients with different IKDs in parallel in the ment membrane, or (iii) nephrotic range proteinuria (>40 mg/h/ same HiSeq (Illumina, San Diego, CA) sequencing run. Third, m2) and steroid resistance. Adult-onset patients (n ¼ 43) were it allows the detection of all types of genetic variants, from included if they fulfilled criteria i or ii. SNVs to CNVs, in the same approach. Fourth, the simulta- All patients with bilateral renal cysts/$2 renal angiomyolipomas neous analysis of this broad range of kidney disease genes or hematuria/proteinuria/CKD as part of a syndromic disease were

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included. In all age groups, we excluded patients with a likely im- 2014/SGR-1441), and the Fundación Renal Iñigo Álvarez de Toledo mune pathogenesis as indicated by development of steroid resistance (FRIAT) in Spain. We thank Instituto de Investigaciones Biomédicas at a later stage of the disease or with primary disease recurrence after Sant Pau Biobank for providing some of the samples. renal transplant. SUPPLEMENTARY MATERIAL Kidney-disease gene panel Supplementary Materials and Methods. We designed a kidney-disease gene panel including 140 genes caus- Figure S1. Schematic visualization of the workflow used for genetic ative or associated with cystic and glomerular IKDs, as well as genes diagnosis of cystic and glomerular inherited kidney diseases (IKDs). that may cause phenocopies in humans or related phenotypes in del, deletions; ins, insertions; MLPA, multiplex ligation-dependent animal models (Supplementary Table S5). All exons and exon-intron probe amplification; SNV, single nucleotide variants. boundaries (plus 20 base pairs at each end) of these genes were Table S1. Clinical and genetic data of patients diagnosed with cystic captured using a custom NimbleGen SeqCap EZ Choice Library inherited kidney diseases (IKDs) in whom disease-causing mutations (NimbleGen; Roche, Madison, WI) for a final targeted region of were identified. 1.05 Mb. Table S2. Novel nontruncating likely pathogenic variants found in genes causative of cystic inherited kidney diseases (IKDs). Library preparation and sequencing Table S3. Clinical and genetic data of patients diagnosed with glomerular inherited kidney diseases (IKDs) in whom disease-causing Libraries were prepared according to the manufacturer’s standard mutations were identified. protocol, NimbleGen SeqCap EZ Library SR version 4.3. Pools of 24 Table S4. Novel nontruncating likely pathogenic variants found in patients were hybridized to the custom NimbleGen SeqCap EZ genes causative of glomerular inherited kidney diseases (IKDs). Choice and sequenced on a HiSeq2500 instrument or NextSeq500 Table S5. Kidney-disease gene panel and mean depth of coverage (Illumina). Data analysis was performed using an open-source in- for targeted genes across individuals. 17,18 fi house pipeline, as previously reported with few modi cations Supplementary material is linked to the online version of this paper at (Supplementary Materials and Methods). Analysis of CNVs was www.kidney-international.org. performed using the CONTRA (Copy Number Targeted Rese- quencing Analysis) tool.44 A schematic representation of the work- fl REFERENCES ow is shown in Supplementary Figure S1. 1. ERA-EDTA Registry: ERA-EDTA Registry Annual Report 2009. Amsterdam, The Netherlands: Academic Medical Center, Department of Medical Filtering and evaluation of the pathogenicity of the variants Informatics, 2011. Variants were included if minor allele frequency was <0.01 and they 2. Alkanderi S, Yates LM, Johnson SA, Sayer JA. Lessons learned from a multidisciplinary renal genetics clinic. QJM. 2017;110:453–457. were not seen as homozygous in any publicly available population 3. Mallett A, Fowles LF, McGaughran J, et al. A multidisciplinary renal databases (Exome Aggregation Consortium, 1000 Genomes, Exome genetics clinic improves patient diagnosis. Med J Aust. 2016;204:58–59. Sequencing Project), except for likely hypomorphic variants. 4. Vivante A, Hildebrandt F. Exploring the genetic basis of early-onset Protein-truncating variants, deletions or insertions involving $5 chronic kidney disease. Nat Rev Nephrol. 2016;12:133–146. 5. Bergmann C. ARPKD and early manifestations of ADPKD: the original amino acids, and canonical splice site variants were considered polycystic kidney disease and phenocopies. Pediatr Nephrol. 2015;30:15–30. definitely pathogenic. In-frame variants were classified as likely 6. Bergmann C, von Bothmer J, Ortiz Brüchle N, et al. Mutations in multiple pathogenic when they fulfilled $2 of the following criteria: (i) they PKD genes may explain early and severe polycystic kidney disease. JAm had been previously reported as disease-causing in individuals with a Soc Nephrol. 2011;22:2047–2056. similar phenotype, (ii) the altered amino acid was highly conserved 7. Cramer MT, Guay-Woodford LM. Cystic kidney disease: a primer. Adv Chronic Kidney Dis. 2015;22:297–305. and the variant was not present in orthologs, and (iii) they were 8. Bierzynska A, Soderquest K, Dean P, et al. MAGI2 mutations cause predicted to be potentially deleterious by $2 in silico tools for congenital nephrotic syndrome. J Am Soc Nephrol. 2017;28:1614–1621. missense variants or by 1 tool for deletions or insertions or both. 9. Kruegel J, Rubel D, Gross O. Alport syndrome—insights from basic and Noncanonical splice site variants were considered likely pathogenic if clinical research. Nat Rev Nephrol. 2013;9:170–178. 10. Savoia A, Pecci A. MYH9-related disorders. In: Adam MP, Ardinger HH, splicing alteration was predicted for most tools. All candidate Pagon RA, et al., eds. GeneReviews . Seattle, WA: University of pathogenic variants were validated by conventional polymerase chain Washington; 2008:1993–2018 [updated July 16, 2016]. Available at: reaction amplification and Sanger sequencing or by multiplex https://www.ncbi.nlm.nih.gov/books/NBK2689/. fi 11. Plaisier E, Ronco P. COL4A1-related disorders. In: Adam MP, Ardinger HH, ligation-dependent probe ampli cation analysis. Segregation analysis was assessed for all the available family members. Lack of segregation Pagon RA, et al., eds. GeneReviews . Seattle, WA: University of Washington; 2009:1993–2018 [updated July 7, 2016]. Available at: excluded the variant as disease-causing. https://www.ncbi.nlm.nih.gov/books/NBK7046/. 12. Santín S, Bullich G, Tazón-Vega B, et al. Clinical utility of genetic testing in DISCLOSURE children and adults with steroid-resistant nephrotic syndrome. Clin J Am – All the authors declared no competing interests. Soc Nephrol. 2011;6:1139 1148. 13. Sadowski CE, Lovric S, Ashraf S, et al. A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome. J Am Soc Nephrol. 2015;26: – ACKNOWLEDGMENTS 1279 1289. We thank the patients for taking part in this study and the referring 14. Trautmann A, Bodria M, Ozaltin F, et al. Spectrum of steroid-resistant and congenital nephrotic syndrome in children: the podoNet registry cohort. physicians who participated. This project was funded by the Instituto Clin J Am Soc Nephrol. 2015;10:592–600. de Salud Carlos III/Fondo Europeo de Desarrollo Regional (FEDER) 15. Malone AF, Phelan PJ, Hall G, et al. Rare hereditary COL4A3/COL4A4 funds (PI13/01731, PI15/01824, PI16/01998, Redes Temáticas de variants may be mistaken for familial focal segmental glomerulosclerosis. Investigación Cooperativa en Salud (RETICS) Red de Investigación Kidney Int. 2014;86:1253–1259. Renal (REDINREN) RD16/0009, PT13/0010/0034), the Agència de 16. Gast C, Pengelly RJ, Lyon M, et al. Collagen (COL4A) mutations are the Gestió d’Ajuts Universitaris i de Recerca (AGAUR) Catalan Government most frequent mutations underlying adult focal segmental Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (AGAUR glomerulosclerosis. Nephrol Dial Transplant. 2016;31:961–970.

8 Kidney International (2018) -, -–- G Bullich et al.: Next-generation sequencing of kidney gene panel clinical investigation

17. Trujillano D, Bullich G, Ossowski S, et al. Diagnosis of autosomal dominant 31. Ars E, Bernis C, Fraga G, et al. Spanish guidelines for the management of polycystic kidney disease using efficient PKD1 and PKD2 targeted next- autosomal dominant polycystic kidney disease. Nephrol Dial Transplant. generation sequencing. Mol Genet Genomic Med. 2014;2:412–421. 2014;29(suppl 4):iv95–iv105. 18. Bullich G, Trujillano D, Santin S, et al. Targeted next-generation 32. Rossetti S, Kubly VJ, Consugar MB, et al. Incompletely penetrant PKD1 sequencing in steroid-resistant nephrotic syndrome: mutations in alleles suggest a role for gene dosage in cyst initiation in polycystic multiple glomerular genes may influence disease severity. Eur J Hum kidney disease. Kidney Int. 2009;75:848–855. Genet. 2015;23:1192–1199. 33. Vujic M, Heyer CM, Ars E, et al. Incompletely penetrant PKD1 alleles 19. Stokman MF, Renkema KY, Giles RH, et al. The expanding phenotypic mimic the renal manifestations of ARPKD. J Am Soc Nephrol. 2010;21: spectra of kidney diseases: insights from genetic studies. Nat Rev 1097–1102. Nephrol. 2016;12:472–483. 34. Cornec-Le Gall E, Audrezet M-P, Chen J-M, et al. Type of PKD1 mutation 20. Ars E, Torra R. Rare diseases, rare presentations: recognizing atypical influences renal outcome in ADPKD. J Am Soc Nephrol. 2013;24:1006– inherited kidney disease phenotypes in the age of genomics. Clin Kidney 1013. https://doi.org/10.1681/ASN.2012070650. J. 2017;10:586–593. 35. Qin W, Kozlowski P, Taillon BE, et al. Ultra deep sequencing detects a low 21. Gee HY, Otto EA, Hurd TW, et al. Whole-exome resequencing rate of mosaic mutations in tuberous sclerosis complex. Hum Genet. distinguishes cystic kidney diseases from phenocopies in renal 2010;127:573–582. ciliopathies. Kidney Int. 2014;85:880–887. 36. Tyburczy ME, Dies KA, Glass J, et al. Mosaic and intronic mutations in 22. Davis EE, Zhang Q, Liu Q, et al. TTC21B contributes both causal and TSC1/TSC2 explain the majority of TSC patients with no mutation modifying alleles across the ciliopathy spectrum. Nat Genet. 2011;43: identified by conventional testing. PLoS Genet. 2015;11:e1005637. 189–196. 37. Morinière V, Dahan K, Hilbert P, et al. Improving mutation screening in 23. Huynh Cong E, Bizet AA, Boyer O, et al. A homozygous missense familial hematuric nephropathies through next generation sequencing. mutation in the ciliary gene TTC21B causes familial FSGS. J Am Soc J Am Soc Nephrol. 2014;25:2740–2751. Nephrol. 2014;25:2435–2443. 38. Benoit G, Machuca E, Antignac C. Hereditary nephrotic syndrome: a 24. Sanyanusin P, Schimmenti LA, McNoe LA, et al. Mutation of the PAX2 systematic approach for genetic testing and a review of associated gene in a family with optic nerve colobomas, renal anomalies and podocyte gene mutations. Pediatr Nephrol. 2010;25:1621–1632. vesicoureteral reflux. Nat Genet. 1995;9:358–364. 39. Voskarides K, Damianou L, Neocleous V, et al. COL4A3/COL4A4 25. Barua M, Stellacci E, Stella L, et al. Mutations in PAX2 associate with mutations producing focal segmental glomerulosclerosis and renal adult-onset FSGS. J Am Soc Nephrol. 2014;25:1942–1953. failure in thin basement membrane nephropathy. J Am Soc Nephrol. 26. Eisenberger T, Decker C, Hiersche M, et al. An efficient and 2007;18:3004–3016. comprehensive strategy for genetic diagnostics of polycystic kidney 40. Torra R, Tazon-Vega B, Ars E, Ballarin J. Collagen type IV (alpha3-alpha4) disease. PLoS One. 2015;10:e0116680. nephropathy: from isolated haematuria to renal failure. Nephrol Dial 27. Mori T, Hosomichi K, Chiga M, et al. Comprehensive genetic testing Transplant. 2004;19:2429–2432. approach for major inherited kidney diseases, using next-generation 41. Fallerini C, Dosa L, Tita R, et al. Unbiased next generation sequencing sequencing with a custom panel. Clin Exp Nephrol. 2017;21:63–75. analysis confirms the existence of autosomal dominant Alport syndrome 28. Mallett AJ, McCarthy HJ, Ho G, et al. Massively parallel sequencing and in a relevant fraction of cases. Clin Genet. 2014;86:252–257. targeted exomes in familial kidney disease can diagnose underlying 42. Mencarelli MA, Heidet L, Storey H, et al. Evidence of digenic inheritance genetic disorders. Kidney Int. 2017;92:1493–1506. in Alport syndrome. J Med Genet. 2015;52:163–174. 29. Al-Hamed MH, Kurdi W, Alsahan N, et al. Genetic spectrum of Saudi Arabian 43. Fallerini C, Baldassarri M, Trevisson E, et al. Alport syndrome: impact patients with antenatal cystic kidney disease and ciliopathy phenotypes of digenic inheritance in patients management. Clin Genet. 2017;92: using a targeted renal gene panel. J Med Genet. 2016;53:338–347. 34–44. 30. Kurschat CE, Muller R-U, Franke M, et al. An approach to cystic kidney 44. Li J, Lupat R, Amarasinghe KC, et al. CONTRA: copy number analysis for diseases: the clinician’s view. Nat Rev Nephrol. 2014;10:687–699. targeted resequencing. Bioinformatics. 2012;28:1307–1313.

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