High NPHP1 and NPHP6 Mutation Rate in Patients With

High NPHP1 and NPHP6 Mutation Rate in Patients with Joubert Syndrome and Nephronophthisis: Potential Epistatic Effect of NPHP6 and AHI1 Mutations in Patients with NPHP1 Mutations

Ka´lma´n Tory,*† Tiphanie Lacoste,*† Lydie Burglen,‡ Vincent Morinie`re,*† ʈ Nathalie Boddaert,§ Marie-Alice Macher, Brigitte Llanas,¶ Hubert Nivet,** Albert Bensman,†† Patrick Niaudet,†‡‡ Corinne Antignac,*†§§ Re´mi Salomon,*†‡‡ and Sophie Saunier*† *INSERM, U574, †Universite´Paris Descartes, Departments of ‡Genetics and Pediatric Neurology and ††Pediatric Nephrology, Hoˆpital Trousseau, AP-HP, Departments of §Pediatric Radiology, ‡‡Pediatric Nephrology, and §§Genetics, ʈ Hoˆpital Necker-Enfants Malades, AP-HP, and Department of Pediatric Nephrology, Hoˆpital Robert Debre´, AP-HP, Paris, ¶Department of Pediatrics, Hoˆpital Pellegrin, Bordeaux, and **Department of Pediatric Nephrology, Hoˆpital Gatien de Clocheville, Tours, France

Joubert syndrome (JS) is an autosomal recessive disorder that is described in patients with cerebellar ataxia, mental retardation, hypotonia, and neonatal respiratory dysregulation. Kidney involvement (nephronophthisis or cystic renal dysplasia) is associated with JS in one fourth of known cases. Mutations in three genes—AHI1, NPHP1, and NPHP6—have been identified in patients with JS. However, because NPHP1 mutations usually cause isolated nephronophthisis, the factors that predispose to the development of neurologic involvement are poorly understood. In an attempt to identify such genetic determinants, a cohort of 28 families with nephronophthisis and at least one JS-related neurologic symptom were screened for mutations in AHI1, NPHP1, and NPHP6 genes. NPHP1 and NPHP6 homozygous or compound heterozygous mutations were found in 13 (46%) and six (21%) unrelated patients, respectively. Two of the 13 patients with NPHP1 mutations carried either a heterozygous truncating mutation in NPHP6 or a heterozygous missense mutation in AHI1. Furthermore, five patients with NPHP1 mutations carried the AHI1 variant R830W, which was predicted to be “possibly damaging” and was found with significantly higher frequency than in healthy control subjects and in patients with NPHP1 mutations without neurologic symptoms (five of 26 versus four of 276 and three of 152 alleles; P < 0.001 and P < 0.002, respectively). In contrast to the variable neurologic and milder retinal phenotype of patients with NPHP1 mutations, patients with NPHP6 mutations presented with a more severe neurologic and retinal phenotype. In conclusion, NPHP1 and NPHP6 are major genes of nephronophthisis associated with JS. Epistatic effects that are provided by heterozygous NPHP6 and AHI1 mutations and variants may contribute to the appearance of extrarenal symptoms in patients with NPHP1 mutations. J Am Soc Nephrol 18: 1566–1575, 2007. doi: 10.1681/ASN.2006101164

oubert syndrome (JS [OMIM 213300]) is an autosomal reces- widened. In particular, retinal dystrophy and kidney involve- sive disorder that was first described in 1969 in four siblings ment (nephronophthisis or cystic renal dysplasia) have been J with cerebellar ataxia, mental retardation, hypotonia, and reported in approximately 17 and 27% of JS cases, respectively neonatal respiratory dysregulation (1). Neuroradiologically, JS is (3). Additional clinical features such as congenital saccade ini- characterized by the “molar tooth sign” (MTS), which reflects tiation failure (Cogan oculomotor apraxia), coloboma, hepatic cerebellar vermis hypoplasia, thickened and elongated superior fibrosis, autism, and other central nervous system malforma- cerebellar peduncles, and an abnormally large interpeduncular tions have been shown to be associated with JS. On the basis of fossa (2). the variable involvement of these organs, several JS-related Since its first description, the range of the JS phenotype has disorders termed cerebello-oculo-renal syndromes have been distinguished and have as a unifying characteristic the presence of cerebellar vermis hypoplasia (4). Nephronophthisis, an au- Received October 27, 2006. Accepted February 19, 2007. tosomal recessive nephropathy that is characterized by polyu- Published online ahead of print. Publication date available at www.jasn.org. ropolydipsia, mild or absent proteinuria, interstitial fibrosis, Address correspondence to: Dr. Corinne Antignac, INSERM U574 and Depart- and cysts at the corticomedullary junction is the most common ment of Genetics, Tour Lavoisier 6° e´tage, Hoˆpital Necker-Enfants Malades, 149 genetic cause of chronic renal failure in childhood. rue de Se`vres, 75015 Paris, France. Phone: ϩ33-1-44-49-50-98; Fax: ϩ33-1-44-49- 02-90; E-mail: [email protected] In patients with JS, three genetic loci have been mapped

Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1805-1566 J Am Soc Nephrol 18: 1566–1575, 2007 NPHP1, NPHP6, and AHI1 Mutations in JS and Nephronophthisis 1567 until recently to 9q34.3 (JBTS1: OMIM213300), to 11p12-q13.3 thermore, the potential epistatic effect of mutations in a second (JBTS2: OMIM608091), and to 6q23 (JBTS3: OMIM608629) locus in patients with NPHP1 mutations and neurologic symp- (5–8). Of these, JBTS3 is the only locus with which a disease- toms has likewise not been investigated. Here we present the specific gene has been identified, AHI1 (9,10). The phenotype mutational analysis of AHI1, NPHP1, and NPHP6 in a cohort of that is linked to JBTS1 and JBTS3 loci was initially found to 28 families with nephronophthisis and JS-related symptoms. be restricted to neurologic and retinal involvement (11); We report for the first time the presence of NPHP6 and AHI1 however, more recently, AHI1 mutations have also been heterozygous mutations in patients with NPHP1 mutations and detected in patients with both JS and nephronophthisis neurologic symptoms. (12,13). JBTS2 was also associated with renal and hepatic abnormalities (6,7). Of the six nephronophthisis genes iden- Materials and Methods tified to date (NPHP1 through 6) (14–22), mutations of Patients NPHP6 (CEP290, JBTS5: OMIM610188) have recently been A total of 28 unrelated families were selected from a worldwide identified in patients with JS, in most cases occurring in cohort of 327 families with nephronophthisis—originating mainly association with nephronophthisis and severe retinopathy from France and North Africa—on the basis of the presence of (21,22). It is interesting that patients with Leber congenital mental retardation and/or cerebellar ataxia in at least one affected amaurosis and without any neurologic or renal phenotype child. Diagnostic criteria of nephronophthisis were (1) a history of polyuria and polydipsia with a progressive decline in kidney func- have been found to carry homozygous intronic mutations in tion leading to ESRD within the expected age range and (2) histology NPHP6 (23). or renal ultrasound compatible with nephronophthisis (24). Of these Mutations of NPHP1 (also called JBTS4: OMIM609583), the 28 families, 19 had only one affected child, whereas nine were majority of which are homozygous deletions, are found in a multiplex (one of which with two first-degree cousins affected), large proportion of patients with isolated nephronophthisis (45 leading to a total of 38 patients. Six of the families were consanguineous. In the 19 families with only one affected child, all children had to 60%) (24,25) and have also been identified in some patients both nephronophthisis and neurologic involvement. In three multi- who present with nephronophthisis associated with JS or plex families (F265, F130, and F23), one of the affected siblings Cogan oculomotor apraxia (25–29). The cerebellar malforma- (F265-2, F130-1, and F23-2) presented with a renal phenotype but tions of patients with JS and NPHP1 mutations were found to without neurologic symptoms, whereas in two of these families be less severe, leading to a mild MTS in most of the patients (F265 and F130), the other affected sibling (F265-1 and F130-2) had a (25,28,29). The NPHP1 deletion that is carried by patients with neurologic phenotype but no renal involvement by the ages of 10 and 7 yr, respectively (Table 1). In one family (F358), patient F358-1 nephronophthisis and JS is not different from that found in had neurologic phenotype but no renal involvement by the age of 8 patients with isolated nephronophthisis (25,28). Therefore, the yr, whereas his first-degree cousin, F358-2, presented with a renal association of extrarenal symptoms with nephronophthisis has and retinal phenotype but without neurologic symptoms, giving the been suggested to be explained by the potential epistatic effect clinical picture of Senior-Løken syndrome (SLS). of heterozygous mutations in other genes of JS (28). Brain imaging was available in at least one affected child in 21 The AHI1 and NPHP6 genes are expressed during early brain families. Genomic DNA was isolated from peripheral blood by stan- dard methods, after obtaining informed consent from the patients or development (10,22). Nephrocystin-1 and -6, the proteins that their parents. Experiments were done in accordance with French ethical are encoded by NPHP1 and NPHP6, are localized in primary committee recommendations and with the Declaration of Helsinki. cilia, basal bodies, and centrosomes, similarly to other proteins that are involved in disorders that overlap with JS, such as polycystic kidney diseases and Bardet-Biedl syndrome NPHP1 Deletion Screening All 28 families were tested for homozygous deletion of the NPHP1 (18,21,30). Nephrocystin-1 and -6 each possess numerous pro- region using the PCR primers 765F2L, 804/6, and 187.41 (37). Patients tein interacting domains that allow them to associate with without homozygous NPHP1 deletion were tested for heterozygous various proteins, such as actin-binding proteins, microtubule- deletion by quantitative multiplex PCR using primers in NPHP1 exons binding proteins, or the retinitis pigmentosa GTPase regulator, 14 and 15 as well as in exon 4 of NPHS2 as a reporter (V. Morinie`re, which is mutated in some forms of retinitis pigmentosa (31–35). INSERM U574, Paris, personal communication, February 12, 2006). Jouberin, the product of AHI1, also contains several protein- interacting domains and has been recently shown to interact Pulse-Field Gel Electrophoresis with nephrocystin-1, which suggests that they lie in a common To identify a potential rearrangement in a family with possible functional pathway (36). The role of jouberin and nephrocys- linkage to the NPHP1 locus and without identified deletion or muta- tin-1 and -6 in brain development is unknown, but the involve- tion, we performed pulse-field gel electrophoresis (PFGE) and Southern ment of nephrocystins in ciliary function suggests a possible blot analyses. Agarose-embedded DNA from patients and control subjects were digested with the following enzymes: SfiI, NotI, HindIII, link between cilia and cerebellar development. ClaI, EcoRI, or EcoRV. PFGE was performed as described previously No comprehensive study has been performed thus far to (38). Hybridizations were performed using probe N123 (38) and cDNA assess the mutation rate of NPHP1, NPHP6, and AHI1 in pa- probes of NPHP1 gene that contained exons 1 to 6, exons 7 to 11, exons tients with nephronophthisis and neurologic symptoms. Fur- 12 to 14, and exons 15 to 20 (Figure 1). 58Junlo h mrcnSceyo Nephrology of Society American the of Journal 1568

Table 1. Genetic and clinical characteristics of the patientsa

NPHP1 Alterations NPHP6 Alterationsb AHI1 Alterationsb Renal Family Individual Origin Function VI OMD At Mental CVA Other Coding Coding Coding (Age in yr) Retardation Nucleotide Sequence Nucleotide Sequence Nucleotide Sequence

F9 F9-1 France — — c.5649 ins A, het L1884fsX1906 c.2488 CϾT, het R830Wd ESRD (11) Bl nys ϩϩ nd c.5850 del T, het F1950fsX1964 c.5649 ins A, het L1884fsX1906 F9-2 ns c.5850 del T, het F1950fsX1964 ns ESRD (25) Bl nys ϩϩ nd F72 F72-1 France — — c.4963–4964 del AG, R1655fsX1659 — — ESRD (19) ϩ nd ϩϩ nd het pot abn transc c.103-13 to -18 del GCTTTT, het F99 F99-1 Italy — — c.2251 CϾT, het R751X — — ESRD (14) Bl nd ϩ nd nd c.6869 del A, het N2290fsX2300 F358 F358-1 France — — c.1645 CϾT, het R549X — — Normal (8) Bl nd ϩϩMTS HD, convulsions c.5649 ins A, het L1884fsX1906 F358-2c France — — c.5649 ins A, het L1884fsX1906 ns ESRD (12) Bl ϪϪ Ϫ nd c.4195-1 GϾA, het abn transc F375 F375-1 France — — c.1361 del G, het G454fsX458 — — CK/UCD (3) ϩϩϩ ϩ MTS c.6869 del A, het N2290fsX2300 F419 F419-1 Argentina — — c.3310-1 GϾC, het abn transc — — CRF (24) Bl nys ϩϩ ϩ c.6271-8 TϾG, het pot abn transc F265 F265-1 France Del, hom no transc c.287 del A, het N96fsX124 — — Normal (10) ϩ nys Ϫϩ ϪAutism, ptosis F265-2 Del, hom c.287 del A, het N96fsX124 — — ESRD (17) ϩϪϪ Ϫ Ϫ F405 F405-1 France Del, hom no transc — — c.989 AϾG, het D330G ESRD (15) Ϫ Cog ϩϩMTS F103 F103-1 France Del, hom no transc — — c.2488 CϾT, het R830Wd CRF (12) Ϫ Cog ϩϩMTS F110 F110-1 France Del, hom no transc — — c.2488 CϾT, het R830Wd ESRD (13) Ϫ Cog ϪϩMTS F312 (cs) F312-1 Morocco Del, hom no transc — — c.2488 CϾT, het R830Wd ESRD (7) ϩ nys Ϫϩ Ve Convulsions F78 F78-1 France Del, hom no transc — — c.2488 CϾT, het R830Wd ESRD (7) ϩ nys ϩϩMTS F168 F168-1 France Del, hom no transc — — c.2488 CϾT, het R830Wd ESRD (9) Ϫ nys Ϫϩ nd F57 F57-1 France Del, hom no transc — — — — ESRD (12) ϪϪϪ ϩ nd F82 F82-1 France Del, het abn — — — — ESRD (8) ϩ Cog ϩϪ nd c.1467 ϩ 1GϾT, het transc F130 (cs) F130-1 Senegal Del, hom no transc — — — — ESRD (8) ϪϪϪ Ϫ nd F130-2 Del, hom — — — — normal (7) Ϫ Cog ϩϩMTS F267 F267-1 France Del, hom no transc — — — — CRF (8) ϩ nys ϩϩMTS F267-2 Del, hom ns ns ESRD (10) ϩ nys ϩϩMTS F443 F443-1 France Del, het abn — — — — UCD (5) ϪϪϩ Ϫ MTS Polydactyly c.1027 GϾA, het. transc F69 (cs) F69-1 Turkey Dupl ex 7–11, hom abn — — — — ESRD (18) ϩ Cog nd ϩ nd transc F171 F171-1 France — — — — — — ESRD (14) Ϫ Cog ϩϪ Ϫ F171-2 ns ns ns ESRD (14) Ϫ Cog ϩϪ nd F171-3 ns ns ns CRF (16) Ϫ Cog ϩϪ nd F23 F23-1 Reunion island — — — — — — ESRD (4) Bl Ϫϩ ϩ Ϫ F23-2 — — — — — — ESRD (14) ϩϪϪ Ϫ nd F42 F42-1 France — — — — — — ESRD (9) ϩ nys ϩϩMTS Strabismus 2007 1566–1575, 18: Nephrol Soc Am J F42-2 — — — — — — ESRD (8) Ϫϩϩ ϩ MTS Strabismus F168 (cs) F168-1 Morocco — — — — — — ESRD (8) Bl ϩϩ ϩ ϩStrabismus, ptosis F168-2 ns ns ns ESRD (Ͻ10) ϩ nd nd ϩϩHepatic fibrosis F222 F222-1 France — — — — — — ESRD (6) ϩϩϩ ϩ ϩStrabismus, ptosis F437 (cs) F437-1 Tunisia — — — — c.2488 CϾT, het R830Wd ESRD (18) ϩϩϩ ϩ ϩPolydactyly F296 (cs) F296-1 France — — — — — — ESRD (9) Bl ϩϪ ϩ AC-I Hypopituitarism F58 F58-1 France — — — — — — ESRD (7) ϩ Op ϩϩMTS Ptosis F126 F126-1 France — — — — — — ESRD (18) ϩϩϩ ϩ MTS Ptosis, pyram sy

aabn, abnormal; AC-I, Arnold-Chiari malformation, type I; At, cerebellar ataxia; Bl, blind; (cs), consanguineous; CK, cystic kidney; Cog, Cogan oculomotor apraxia; CRF, chronic renal failure; CVA, cerebellar vermis aplasia/hypoplasia; del, deletion; dupl, duplication; HD, Hirschsprung disease; het, heterozygous; hom, homozygous; MTS, molar tooth sign; nd, no data available; ns, not sequenced; nys, nystagmus; OMD, oculomotor disorder; Op, ophthalmoplegia; pot, potentially; pyram sy, pyramidal syndrome; transc, transcript; UCD, urine concentration defect; VI, visual impairment; Ve, enlarged fourth ventricle. bMutations and damaging variants. cFirst-degree cousin of patient F358-1. d Possibly damaging variant. J Am Soc Nephrol 18: 1566–1575, 2007 NPHP1, NPHP6, and AHI1 Mutations in JS and Nephronophthisis 1569

investigated in all families except for families without available parental DNA (F99 and F419). The nonsilent nucleotide changes were tested in Ͼ230 ethnically matched control chromosomes and in Ͼ150 chromosomes of unrelated patients with NPHP1 mutations but without neurologic symptoms. The detection of a predicted abnormal transcript in patients F72-1 and F419-1 was not possible because of a lack of RNA availability from these patients.

Results NPHP1 Mutations We identified homozygous or compound heterozygous NPHP1 mutations in 13 families (Table 1). Thirteen patients from 10 families carried homozygous deletions of the whole gene, two unrelated patients a heterozygous NPHP1 deletion and a consensus splice-site mutation (F82-1: c.1467 ϩ 1GϾT; F443-1: c.1027 GϾA), and one patient (F69-1) a homozygous Figure 1. Homozygous duplication of NPHP1 exons 7 to 11 in patient F69-1. (A) Schematic representation of the NPHP1 re- rearrangement within the NPHP1 region (Table 1). This novel gion. The repeated regions flanking the NPHP1 gene are pre- rearrangement was identified by PFGE analysis; the SfiI-di- sented by dashed boxes; the marker N123 covering 149 bp in gested fragment that contained the whole NPHP1 gene was this duplicated area as well as the restriction sites SfiI (S) and found to be approximately 10 kb larger in this patient than in a HindIII (H) are indicated. The insertion of an approximately control subject. Further analyses showed the duplication of an 8-kb genomic region that contains exon 7 to exon 11 within the 8-kb region that contained exons 7 to 11 within the NPHP1 135-kb SfiI fragment is represented. (B) Pulse-field gel electro- region (Figure 1). phoresis analysis of SfiI-digested DNA hybridized with probe N123. An abnormal fragment of approximately 145 kb was detected in patient F69-1, compared with the normal 135-kb NPHP6 Mutations fragment detected in the control (ctl), whereas no 135-kb frag- Ten NPHP6 mutations and two variants in the consensus ment was visualized in a patient who carried the frequent sequence of the 3Ј splice-site were identified in seven families homozygous NPHP1 deletion (del). (C) Southern blot analysis (Table 1). Of the 10 mutations, eight were truncating (six frame- of HindIII-digested DNA hybridized with a cDNA probe that shift and two nonsense) and two were splice-site mutations that contained NPHP1 exons 7 to 11. As expected, the normal 7-kb affected the obligatory G at position Ϫ1 (c.4195-1 GϾA and HindIII fragment detected in the control subject (ctl) and in c.3310-1 GϾC; Table 1). These mutations were found to segre- patient F69-1 was not present in a patient with the homozygous gate with the disease. The two heterozygous variants in the NPHP1 deletion (del). An additional 13-kb fragment was de- consensus sequence of the 3Ј splice site (c.6271-8 TϾGor tected in patient F69-1. Restriction pattern detected with probes c.103-13 to -18 del GCTTTT) were also considered to be likely that hybridized NPHP1 exons 1 to 6, 12 to 14, and 15 to 20 was normal in patient F69-1 (data not shown). These data suggest pathogenic, because they were found to decrease the scores of the presence of a duplication of an approximately 8-kb genomic the normal junction sequences from 0.723 to 0.434 and from region from intron 6 to intron 11 and its insertion within 0.939 to 0.688, respectively (NetGene2) and were not detected in NPHP1 region. Ͼ230 control chromosomes. Both patients (F72-1 and F419-1) carried in addition to the 3Ј splice-site mutation a deleterious NPHP6 mutation, and in patient F72-1, in whom parental DNA Mutational Analysis was available, these two mutations were shown to be present Mutational screening of AHI1, NPHP1, and NPHP6 was performed on different alleles. by direct sequencing of the coding exons and the adjacent intronic In six families, the eight probands were compound heterozy- junctions as well as of the NPHP6 intronic region that contained the gous for NPHP6 mutations. In one of these families (F358), the c.2991 ϩ 1655 base, whose mutation was identified in patients with two first-degree affected cousins carried a common mutation Leber amaurosis (23) (primer sequences available on request). PCR on one allele and one of two different mutations in the other products were treated with Exo-SAP IT (GE Healthcare, Buckingham- allele. In the last family (F265), only one NPHP6 heterozygous shire, UK), and both strands were sequenced using the dideoxy chain frameshift mutation (c.287delA) was found in the two siblings, termination method on a 3130 XL DNA sequencer (Applied Biosys- who interestingly also harbored the NPHP1 homozygous dele- tems, Foster City, CA) and analyzed with Sequencher 3.1 program tion (Table 1). Of note, the intronic mutation c.2991 ϩ 1655 (Genecodes, Ann Arbor, MI). Amino acid conservation at the missense AϾG identified previously in patients with Leber amaurosis mutations and their possible damaging effect were assessed using SIFT (23) was not detected in any patient. (39) and Polyphen softwares (40). Donor and acceptor sites for splicing were predicted by NetGene2 (41). Of patients who carried NPHP1 mutations, four heterozy- At least one child was screened in each family. In addition, for gous NPHP6 nonsilent variants/polymorphisms were iden- identification of mutations with an epistatic effect, sibling pairs with tified (Table 2). When we compared the frequencies of these different neurologic or retinal phenotypes both were sequenced (Table variants in (1) patients with NPHP1 mutation with neuro- 1, F265, F130, F23, and F42). Segregation of the identified mutations was logic symptoms, (2) patients with NPHP1 mutation without 1570 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1566–1575, 2007

neurologic symptoms, and (3) healthy control subjects, no significant differences were found among these three groups (Table 2).

AHI1 Mutations PolyPhen Truncating AHI1 mutations were not identified in this pop- Prediction of (PSIC: 1.575) (PSIC: 1.8) ulation. Two heterozygous nonsilent variants (c.989 AϾG [D330G] and c.2488 CϾT [R830W]) were detected (Tables 1 and 2). The variant D330G was detected in one patient with a homozygous NPHP1 deletion (F405-1) and was found neither

— Unknown in 264 chromosomes of healthy individuals nor in 156 chromosomes of patients who carried NPHP1 mutations without neurologic symptoms. The aspartic acid at this position is conserved in mammals, and its change to glycine was predicted by the software program PolyPhen to be “possibly damaging” (Table 2). Therefore, this nucleotide change was considered to be a missense mutation. The other variant, R830W, was detected in one patient with Domain Conserved in NPHP6 mutations (F9-1), in one patient without NPHP1 or

CCIV NPHP6 mutation (F437-1), and in five patients with NPHP1 mutations (F103-1, F110-1, F312-1, F78-1, and F168--1). Thus, its frequency was higher in patients with NPHP1 mutations and neurologic symptoms (five of 26 chromosomes) compared either with control subjects (four of 276 chromosomes; P Ͻ 0.001) or with patients who had NPHP1 mutations and lacked neurologic symptoms (three of 152 chromosomes; P Ͻ 0.002). The Frequency in affected arginine, located in the WD40 repeat domain (3), is a Controls (Alleles) conserved in vertebrates, and its change to tryptophan is also predicted to be “possibly damaging” by PolyPhen (Table 2). In total, NPHP1 and NPHP6 mutations were found in 19

mutations (67%) of 28 families. In the remaining nine (33%) families, no mutations and neurologic symptoms. Mutations mutation was found in NPHP1, NPHP6, and AHI1. Of the 13

3/152 4/276 WD40 repeatunrelated Vertebrates patients Possibly damaging with NPHP1 mutations, two carried either a Symptoms NPHP1 NPHP1 heterozygous NPHP6 truncating mutation or an AHI1 missense NPHP1 mutation (D330G), and five carried the “possibly damaging” Without Neurologic AHI1 variant R830W. (Alleles) Phenotype–Genotype Correlation Seven of eight patients from six families with two NPHP6 mutations presented cerebellar ataxia and mental retardation

(F103-1, (F9-1, F9-2, F72-1, F99-1, F358-1, F375-1, and F419-1). In two

b,c patients with available cerebral imaging (F358-1 and F375-1), With Neurologic F110-1, F312-1, F78-1, F168-1) agenesia/dysplasia of the superior vermis and MTS were Symptoms (Patients) Frequency in Patients with detected (Figure 2). The first-degree cousin of patient F358-1 with JS presented with a phenotype that was restricted to renal and retinal involvement, giving the clinical picture of

nonsilent variants in patients with SLS (F358-2). He carried one of the two mutations of his cousin (5649insA and L1884fsX1906) and a different second Alteration Amino Acid NPHP6 mutation that affected a consensus splice site Ͼ NPHP6 (c.4195-1 G A; Table 1). All eight patients with NPHP6 mutations presented with severe retinopathy that in six pa-

and tients led to complete loss of vision. Five of eight patients G5237AA989GC2488T R1746Q D330G 1/26 (F312-1) R830W 1/26 (F405-1) 5/26 2/160 0/156 3/264 0/264 CC XII — Mammals Benign Mammals Possibly damaging G829CA1991GA2512G E277Q D664G K838E 2/26 (F110-1, 1/26 F168-1) (F69-1) 2/26 (F443-1, F69-1) 4/164 ne 1/154 8/284developed 15/292 CC I 2/308 ESRD Homology to SMC, CC II at the mean Mammals age Unknown of Mammals 16 Unknown yr (range 11 to 25 yr). Alteration Nucleotide Two patients had a normal GFR at the ages of 3 and 8 yr 0.002, difference between patients with and without neurologic symptoms. 0.001, difference between control subjects and patients with Ͻ

Ͻ (F375-1 and F358-1, respectively), and one patient had P CC, coiled coil; ne, not examined; SMC, chromosome segregation ATPases for structural maintenance of chromosomes. P

a b c chronic renal failure at the age of 24 yr (F419-1; Table 1). Gene AHI1 NPHP6 Table 2. AHI1 In contrast to the patients with NPHP6 mutations, most of the 16 J Am Soc Nephrol 18: 1566–1575, 2007 NPHP1, NPHP6, and AHI1 Mutations in JS and Nephronophthisis 1571

Figure 2. Axial and midline sagittal images in a control individual (A and B), in patients with NPHP1 mutations (C through Q), and in patients with NPHP6 mutations (R through T). Axial sections demonstrate the elongation of the superior cerebellar peduncles (white arrow in C), resulting in a mild molar tooth sign (MTS) in patients with NPHP1 mutations (C, G, K, O, E, I, M, and Q). The superior cerebellar peduncles are more thickened in patients with NPHP6 mutations, leading to a more explicit MTS (R and T). On sagittal plane, a superiorly positioned and enlarged fourth ventricle (open arrow in D), median and inferior vermis hypoplasia (black arrows in D), and superior vermis “agenesia/dysplasia” (white arrows in S and H) are noted. None of the patients had any supratentorial abnormality. patients with NPHP1 mutations presented with a variable neuro- NPHP6 heterozygous frameshift mutation, presented with the logic phenotype and a milder retinal involvement. Mental retar- clinical phenotype of JS with hypotonia, autism, mental retardation and oculomotor disorder were their most frequent neuro- dation, and retinopathy. His sister, F265-2, who carried the logic symptoms, both involving 12 of 16 patients. Eight of 15 same NPHP1 and NPHP6 mutations, however, presented with patients with available clinical information developed cerebellar a milder clinical phenotype, with retinopathy but neither au- ataxia, of whom all seven patients with available brain imaging tism nor mental retardation (Table 1). showed elongation of the superior cerebellar peduncles resulting in a mild MTS on axial images (Figure 2). The cerebellar vermis Discussion was found to be dysplastic in its superior part and hypoplastic in Here we report a high NPHP1 and NPHP6 mutation rate in JS its middle and inferior segments in all of these patients (Figure 2, associated with nephronophthisis and the potential epistatic sagittal images). Furthermore, retinopathy affected only half of the effect of heterozygous NPHP6 and AHI1 mutations and vari- patients with NPHP1 mutations and did not lead to complete loss ants in patients who carry NPHP1 mutations with neurologic of vision in any of them. Eleven of 16 patients developed ESRD at symptoms. the mean age of 11 yr (range 7 to 18 yr). Three patients with NPHP1 mutations had a normal GFR at the ages of 5, 7, and 10 yr High NPHP1 and NPHP6 Mutation Rate (F443-1, F130-2, and F265-1, respectively), and two patients had Both NPHP1 and NPHP6 were found to be major genes in JS chronic renal failure at the ages of 8 and 12 yr (F267-1 and F103-1, with nephronophthisis. Nineteen (67%) of 28 unrelated patients respectively; Table 1). were homozygous or compound heterozygous for NPHP1 Two of three sibling pairs who carried the NPHP1 homozy- (46%) or NPHP6 (21%) mutations. gous deletion (F265 and F130) presented marked intrafamilial The high proportion of NPHP1 mutations in this population variability of neurologic symptoms. For instance, patient was unexpected on the basis of previous studies that showed F265-1, who carried an NPHP1 homozygous deletion and an an NPHP1 mutation rate of 4 to 8% in patients with JS and 1572 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1566–1575, 2007 retinal or renal involvement (28,29). This striking difference can vious studies, cerebellar malformations on brain magnetic res- be at least partly explained by the fact that in the previous onance imaging were found in seven of eight unrelated patients studies, only a small proportion of patients presented with with neurologic symptoms and NPHP1 mutations (25,28,29). nephronophthisis (20% [28] and 25% [29]), whereas in our However, most of these patients showed less severely affected study, the presence of nephronophthisis was a selection crite- superior cerebellar peduncles associated with a mild MTS on rion. Taking into account only patients with JS and nephron- axial images (25,28,29). Consistent with these findings, in our ophthisis in the cohort of Parisi et al. (28), the proportion of series, the cerebellar malformations that were seen in patients patients with NPHP1 mutations (two of five patients) is similar with NPHP1 mutations and cerebellar ataxia seemed to be to our result. By contrast, we confirm that AHI1 is not a major similar but less severe compared with the malformations that gene in nephronophthisis that is associated with JS, in agree- were found in patients with NPHP6 mutations, either in the ment with the study of Valente et al. (3), who did not find any present or in a previous study (22). The superior cerebellar AHI1 mutations in 37 patients with JS and kidney involvement. peduncles in patients with NPHP1 mutations were found to be elongated but in most of them not as thickened as in patients Phenotype of Patients with NPHP6 Mutations with NPHP6 mutations, leading to a less explicit MTS. The The retinal and neurologic involvement was severe in all neurologic phenotype of patients with NPHP1 mutations, sim- patients with JS and NPHP6 mutations, which is in agreement ilar to previous data (25,28,29), was found to be highly variable, with previous findings (21,22). Their renal function seemed to even in the same family, fulfilling rarely the complete picture of decline less rapidly compared with patients with NPHP1 mu- JS. The association of neurologic symptoms cannot be ex- tations, because none of the patients with NPHP6 mutations plained by a contiguous gene deletion syndrome because (1) developed ESRD by the age of 10 yr, whereas half of the the extent of the NPHP1 deletion was found to be the same in patients with NPHP1 mutations did. However, in the cohort of patients with or without extrarenal symptoms (28,29), (2) sib- Sayer et al. (21), most patients with NPHP6 mutations devel- lings who carry the same deletion may present with dramati- oped ESRD already at approximately the age of 11 yr, and the cally different extrarenal involvement, and (3) patients who number of patients with NPHP6 mutations in our study is too carried heterozygous deletion and point mutation or homozy- low to assess properly the age at the onset of ESRD. gous partial duplication in this study also developed similar Patient F358-2 with SLS, in contrast to his cousin who had JS neurologic symptoms. Therefore, the presence of neurologic (F358-1) and carried two truncating NPHP6 mutations, har- symptoms in a minority of patients with NPHP1 mutations and bored along with the common frameshift truncating mutation their inter- and intrafamilial variability are puzzling. An of- in exon 41 a consensus splice-site mutation of exon 33. Simi- fered explanation could be the coexistence of a “third” muta- larly, a homozygous consensus splice-site mutation of exon 22 tion exerting an epistatic effect in another JS-related locus (28); was found by Sayer et al. (21) in a family with SLS. Considering however, such an epistatic effect has never been reported. We our findings and those of Sayer et al., one can hypothesize that detected an NPHP6 heterozygous truncating mutation in one these splice-site mutations do not lead to frameshift and protein sibling pair and an AHI1 missense mutation (D330G) in one truncation but rather to partial deletion of one of the coiled-coil patient, all simultaneously carrying NPHP1 homozygous dele- domains. This mutated protein allows normal neurologic de- tion. Furthermore, the AHI1 variant R830W was enriched in velopment but results in retinal and renal defects. Reinforcing patients who harbored NPHP1 mutations with neurologic this hypothesis is the congenic mouse strain rd16, which devel- symptoms compared with those without neurologic symptoms ops pigmentary retinopathy as a result of a homozygous in- or with healthy control subjects. The R830W variant affects an frame mutation of the murine ortholog of Nphp6 that leads to a evolutionarily conserved amino acid located in the WD40 re- lack of exons 26 to 29 (35). The absence of this region seems to peat domain, a domain that is known to allow specific protein disturb the interaction of nephrocystin-6 with retinitis pigmen- complexes to assemble. A missense mutation within this do- tosa GTPase regulator and may explain the isolated retinal main has already been reported in a patient who presented phenotype without neurologic or renal involvement in the rd16 with JS (3). Although the size of the population studied is not mice. Thus, the type of NPHP6 mutation seems to influence adequately large enough to assess properly the enrichment of largely the degree of neurologic involvement, leading either to R830W in patients with neurologic symptoms, these data sug- an apparently complete neurologic phenotype of JS or to the gest that this variant makes the patients with NPHP1 mutations lack of neurologic symptoms as in SLS. Further functional more susceptible to develop neurologic symptoms. The identi- studies are needed to clarify the effects of these identified fication of second-locus mutations and variants in half of the splice-site mutations on their resultant proteins. patients with NPHP1 mutations reinforce the hypothesis that the epistatic effect that is provided by these genes contributes to Phenotype of Patients with NPHP1 Mutations: Epistatic the appearance of neurologic symptoms in patients with Effect of NPHP6 and AHI1 Mutations and Variants NPHP1 mutations. In our cohort of 152 patients with NPHP1 mutations from 104 As can be expected on the basis of the wide range of pheno- families, 14 (9%) patients from 13 (12%) families presented with types, the identified “third” mutations and variants in a second JS-related neurologic symptoms. Similarly, Caridi et al. (25) locus are not sufficient to account for all phenotypic variability, found JS-related symptoms in five (9%) of 56 patients with suggesting that other genes are involved. The neurologic status NPHP1 homozygous deletions. Combining the results of pre- of the sibling pair with a homozygous NPHP1 deletion and a J Am Soc Nephrol 18: 1566–1575, 2007 NPHP1, NPHP6, and AHI1 Mutations in JS and Nephronophthisis 1573 heterozygous NPHP6 truncating mutation was dramatically flug-Tanguy, Christine Pietrement, Claire Gazengel, Jean-Luc Andre´, different; only their similar retinopathy phenotype can be ex- Franc¸ois Berthoux, Gian-Franco Rizzoni, Djamil Hachicha, and Isabelle plained by the epistatic effect of their common NPHP6 muta- Desguerre. tion. Similarly, patients who harbored both NPHP1 mutations and the AHI1 R830W variant did not present with the same Disclosures neurologic phenotype. Of note, because similarly discordant None. phenotypes have been found in monozygotic twins with JS— with autism and severe mental retardation in one and no neu- References rologic symptom in the other twin (42)—nongenetic factors 1. Joubert M, Eisenring JJ, Robb JP, Andermann F: Familial must also be taken into consideration. agenesis of the cerebellar vermis: A syndrome of episodic It is interesting that a similar epistatic effect was found in hyperpnea, abnormal eye movements, ataxia, and retarda- patients with Bardet-Biedl syndrome (BBS), another cilia-re- tion. Neurology 19: 813–825, 1969 lated disease that is characterized by cystic kidney disease, 2. Maria BL, Hoang KB, Tusa RJ, Mancuso AA, Hamed LM, pigmentary retinal dystrophy, and mental retardation (30). In- Quisling RG, Hove MT, Fennell EB, Booth-Jones M, Ring- deed, the association of a homozygous mutation in one gene dahl DM, Yachnis AT, Creel G, Frerking B: “Joubert syn- and a heterozygous mutation in a second gene has been re- drome” revisited: Key ocular motor signs with magnetic ported in some patients with BBS. Moreover, inter- or intrafa- resonance imaging correlation. J Child Neurol 12: 423–430, milial variability is observed in these patients, suggesting a 1997 3. Valente EM, Brancati F, Silhavy JL, Castori M, Marsh SE, more complex mechanism of inheritance (43,44). Barrano G, Bertini E, Boltshauser E, Zaki MS, Abdel-Aleem The nephrocystin proteins are known to be part of complexes A, Abdel-Salam GM, Bellacchio E, Battini R, Cruse RP, that are localized to basal bodies/centrosomes (18,21,34), and Dobyns WB, Krishnamoorthy KS, Lagier-Tourenne C, Ma- nephrocystin-1 has been shown to interact with jouberin (36). It gee A, Pascual-Castroviejo I, Salpietro CD, Sarco D, Dal- is tempting to assume that jouberin and the nephrocystins may lapiccola B, Gleeson JG; International JSRD Study Group: be part of the same basal body/centrosomal complex and par- AHI1 gene mutations cause specific forms of Joubert syn- ticipate in concert with ciliary function. However, the effects drome-related disorders. Ann Neurol 59: 527–534, 2006 that are exerted by the identified heterozygous NPHP6 and 4. Satran D, Pierpont ME, Dobyns WB: Cerebello-oculo-renal AHI1 variants on ciliary function that are already affected by syndromes including Arima, Senior-Loken and COACH the lack of nephrocystin-1 remain to be investigated. syndromes: More than just variants of Joubert syndrome. Altogether, our results suggest that a classical mendelian Am J Med Genet 86: 459–469, 1999 5. Saar K, AL-Gazali L, Sztriha L, Rueschendorf F, Nur EK, inheritance is not sufficient to explain all cases of JS and Reis A, Bayoumi R: Homozygosity mapping in families nephronophthisis. An oligogenic model of inheritance might be with Joubert syndrome identifies a locus on chromosome more relevant in some patients. 9q34.3 and evidence for genetic heterogeneity. Am J Hum Genet 65: 1666–1671, 1999 Conclusion 6. Valente EM, Salpietro DC, Brancati F, Bertini E, Galluccio NPHP1 and NPHP6 are major genes of nephronophthisis T, Tortorella G, Briuglia S, Dallapiccola B: Description, nomenclature, and mapping of a novel cerebello-renal syn- associated with JS-related neurologic symptoms, accounting for drome with the molar tooth malformation. Am J Hum Genet roughly two thirds of the cases. In general, patients with 73: 663–670, 2003 NPHP1 mutations tend to have a variable neurologic and 7. Keeler LC, Marsh SE, Leeflang EP, Woods CG, Sztriha L, milder retinal phenotype, whereas patients with NPHP6 muta- Al-Gazali L, Gururaj A, Gleeson JG: Linkage analysis in tions tend to develop a severe neurologic and retinal pheno- families with Joubert syndrome plus oculo-renal involve- type. Our finding that several patients with NPHP1 mutations ment identified the CORS2 locus on chromosome 11p12– and neurologic symptoms carry either a mutation in a second q13.3. Am J Hum Genet 73: 656–662, 2003 locus or the possibly damaging AHI1 variant R830W strongly 8. Lagier-Tourenne C, Boltshauser E, Breivik N, Gribaa M, supports the notion that epistatic effects that are provided by Betard C, Barbot C, Koenig M: Homozygosity mapping of these genes or other genes as yet uncharacterized contribute to a third Joubert syndrome locus to 6q23. J Med Genet 41: the appearance of extrarenal symptoms. 273–277, 2004 9. Ferland RJ, Eyaid W, Collura RV, Tully LD, Hill RS, Al- Nouri D, Al-Rumayyan A, Topcu M, Gascon G, Bodell A, Acknowledgments Shugart YY, Ruvolo M, Walsh CA: Abnormal cerebellar This work was supported by the Institut National de la Sante´etdela development and axonal decussation due to mutations in Recherche Me´dicale, the Fondation Recherche Medicale (grant to K.T.), AHI1 in Joubert syndrome. Nat Genet 36: 1008–1013, 2004 the Association pour l’Utilization du Rein Artificiel, and the GIS-Mal- 10. Dixon-Salazar T, Silhavy JL, Marsh SE, Louie CM, Scott LC, adies Rares. Gururaj A, Al-Gazali L, Al-Tawari AA, Kayserili H, Sztriha We thank the patients and their families for participation. We ac- L, Gleeson JG: Mutations in the AHI1 gene, encoding jou- knowledge Anne Legall for excellent technical assistance and Marie- berin, cause Joubert syndrome with cortical polymicro- Claire Gubler and Nathan Hellman for careful reading of the article. We gyria. Am J Hum Genet 75: 979–987, 2004 are grateful to the following physicians for contribution of material and 11. Valente EM, Marsh SE, Castori M, Dixon-Salazar T, Bertini clinical data from patients: Chantal Loirat, Anne Maisin, Odile Boesp- E, Al-Gazali L, Messer J, Barbot C, Woods CG, Boltshauser 1574 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1566–1575, 2007

E, Al-Tawari AA, Salpietro CD, Kayserili H, Sztriha L, Kusakabe T, Tsuda M, Ma L, Lee H, Larson RG, Allen SJ, Gribaa M, Koenig M, Dallapiccola B, Gleeson JG: Distin- Wilkinson CJ, Nigg EA, Shou C, Lillo C, Williams DS, guishing the four genetic causes of Jouberts syndrome- Hoppe B, Kemper MJ, Neuhaus T, Parisi MA, Glass IA, related disorders. Ann Neurol 57: 513–519, 2005 Petry M, Kispert A, Gloy J, Ganner A, Walz G, Zhu X, 12. Utsch B, Sayer JA, Attanasio M, Pereira RR, Eccles M, Goldman D, Nurnberg P, Swaroop A, Leroux MR, Hilde- Hennies HC, Otto EA, Hildebrandt F: Identification of the brandt F: The centrosomal protein nephrocystin-6 is mu- first AHI1 gene mutations in nephronophthisis-associated tated in Joubert syndrome and activates transcription fac- Joubert syndrome. Pediatr Nephrol 21: 32–35, 2006 tor ATF4. Nat Genet 38: 674–681, 2006 13. Parisi MA, Doherty D, Eckert ML, Shaw DW, Ozyurek H, 22. Valente EM, Silhavy JL, Brancati F, Barrano G, Krish- Aysun S, Giray O, Al Swaid A, Al Shahwan S, Dohayan N, naswami SR, Castori M, Lancaster MA, Boltshauser E, Bakhsh E, Indridason OS, Dobyns WB, Bennett CL, Chance Boccone L, Al-Gazali L, Fazzi E, Signorini S, Louie CM, PF, Glass IA: AHI1 mutations cause both retinal dystrophy Bellacchio E; Related Disorders Study Group IJ; Bertini E, and renal cystic disease in Joubert syndrome. J Med Genet Dallapiccola B, Gleeson JG: Mutations in CEP290, which 43: 334–339, 2006 encodes a centrosomal protein, cause pleiotropic forms of 14. Hildebrandt F, Otto E, Rensing C, Nothwang HG, Vollmer Joubert syndrome. Nat Genet 38: 623–625, 2006 M, Adolphs J, Hanusch H, Brandis M: A novel gene en- 23. den Hollander AI, Koenekoop RK, Yzer S, Lopez I, Arends coding an SH3 domain protein is mutated in nephronoph- ML, Voesenek KE, Zonneveld MN, Strom TM, Meitinger T, thisis type 1. Nat Genet 17: 149–153, 1997 Brunner HG, Hoyng CB, van den Born LI, Rohrschneider 15. Saunier S, Calado J, Heilig R, Silbermann F, Benessy F, K, Cremers FP: Mutations in the CEP290 (NPHP6) gene are Morin G, Konrad M, Broyer M, Gubler MC, Weissenbach J, a frequent cause of Leber congenital amaurosis. Am J Hum Antignac C: A novel gene that encodes a protein with a Genet 79: 556–561, 2006 putative src homology 3 domain is a candidate gene for 24. Salomon R, Gubler MC, Antignac C: Nephronophthisis. In: familial juvenile nephronophthisis. Hum Mol Genet 6: 2317– Oxford Textbook of Clinical Nephrology, 3rd Ed., edited by 2323, 1997 Davison AM, Cameron JS, Gru¨nfeld JP, Ponticelli C, Ritz E, 16. Mollet G, Salomon R, Gribouval O, Silbermann F, Bacq D, Winearls CG, van Ypersele C, Oxford, Oxford University Landthaler G, Milford D, Nayir A, Rizzoni G, Antignac C, Press, 2005, pp 2325–2333 Saunier S: The gene mutated in juvenile nephronophthisis 25. Caridi G, Dagnino M, Rossi A, Valente EM, Bertini E, Fazzi type 4 encodes a novel protein that interacts with nephro- E, Emma F, Murer L, Verrina E, Ghiggeri GM: Nephron- cystin. Nat Genet 32: 300–305, 2002 ophthisis type 1 deletion syndrome with neurological 17. Otto E, Hoefele J, Ruf R, Mueller AM, Hiller KS, Wolf MT, symptoms: Prevalence and significance of the association. Schuermann MJ, Becker A, Birkenhager R, Sudbrak R, Kidney Int 70: 1342–1347, 2006 Hennies HC, Nurnberg P, Hildebrandt F: A gene mutated 26. Saunier S, Morin G, Calado J, Benessy F, Silbermann F, in nephronophthisis and retinitis pigmentosa encodes a Antignac C: Large deletions of the NPH1 region in Cogan novel protein, nephroretinin, conserved in evolution. Am J syndrome (CS) associated with familial juvenile nephron- Hum Genet 71: 1161–1167, 2002 ophthisis [Abstract]. Am J Hum Genet 61: A346, 1997 18. Otto EA, Schermer B, Obara T, O’Toole JF, Hiller KS, 27. Betz R, Rensing C, Otto E, Mincheva A, Zehnder D, Lichter Mueller AM, Ruf RG, Hoefele J, Beekmann F, Landau D, P, Hildebrandt F: Children with ocular motor apraxia type Foreman JW, Goodship JA, Strachan T, Kispert A, Wolf Cogan carry deletions in the gene (NPHP1) for juvenile MT, Gagnadoux MF, Nivet H, Antignac C, Walz G, Drum- nephronophthisis. J Pediatr 136: 828–831, 2000 mond IA, Benzing T, Hildebrandt F: Mutations in INVS 28. Parisi MA, Bennett CL, Eckert ML, Dobyns WB, Gleeson encoding inversin cause nephronophthisis type 2, linking JG, Shaw DW, McDonald R, Eddy A, Chance PF, Glass IA: renal cystic disease to the function of primary cilia and The NPHP1 gene deletion associated with juvenile left-right axis determination. Nat Genet 34: 413–420, 2003 nephronophthisis is present in a subset of individuals with 19. Olbrich H, Fliegauf M, Hoefele J, Kispert A, Otto E, Volz A, Joubert syndrome. Am J Hum Genet 75: 82–91, 2004 Wolf MT, Sasmaz G, Trauer U, Reinhardt R, Sudbrak R, 29. Castori M, Valente EM, Donati MA, Salvi S, Fazzi E, Pro- Antignac C, Gretz N, Walz G, Schermer B, Benzing T, copio E, Galluccio T, Emma F, Dallapiccola B, Bertini E; Hildebrandt F, Omran H: Mutations in a novel gene, Italian MTS Study Group: NPHP1 gene deletion is a rare NPHP3, cause adolescent nephronophthisis, tapeto-retinal cause of Joubert syndrome related disorders. J Med Genet degeneration and hepatic fibrosis. Nat Genet 34: 455–459, 42: e9, 2005 2003 30. Watnick T, Germino G: From cilia to cyst. Nat Genet 34: 20. Otto EA, Loeys B, Khanna H, Hellemans J, Sudbrak R, Fan 355–356, 2003 S, Muerb U, O’Toole JF, Helou J, Attanasio M, Utsch B, 31. Benzing T, Gerke P, Hopker K, Hildebrandt F, Kim E, Walz Sayer JA, Lillo C, Jimeno D, Coucke P, De Paepe A, Rein- G: Nephrocystin interacts with Pyk2, p130(Cas), and tensin hardt R, Klages S, Tsuda M, Kawakami I, Kusakabe T, and triggers phosphorylation of Pyk2. Proc Natl Acad Sci Omran H, Imm A, Tippens M, Raymond PA, Hill J, Beales USA98: 9784–9789, 2001 P, He S, Kispert A, Margolis B, Williams DS, Swaroop A, 32. Donaldson JC, Dempsey PJ, Reddy S, Bouton AH, Coffey Hildebrandt F: Nephrocystin-5, a ciliary IQ domain pro- RJ, Hanks SK: Crk-associated substrate p130(Cas) interacts tein, is mutated in Senior-Loken syndrome and interacts with nephrocystin and both proteins localize to cell-cell with RPGR and calmodulin. Nat Genet 37: 282–288, 2005 contacts of polarized epithelial cells. Exp Cell Res 256: 168– 21. Sayer JA, Otto EA, O’Toole JF, Nurnberg G, Kennedy MA, 178, 2000 Becker C, Hennies HC, Helou J, Attanasio M, Fausett BV, 33. Donaldson JC, Dise RS, Ritchie MD, Hanks SK: Nephro- Utsch B, Khanna H, Liu Y, Drummond I, Kawakami I, cystin-conserved domains involved in targeting to epithe- J Am Soc Nephrol 18: 1566–1575, 2007 NPHP1, NPHP6, and AHI1 Mutations in JS and Nephronophthisis 1575

lial cell-cell junctions, interaction with filamins, and estab- Mutational mechanism involved in deletions in familial juve- lishing cell polarity. J Biol Chem 277: 29028–29035, 2002 nile nephronophthisis. Am J Hum Genet 66: 778–789, 2000 34. Mollet G, Silbermann F, Delous M, Salomon R, Antignac C, 39. Reese MG, Eeckman FH, Kulp D, Haussler D: Improved Saunier S: Characterization of the nephrocystin/nephro- splice site detection in Genie. J Comput Biol 4: 311–323, cystin-4 complex and subcellular localization of nephro- 1997 cystin-4 to primary cilia and centrosomes. Hum Mol Genet 40. Ramensky V, Bork P, Sunyaev S: Human non-synonymous 14: 645–656, 2005 SNPs: Server and survey. Nucleic Acids Res 30: 3894–3900, 35. Chang B, Khanna H, Hawes N, Jimeno D, He S, Lillo C, 2002 Parapuram SK, Cheng H, Scott A, Hurd RE, Sayer JA, Otto 41. Brunak S, Engelbrecht J, Knudsen S: Prediction of human EA, Attanasio M, O’Toole JF, Jin G, Shou C, Hildebrandt F, mRNA donor and acceptor sites from the DNA sequence. Williams DS, Heckenlively JR, Swaroop A: In-frame dele- J Mol Biol 220: 49–65, 1991 tion in a novel centrosomal/ciliary protein CEP290/ 42. Raynes HR, Shanske A, Goldberg S, Burde R, Rapin I: NPHP6 perturbs its interaction with RPGR and results in Joubert syndrome: Monozygotic twins with discordant early-onset retinal degeneration in the rd16 mouse. Hum phenotypes. J Child Neurol 14: 649–654, 1999 Mol Genet 15: 1847–1857, 2006 36. Sayer JA, Otto EA, Hildebrandt F: Protein interaction part- 43. Badano JL, Kim JC, Hoskins BE, Lewis RA, Ansley SJ, ners of nephrocystin-1 using yeast-2-hybrid analysis [Ab- Cutler DJ, Castellan C, Beales PL, Leroux MR, Katsanis N: stract]. J Am Soc Nephrol 17: 517A, 2006 Heterozygous mutations in BBS1, BBS2 and BBS6 have a 37. Konrad M, Saunier S, Heidet L, Silbermann F, Benessy F, potential epistatic effect on Bardet-Biedl patients with two Calado J, Le Paslier D, Broyer M, Gubler MC, Antignac C: mutations at a second BBS locus Hum Mol Genet 12: 1651– Large homozygous deletions of the 2q13 region are a major 1659, 2003 cause of juvenile nephronophthisis. Hum Mol Genet 5: 367– 44. Badano JL, Leitch CC, Ansley SJ, May-Simera H, Lawson S, 371, 1996 Lewis RA, Beales PL, Dietz HC, Fisher S, Katsanis N: 38. Saunier S, Calado J, Benessy F, Silbermann F, Heilig R, Weis- Dissection of epistasis in oligogenic Bardet-Biedl syn- senbach J, Antignac C: Characterization of the NPHP1 locus: drome. Nature 439: 326–330, 2006

Access to UpToDate on-line is available for additional clinical information at http://www.jasn.org/