# The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies

Friederike Petzold, Katy Billot, Xiaoyi Chen, Charline Henry, Emilie Filhol, Yoann Martin, Marina Avramescu, Maxime Douillet, Vincent Morinière, Pauline Krug, et al.

► To cite this version:

Friederike Petzold, Katy Billot, Xiaoyi Chen, Charline Henry, Emilie Filhol, et al. The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies. Kidney International, 2023, 104 (2), pp.378-387. 10.1016/j.kint.2023.05.007. hal-04339478

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2 The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies.

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Friederike Petzold, MD*¹,², Katy Billot, PhD*¹, Xiaoyi Chen PhD*³,⁴, Charline Henry¹, Emilie Filhol¹,

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Yoann Martin¹, Marina Avramescu MD¹,⁶, Maxime Douillet³,⁴, Vincent Morinière⁵, Pauline Krug,

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MD¹,⁶, Cécile Jeanpierre, PhD¹, Kalman Tory, MD, PhD⁷, Olivia Boyer, MD, PhD⁶,⁸, Anita Burgun, MD,

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PhD⁴,⁹,¹⁰, Aude Servais, MD, PhD⁸, Remi Salomon¹,⁶,¹¹, Alexandre Benmerah, PhD¹, Laurence Heidet,

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MD, PhD¹,⁶,⁸, Nicolas Garcelon, PhD³,⁴, Corinne Antignac, MD, PhD¹, Mohamad Zaidan, MD, PhD§¹²,¹³

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and Sophie Saunier, PhD§¹

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+ INSERM-Necker Hospital NPH collaborative group

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* These authors contributed equally

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§ These authors contributed equally

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13 Affiliations:

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¹Laboratory of Hereditary Kidney Diseases, Université de Paris, Imagine Institute, INSERM UMR 1163, Paris, France.

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²Division of Nephrology, Department of Endocrinology, Nephrology, and Rheumatology, University Hospital Leipzig,
Leipzig, Germany.

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³ Université de Paris, Imagine Institute, Data Science Platform, INSERM UMR 1163, Paris, France.

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⁴Centre de Recherche des Cordeliers, Sorbonne Université, INSERM, Université de Paris, Paris, France.

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⁵APHP, Génétique moléculaire, Hôpital universitaire Necker-Enfants malades, Paris, France.

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⁶Department of Pediatrics, Necker Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.

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⁷Ist Department of Pediatrics, Semmelweis University, 1083 Budapest, Hungary.

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⁸Nephrology and Transplantation Department, Centre de référence des Maladies Rénales Héréditaires de l'Enfant et de l'Adulte, Necker Hospital, APHP, Université de Paris, France.

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⁹Hôpital Necker-Enfants Malades, Department of Medical Informatics, AP-HP, Paris, France.

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¹⁰PaRis Artificial Intelligence Research InstitutE (PRAIRIE), France.

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¹¹Université de Paris, Paris, France.

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¹²Assistance Publique des Hôpitaux de Paris (AP-HP), Université Paris-Saclay, Hôpital de Bicêtre, Service de Néphrologie et Transplantation, Le Kremlin-Bicêtre, France

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29 ¹³Centre de Compétence Maladies Rares « Syndrome Néphrotique Idiopathique », Hôpital de Bicêtre, Le Kremlin-

30 Bicêtre, France

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32 **Corresponding author:**

33 Sophie Saunier, PhD

34 Inserm U1163—Institut Imagine

35 Laboratory of Hereditary Kidney Diseases

36 24 Boulevard du Montparnasse

37 75015 Paris, France

38 Tel: +33 1 42 75 43 44

39 Fax: +33 1 42 75 42 25

40 Email: sophie.saunier@inserm.fr
41 Abstract

42 Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most 43 frequent causes of kidney failure in childhood characterized by a broad clinical and genetic 44 heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic 45 analysis encompassing targeted and whole exome sequencing identified disease-causing 46 variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic 47 variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) 48 bore biallelic pathogenic variants in *NPHP1*. NPH-causing gene alterations affected all ciliary 49 modules defined by structural and/or functional subdomains. Seventy six percent of these 50 patients had progressed to kidney failure, of which 18% had an infantile form (under five 51 years) and harbored variants affecting the Inversin compartment or intraflagellar transport 52 complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late- 53 onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition 54 Zone module. Furthermore, while more than 85% of patients with an infantile form presented 55 with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye 56 involvement represented a predominant feature, followed by cerebellar hypoplasia and other 57 brain abnormalities, liver and skeletal defects. The phenotypic variability were in a large part 58 associated with mutation types, genes and corresponding ciliary modules with hypomorphic 59 variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile- 60 to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH 61 suggesting an underdiagnosis in adult chronic kidney disease. 62

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63 **Keywords:** ciliopathy, cilium, end-stage kidney disease, nephronophthisis, tubulointerstitial nephritis.
64 **Lay summary**

65 Nephronophthisis (NPH) is a rare genetic disorder causing end-stage kidney disease in children and
66 young adults. It is based on dysfunction of the primary cilium, which is an antenna-like structure
67 playing an important role in many developmental processes. As it is located at the surface of almost
68 all cells, many patients present with additional extrarenal features affecting the eye, central nervous
69 system, liver, skeleton, and heart resulting in a broad clinical presentation. For a better
70 understanding of this clinical and genetic complexity, we analyzed a large cohort of 600 NPH patients
71 to classify genetic variants and to subgroup symptom complexes according to the gene and the
72 belonging ciliary structure. Using this approach, our study helps to assess families and patients to
73 predict their course of disease that will hopefully contribute to explore new therapeutic strategies.

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Introduction

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Ciliopathies are a large group of rare and severe diseases caused by genetic alterations that disrupt the function of the primary cilium. More than 180 genes have been associated with ciliopathies affecting up to one per 2000 persons.¹ Primary cilia are cellular organelles present at the surface of almost all cells in vertebrates, that sense extracellular cues and transmit signals to the cell body to orchestrate cell proliferation, tissue differentiation and homeostasis, as well as organ development and function.² Ciliopathies hence display a wide phenotypic spectrum affecting different organs including the kidneys, eyes, central nervous system (CNS), liver, bone and heart.

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One of the most common manifestations is kidney involvement comprising polycystic kidney disease and nephronophthisis (NPH), an autosomal recessive form of chronic tubulointerstitial nephritis representing one of the most frequent causes of end-stage kidney disease (ESKD) in children.³ Three clinical forms of NPH are distinguished according to the age of ESKD. While the infantile form manifests in patients younger than five years, juvenile form ranges between five and 15 years, and late-onset form emerge with ESKD older than 15 years to the adulthood.⁴,⁵ NPH can present either as an isolated kidney disease or as syndromic form associated with other organ manifestations. Extrarenal manifestations include retinitis pigmentosa, Leber amaurosis (Senior-Løken Syndrome, SLSN, MIM 266900), cerebellar vermis hypoplasia (Joubert syndrome, JBTS, MIM 609583), skeletal dysplasia (Jeune asphyxiating thoracic dystrophy syndrome, JATD, MIM 208500; Mainzer-Saldino syndrome, MZSDS, MIM 266920; cranioectodermal dysplasia, CED, MIM 218330), developmental delay, obesity, polydactyly (Bardet Biedl syndrome, BBS, MIM 209900), or encephalocele, and ductal plate malformations (Meckel syndrome, MKS, MIM 611561).

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Most nephronophthisis gene protein products play a key role in the structure or function of the cilium, in which they belong to different ciliary modules including the Basal Body (BB), the Distal Appendages (DA), the Transition Zone (TZ), the Inversin compartment (INVS), the Intraflagellar transport (IFT) A and B complexes, and the Bardet Biedl protein complex (BBS).

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Noteworthy, each module is defined by its localization within the cilium as well as the function and interaction of its constituting proteins (Figure 1a). However, the functional

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roles of proteins from a given module differ from each other, which explains the differential
phenotypic spectrum which can be observed in the context of mutations of genes encoding
proteins from a same module.<sup>2,6,7,8,9,10</sup>

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Briefly, primary cilia are assembled from the mother centriole of the centrosome, then forming the BB. Indeed, while DA at the distal part of the mother centriole initiate ciliogenesis allowing docking to cellular membranes,¹¹ the elongation of the distal ends of centriolar microtubules gives rise to the microtubule-based ciliary axoneme.⁹ Above the BB lies the TZ that is characterized by Y-shaped fibers connecting the microtubule doublets of the axoneme to the overlying ciliary membrane.⁹ The TZ controls both protein entry and exit from the cilium, thus exerting a gatekeeper role for ciliary composition. It includes two biochemically distinct domains: the TZ-JBTS/MKS and the TZ-NPH subcomplexes.¹² Distal to the TZ, is the INVS compartment, which regulates the Wnt pathway and is particularly involved in response to flow in different cell types, notably the renal tubular epithelial cells.¹³ The bidirectional transport of cargo across TZ and along the microtubules of the axoneme follows an anterograde (IFT-B) and retrograde (IFT-A) direction. IFT is necessary for both cilia growth and maintenance as well as for the dynamic transport of signaling components. Of note, IFT-B interacts with the cargo adapter BBSome to connect the IFT machinery to ciliary membrane proteins allowing their trafficking in and out the cilium.⁷,¹⁴

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So far, only a few NPH cohorts have been reported. One US American cohort comprised 142 patients carrying mutations in 13 NPH genes, foremost *NPHP1*, *NPHP4*, and *CEP290*; ¹⁵ a Dutch cohort collected 28 genetically solved patients (mainly *NPHP1*, *NPHP4*, *WDR35*); ¹⁶ a Chinese study recruited 60 patients (mainly *NPHP1*, *NPHP3*);¹⁷ finally, a clinical registry with an online network database included 383 NPH patients and identified the main variants in *NPHP1*, *NPHP3* and *TMEM67*.¹⁸

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In the present study, we report the genetic landscape and clinical spectrum of one of the worldwide largest cohorts of NPH patients collected over the last three decades at the INSERM/Imagine research laboratories and Genetic Department of Necker Hospital campus in Paris. With this unique cohort comprising 600 NPH patients showing a remarkably clinical and genetic complexity, we aimed at better defining the involvement of known ciliopathy genes in NPH patients, classifying mutation types, including hypomorphic variants, in terms of pathogenicity especially in late-onset NPH forms. We also outlined phenotypic groups to improve the clinical and molecular diagnosis as well as the prediction of kidney disease prognosis and pattern of extrarenal defects.

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149 **Methods**

150 **Patient recruitment**

151 Patients were recruited at the INSERM Research laboratories and Genetics Department of Necker
152 University Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France between 1990 and
153 2020. Written informed consent from all adult patients or legal guardians was obtained. DNA
154 samples were included in the Institut Imagine Biocollection declared to the French Minister of
155 Research (DC-2020-3994) and approved by the French Ethics committee for research at Assistance
156 Publique-Hôpitaux de Paris (CERAPHP) (IRB registration number #00011928). Detailed inclusion
157 criteria are listed in Supplementary File S1.

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159 **Data collection**

160 Detailed description of data curation and collection are described in Supplementary File S1.

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162 **Genetic testing**

163 Genomic DNA was extracted from peripheral blood samples by standard methods. Genetic analyses
164 were performed using various techniques including conventional PCR, QMPSF (quantitative multiplex
165 PCR of short fluorescent fragments) to detect large homozygous *NPHP1* deletions,<sup>19,20</sup>targeted
166 Sanger sequencing, NGS gene panel, referred as "ciliome" targeting the known ciliopathy genes<sup>21</sup>,
167 and Whole Exome Sequencing (WES). Detailed variant analysis are described in Supplementary File
168 S1.

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170 **Statistics**

171 Statistical tests and programs are listed in Supplementary File S1.

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173 **Results**

174 **The genetic landscape of NPH and related ciliopathies**

175 Over the last three decades, 834 patients from 694 families with clinically suspected NPH were
176 referred to our institution for genetic testing. Males and females were equally represented (50.5 vs.
177 49.5%). Half of the patients were European (50%), 33% were African, and 16% were Asian.
178 Consanguinity was noted in 19% of families.

179 Genetic analysis identified causal variants in 600 affected individuals from 493 families. The
180 subsequent detection rate was 71% (Supplementary Figure S1). Overall, 788 pathogenic variants in
40 ciliopathy genes were identified (Figure 1a and 1b). Among them, 18% are novel variants. The majority of patients for which causal variants could be identified bore biallelic variants in *NPHP1* (53%) with the common *NPHP1* deletion<sup>22(p1)</sup> being by far the main disease-causing event. Biallelic *TTC21B* variants were present in 6% of cases including 5% who carried the *TTC21B* p.P209L variant.<sup>23</sup> Biallelic variants in *NPHP4* or *NPHP3* each accounted for 5%, whereas variants in *IQCB1* amounted to 4%, including 1% with the *IQCB1* p.F142Pfs*5 variant<sup>24</sup> and *CEP290* accounted individually for 4% of patients. Finally, 3% of patients exhibited *INVS* variants, with the *INVS* p.R907* variant<sup>25</sup> found in 1% of cases (Supplementary Figure S2). Each remaining gene represented less than 3% of cases.

In addition to the genes classically known to be associated with NPH, 5% of patients carried pathogenic variants in ciliopathy genes which were not considered as NPH-associated genes so far, but for which variants were reported in individuals presenting with an NPH-like disorder. In 14 patients (2%), we identified causal variants in BBS genes (*BBS1*, *BBS2*, *BBS4*, *BBS5*, *BBS7*, *BBS10*, *BBS12*). We also related the clinical spectrum of 19 patients with NPH to five other ciliopathy genes (*OFD1*, *SCLT1*, *WDR35*, *IFT43*, *CPLANE1*). Finally, three patients displayed biallelic variants in two different ciliopathy genes indicating oligogenic inheritance (*NPHP4* and *TMEM237*, *WDR19* and *TEKT1*, *OFD1* and *INVS*; Supplementary File S2).

Overall, NPH-causing gene variants affected all ciliary modules with the TZ-NPH module representing 65% of pathogenic variants, followed by the IFT-A and the INVS compartment, representing 12% and 9%, respectively (Figure 1a). Noteworthy, the genes involved in the IFT-B complex, BBS, BB and DA each represented less than 3% of NPH-causing genetic variants.

Interestingly, biallelic loss-of-function (LoF) variants were predominant in genes of the TZ-NPH (93%), as well as the INVS (77%) and BB (60%) compartment. By contrast, biallelic LoF variants were observed in a minority fraction in IFT (IFT-A 33%, and IFT-B 19%) and in TZ-JBTS/MKS genes (5%; Supplementary Figure S3), probably reflecting the lethal consequences of biallelic LoF variants in the IFT genes as described in animal studies.<sup>26</sup>

## NPH-related CKD profile and progression towards chronic kidney failure

At the time of data collection, most patients (92%) had CKD (11.7 ± 7.5 years), of whom 83% had progressed to ESKD (13.2 ± 8.3 years). 18% of patients with ESKD had an infantile form whereas nearly half (48%) exhibited juvenile NPH and the remaining 34% showed a late-onset disease (Figure 2a). 16% of patients have not yet progressed to ESKD (12.0 years ± 8.8 years).
Patients with infantile NPH mainly harbored variants affecting the INVS compartment followed by the IFT-A complex, while patients with juvenile and late-onset form bore mostly variants belonging to the TZ-NPH module. Notably, *NPHP1* variants caused less than 6% of infantile forms, but 67% in juvenile and late-onset patients. (Figure 2a). Overall, patients carrying variants in genes encoding proteins of the TZ modules had a better kidney survival compared to those with variants in the INVS, DA, and IFT compartments (15.2 ± 7.2 years vs. 8 years ± 9.2; Figure 2b, Supplementary Figure S3).

In addition to the data on kidney function, information on renal ultrasounds, biopsies, and proteinuria were also accessible for some of the patients. Kidney ultrasound data were available for 216 patients and revealed hyperechogenicity (56%), cysts (43%), small kidney size (32%) and corticomedullary dedifferentiation (26%).

Detailed information on histopathological analysis of kidney biopsy specimens was available in 185 cases. Chronic tubulointerstitial nephropathy suggestive for NPH was described in 92% of biopsies; tubular membrane thickness was reported in 21% of cases that were mainly caused by *NPHP1* and *TTC21B* alterations; cortical microcysts were described in 15% of biopsied patients carrying mostly *INVS* or *NPHP3* variants; and glomerular basement membrane alterations was seen in 11% of patients that bore predominantly *TTC21B* and *NPHP4* variants.²³

Records for urinary abnormalities were available for 129 patients. 18% displayed significant proteinuria (> 0.5 g/d) and carried frequently variants belonging to the IFT-A module; most of which harbored the *TTC21B p.209L* variant (30%).

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## Ciliopathies-related extrarenal features and syndromic forms

Extrarenal manifestations were observed in 57% of patients (Figure 4a), including retinopathies, CNS defects, liver pathologies, skeletal disorders, heterotaxia (situs inversus) and male infertility (Figure 3). Patients with infantile NPH presented with extrarenal manifestations in more than 85% of cases, whereas juvenile and late-onset forms showed up as syndromic disease in 58% and 50%, respectively (Figure 4b-4d).

Eye involvement represented by far the predominant feature (65% of the syndromic cases). Retinal dystrophy and Leber Congenital Amaurosis were the most frequent manifestations. SLSN represented the most frequent syndromic form (35%), however, retinal involvement was also found in association with other syndromes such as JBTS, MZSDS, or BBS. Overall, *NPHP1*, *IQCB1*, and *CEP290* were the most frequently involved genes in these retinopathy patients (Figure 4a).
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The second major extrarenal feature was alterations of the CNS (55% of syndromic patients), with cerebellar hypoplasia corresponding to JBTS in 25% of syndromic cases. *NPHP1*, *CEP290*, and *TMEM67* were identified as the main causative genes. Besides the characteristic cerebellar involvement associated with JBTS, other neurodevelopmental defects including intellectual disability, autism, and cortical atrophy were described in an additional 30% of syndromic patients who predominantly bore *NPHP1*, *CEP83*, and *NPHP3* variants.

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30% of syndromic cases exhibited liver abnormalities, comprising altered liver function, hepatomegaly, bile duct dilatation, liver fibrosis and cirrhosis; 6% presented with congenital hepatic fibrosis, which is the typical ductal plate malformation seen in liver ciliopathies. *NPHP3*, *TTC21B*, and *IFT140* were identified as the main causative genes. Of note, liver abnormalities were also found in the context of other syndromic NPH, notably skeletal ciliopathies, JBTS, and BBS.

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Skeletal malformations are common features in ciliopathies and involved mainly genes encoding IFT-A and B components. In our cohort, well-characterized skeletal defects accounted for 11% of syndromic NPH cases, and included MZSDS (8%), CED (3%), and JATD (1%). Skeletal anomalies were mainly related to hands and feet (13% of syndromic patients), cranial and thorax abnormalities (4% each). Besides the typical bone lesions, MZSDS patients also displayed liver abnormalities in many cases (83%) and frequently retinal dystrophy (63%). *TTC21B*, *IFT140*, and *TRAF3IP1* were identified as the main causative genes. Beside skeletal, liver and eye involvements (100%, 67%, and 42%, respectively), CED and JATD patients also exhibited CNS defects (25%), and harbored most frequently *WDR35* and *IFT140* variants.

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Of note, 5% of syndromic patients displayed post-axial polydactyly, and were considered apart from those with the above-mentioned skeletal ciliopathies. Indeed, most of them were diagnosed with BBS, based on the presence of additional typical features and harbored, as expected, variants in BBS genes.

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Heart involvement was reported in 8% of syndromic cases as the unique extrarenal manifestation or in association with liver abnormalities, CNS involvement, and skeletal defects.

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Finally, 3% of patients displayed laterality defects as situs inversus. Heart involvement and situs inversus were both mainly related to *INVS* and *TTC21B* variants. Fertility disorders including infertility, hypofertility, hypogonadism, and cryptorchidism were reported in 13 male patients and associated to variants in *NPHP1* (hypo-/infertility), *BBS7*, and *NPHP3*. Possible causes are defects in the spermatogenesis as demonstrated in *NPHP1* and *NPHP4* murine and human studies²⁷,²⁸,²⁹ as well as primary testicular atrophy or hypothalamic-pituitary dysfunction in BBS patients.³⁰

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276 Genotype-Phenotype correlations with the mutation type

277 Genotype-Phenotype correlations have been described previously for some ciliopathy genes, such as
278 the TZ genes *RPGRIP1L* and *TMEM67*, and for the INVS compartment component *NPHP3*.³¹,¹⁸ Hereby,
279 *TMEM67* missense mutations were associated with a JBTS phenotype, while truncating variants
280 caused severe MKS forms.³¹ In our study, we confirmed similar observations for genes from other
281 ciliary modules. For both IFT-A and IFT-B genes, there was a clear genotype-phenotype correlation,
282 as patients with homozygous or compound heterozygous missense mutations developed
283 juvenile/late-onset ESKD at 14 ± 23.9 years, while patients carrying missense in association with LoF
284 variants progressed to ESKD at a younger age (9 ± 7.2 years) (Figure 2c).

285 Similarly, the DA plays an essential role in ciliogenesis by anchoring the mother centriole to the
cellular membrane. As a severe dysfunction of those DA proteins would prevent cilia formation
286 leading to fetal developmental phenotypes,³² the DA genes are characterized by the absence of
287 biallelic LoF variants. All *CEP83* patients displayed a severe syndromic phenotype with ESKD at mean
288 age of four years and bore at least one missense mutation or an in-frame deletion likely resulting in a
289 partially preserved protein function.³³
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291 Finally, seven male patients bore hemizygous *OFD1* variants that are associated with variable OFD
292 syndrome and polycystic kidneys.³⁴ Five had an infantile NPH (4.4 ± 0.4 years) and displayed variants
293 that affected exon splicing (*c.2260+1G>A* or *c.2260+1G>T*) or indels (*c.1655-1_1655delinsAG*). These
294 variants were predicted to cause the skipping of exon 16 resulting in an in-frame deletion with a
295 potential milder impact than LoF variants (Supplementary Table S1; Supplementary Figure S2). In this
296 line, a sixth case presented with ESKD at 2.3 years and carried a stop variant *p.Glu584**, which was
297 also predicted to favor skipping of exon 16. The seventh *OFD1* patient carried a hemizygous variant
298 *p.K976T* within exon 21 and developed ESKD aged 14 years. Six patients showed additional retinal
299 defects. Altogether, these observations suggest that a subset of hypomorphic *OFD1* variants, mainly
300 corresponding to skipping of exon 16, could be specifically involved in NPH and retinal degeneration.

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302 Genotype-Phenotype correlations based on ciliary modules

303 The establishment of genotype-phenotype correlations was complicated by a great variability of
304 symptoms, an important overlap between the main ciliopathy syndromes, and the high number of
305 genes identified in the present cohort.
We performed an unsupervised projection (UMAP) for patients with extrarenal manifestations, and then applied a density-based spatial clustering algorithm (DBSCAN). Five phenotypic clusters could be identified recapitulating the main ciliary modules (Figure 5a and 5b). In the first (C1, n=65) and second (C2, n=30) cluster, most of the patients had kidney and CNS affection, with (C1) or without (C2) eye involvement. These clusters comprised together 95 patients of whom 78% carried variants in TZ modules. The third cluster (C3, n=47) contained patients with kidney and liver abnormalities, while half of them harbored variants encoding INVS compartment components. The fourth (C4) represented the biggest cluster that included 116 patients presenting with eye involvement, 78% of whom harboring variants in the TZ-NPH module. Located in between C3 and C4 clusters, the fifth cluster (C5; n=76) was more heterogenous, presenting basically kidney, eye, liver and bone involvement, including 64% of the patients with variants in the IFT-A genes, and 86% of the patients with IFT-B gene variants. In contrast to other modules, patients carrying variants in DA and BBS modules did not assemble close to each other but were scattered among the different clusters, reflecting the high phenotypic heterogeneity of these groups.

A similar clustering analysis for all the 834 patients, including patients with isolated kidney disease, as well as patients without molecular diagnosis, revealed that patients with similar phenotypes for whom a single pathogenic variant was identified frequently carried a variant in a gene of the same module. This is particularly true for undiagnosed patients with liver abnormalities who carried a heterozygous pathogenic variant in an INVS compartment gene (Supplementary Figure S4). This suggests that a second pathogenic variant in the same gene was likely missed during the analysis, probably located in non-coding regions.

Next, we investigated the proportion of patients presenting with extrarenal phenotypes in each module (Figure 5c). Hierarchical clustering showed that the IFT-B and BBS modules were associated with the broadest phenotypic spectrum mixing kidney, eye, liver, and bone involvement, together with obesity and recurrent respiratory infections. IFT-A and INVS modules were both characterized by predominant syndromic forms including liver involvement. BB, TZ-JBTS/MKS, and DA modules, were associated with a more restricted phenotype including kidney, eye and CNS features, while TZ-NPH was phenotypically distinct with a low percentage of extrarenal features mainly affecting the eye.

## Discussion
The present study depicts the genetic landscape and clinical spectrum of a large cohort of patients with NPH and associated ciliopathies. The recruitment of NPH patients changed during the period of over 30 years. Initially, in 1990, mainly families with infantile NPH and additional retinitis pigmentosa (SLSN), later familiar cases with cerebellar atrophy (JBTS) and cone-shaped epiphysis (MZSDS) were enrolled. With the increasing knowledge of the pathophysiological role of the cilium, and the introduction of targeted gene panels, more sporadic and cases with other extrarenal symptoms were considered for analysis increasing the diagnostic yield. Using NGS-based genetic analysis, we identified NPH-causing variants in 40 ciliopathy genes, extended the number of disease-causing variants and delineated genotype-phenotype correlations in these complex disorders.

Notably, we added 168 novel disease-causing variants to the HGMD library for NPH genes,³⁵ and variants in genes usually associated with skeletal ciliopathies and in BBS genes as a potential cause of NPH. Despite an extensive analysis, we failed to identify a molecular diagnosis in 29% of clinical NPH cases. However, 38% of unresolved cases were analyzed only with Sanger or QMPSF methodologies and not for all known ciliopathy genes. For the remaining 62%, analyzed by panel and WES sequencing, it highlights the possibility of phenocopies caused by non-ciliary genes and the pitfalls of the current strategy to detect complex or intronic genetic variations in known genes.³⁶

NPH is a peculiar cause of autosomal recessive chronic tubulointerstitial nephritis, which is characterized by a great variability in CKD progression. In particular, variants in *NPHP1* and *IQCB1* showed a wide range of age of onset of ESKD ranging from juvenile to adult forms, while variants in INVS and DA modules were predominantly associated with infantile NPH. For IFT genes, there is a clear genotype-phenotype correlation of missense variants causing late-onset ESKD, while LoF variants lead to juvenile ESKD. Finally, a large proportion of the NPH patients showed a late-onset form of disease that was mainly associated to *NPHP1*, *NPHP4*, and *IQCB1* variants. These latter data are in line with recent reports describing adult patients with isolated NPH,³⁷,¹⁸,³⁸ underscoring the difficulty of an accurate diagnosis of late-onset NPH.

Beyond the variability in CKD progression, renal ciliopathies are also characterized by a great heterogeneity of associated extrarenal manifestations. For instance, while a majority of *NPHP1* patients presented with an isolated NPH, one-third exhibited a syndromic form, mostly SLSN and JBTS. Thus, other yet-unidentified modifiers,³⁹ including the mutational burden of ciliary proteome,⁴⁰ are factors that likely determine the phenotypic variability at the patient's level.

However, our study has shown that the nature of the variant may partly account for the clinical heterogeneity, for instance, in ciliary genes playing a role in early ciliogenesis, such as *SCLT1* and *OFD1*, hypomorphic variants, variants with low impact on splicing or in-frame exon skipping were
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associated with late-onset and mild-to-moderate forms, while biallelic LoF variants in these genes lead usually to very severe and, sometimes lethal phenotypes.⁴¹ We therefore suggest that mild damaging variants in critical ciliary modules should be considered in the genetic analysis, particularly in patients with late-onset NPH.

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Interestingly, kidney survival analysis and UMAP clustering revealed that genetic alterations in different components of a same ciliary module shared phenotypic similarities, suggesting a ciliary module-based genotype-phenotype correlation. We identified five clusters corresponding to phenotypic extrarenal groups, three of which were characterized by an overlap, which was consistent with their functional role in ciliogenesis and interaction with each other. Indeed, genetic alterations in the BB and DA share a major role in the initiation of ciliogenesis,⁴² while the IFT is of pivotal importance for the assembly and maintenance of cilia.⁴³

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381

By applying a ciliary module-based approach, we provide a practical view for clinicians to handle the clinical and genetic complexity of NPH-related ciliopathies, as genetic counseling may allow moreover to an accurate diagnosis, the evaluation of the renal prognosis and the extent of the disease. This study will help to better define and extend NPH causing genes beyond the 23 classically well-admitted ones²⁰ and classify variants type.

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Nevertheless, identifying the genetic background of the unsolved families, detecting late-onset patients, and investigating additional factors that account for the disease variability remains challenging. A broader use of new sequencing tools combined with transcriptomic and functional studies will undoubtedly help to better understand the great heterogeneity of renal ciliopathies. This will lead to novel approaches for stratifying risk patients, better predicting disease course, and repurposing potentially common therapeutic targets.⁴⁴

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393
394 Disclosures

395 This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM),
396 by a State funding from the Agence Nationale de la Recherche under "Investissements d'avenir"
397 program (ANR-10-IAHU-01) and by a public grant "RHU-C'IL-LICO" overseen by the French National
398 Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-
399 17-RHUS-0002). FP is funded by the German Research Foundation (Deutsche
400 Forschungsgemeinschaft, DFG) grant PE 3135/1-1. OB has received consulting fees from Alnylam and
401 Vifor/CSL and Support for attending meetings from Biocodex.

402 The authors declare that they have no potential conflicts of interests.

403

404 Acknowledgements

405 The authors sincerely thank the affected individuals and their families for participation, and the
406 clinicians, for their contribution to this study. We greatly acknowledge members of the bioinformatic
407 and genomic facilities of the Imagine Institute.

408

409 Data availability

410 The data that support the findings of this study are available from the corresponding author upon
411 reasonable request.

412

413 Appendix

414 Contributing clinicians of the INSERM-Necker Hospital NPH collaborative group are listed as follows:

415 Tania Attié-Bitach, Valerie Comier-Daire, Jean-Michel Rozet, Yaacov Frishberg, Brigitte Llanas, Michel
416 Broyer, Nabil Mohsin, Marie-Alice Macher, Nicole Philip, Véronique Baudouin, Damian Brackman,
417 Chantal Loirat, Marina Charbit, Maud Dehennault, Claude Guyot, Pierre Bataille, Mariet Elting,
418 Georges Deschenes, Andrea Gropman, Geneviève Guest, Marie-France Gagnadoux, Philippe Nicoud,
419 Pierre Cochat, Bruno Ranchin, Albert Bensman, Anne-Marie Guerrot, Bertrand Knebelmann, Ilmay
420 Bilge, Danièle Bruno, Stéphane Burtey, Caroline Rousset Rouvière, Valérie Caudwell, Denis Morin,
421 Hélène Dollfus, Anne Maisin, Christian Hamel, Eric Bieth, Sophie Gie, Judith Goodship, Gwenaelle
422 Roussey, Hermine La Selve, Hubert Nivet, Lucie Bessenay, Mathilde Caillez, Jean Bernard Palcoux,
423 Stéphane Benoît, Philippe Dubot, Marc Fila, Fabienne Giuliano, Daouya Iftene, Michele Kessler,
424 Theresa Kwon, Anine Lahoche, Audrey Laurent, Anne-Laure Leclerc, David Milford, Thomas Neuhaus,
425 Sylvie Odent, Philippe Eckart, Dominique Chauveau, Patrick Niaudet, Horacio Repetto, Sophie Taque,
426 Alexandra Bruel, Alexandra Noel-Botte, Emma Allain Launay, Lisa Allard, Dany Anlicheau, Anne-Laure
427 Adra, Arnaud Garnier, Arvind Nagra, Remy Baatard, Justine Bacchetta, Banu Sadikoglu, Christine
428 Barnerias, Anne Barthelemy, Lina Basel, Nader Bassilios, Hedi Ben Maiz, Fatma Ben Moussa, Faïza
429 Benmati, Romain Berthaud, Aurélia Bertholet, Dominique Blanchier, Jean Jacques Boffa, Karim
430 Bouchireb, Ihab Bouhabel, Zakaria Boukerroucha, Guylhène Bourdat-Michel, Odile Boute, Karine
431 Brochard, Roseline Caumes, Siham Chafai Elalaoui, Bernard Chamontin, Marie Caroline Chastang,
432 Christine Pietrement, Christine Richer, Christophe Legendre, Karin Dahan, Fabienne Dalla-Vale,
433 Damien Thibaudin, Maxime Dauvergne, Salandre Davourie, Martin Debeukelaer, Jean Daniel Delbet,
434 Constantinos Deltas, Denis Graber, Nadège Devillars, Boucar Diouf, Martine Doco Fenzy, Jean-Luc
435 André, Dominique Joly, Alan Fryer, Laetitia Albano, Elisabeth Cassuto, Aline Pincon, Ana Medeira,
436 Annabelle Chaussenot, Anne Mensire-Marinier, Francois Bouissou, Stephane Decramer, Armand
437 Bottani, Aurélie Hummel, Alexandre Karras, Avi Katz, Christine Azema, Bénédicte Janbon, Bernard
438 Roussel, Claude Bonniol, Christiophe Mariat, Gérard Champion, Deborah Chantreuil, Nicolas
439 Chassaing, Christiane Mousson, Christine Baudeau, Delphine Hafdar Cuntz, Cyril Mignot, Laurene
440 Dehoux, Didier Lacombe, Thierry Hannedouche, Elodie Mérieau, Emmanuelle Charlin, Eric Gauthier,
441 Florent Plasse, Stanislas Faguer, Fanny Lebas, Florence Demurger, Francesco Emma, François
442 Cartault, Geneviève Dumont, Nathalie Godefroid, Vincent Guigonis, Sophie Hillaire, Jaap Groothoff,
443 Jan Dudley, Noémie Jourde-Chiche, Khalil El Karoui, Saoussen Krid, Krier Coudert, Larbi Bencheick,
444 Laurent Yver, Marie-Pierre Lavocat, Le Monies De Sagazan, Valerie Leroy, Lise Thibaudin, Liz Ingulli,
445 Lorraine Gwanmesia, Lydie Burglen, Marie-Hélène Saïd-Menthon, Marta Carrera, Mathilde Nizon,
446 Catherine Melander, Michel Foulard, Monique Blayo, Jacques Prinseau, Nadine Jay, Nathalie Brun,
447 Nicolas Camille, François Nobili, Olivier Devuyst, Ouafa Ben Brahim, Paloma Parvex, Laurence Perrin
448 Sabourin, Philippe Blanc, Philippe Vanhille, Pierre Galichon, Sophie Pierrepont, Vincent Planquois,
449 Gwenaelle Poussard, Claire Pouteil Noble, Radia Allal, Raphaelle Bernard, Raynaud Mounet, Rémi
450 Cahen, Renaud Touraine, Claire Rigothier, Amélie Ryckewaert, Mathieu Sacquepee, Salima El
451 Chehadeh, Charlotte Samaille, Shuman Haq, Ari Simckes, Stéphanie Lanoiselée, Stephanie Tellier,
452 Jean-François Subra, Sylvie Cloarec, Julie Tenenbam, Thomas Lamy, Valérie Drouin Garraud, Huguette
453 Valette, Vanina Meyssonnier, Rosa Vargas-Poussou, Yves Snajer, Sandrine Durault, Emmanuelle
454 Plaisier, Etienne Berard, Fadi Fakhouri, Ferielle Louillet, Paul Finielz, Michel Fischbach, Bernard
455 Foliguet, Hélène Francois-Pradier, Florentine Garaix, Marion Gerard, Gianfranco Rizzoni, Brigitte
456 Gilbert, Denis Glotz, Astrid Godron Dubrasquet, Jean-Pierre Grünfeld, Guillaume Bollee, Michelle
457 Hall, Sverker Hansson, Damien Haye, Hélène Taffin, Friedhelm Hildebrandt, Maryvonne Hourmand,
458 Hümya Kayserili, Ivan Tack, Marie Line Jacquemont, Jennifer Fabre-Teste, Cliff Kashtan, Kkoen Van
459 Hoeck, Alexandre Klein, Yannick Knefati, Nine Knoers, Martin Konrad, Alain Lachaux, Isabelle Landru,
460 Gilbert Landthaler, Philippe Lang, Patrick Le Pogamp, Tristan Legris, Catherine Didailler, Thierry
461 Lobbedez, Loïc de Parscau, Lucile Pinson, Hervé Maheut, Marc Duval-Arnould, Marlène Rio, Marie-
462 Claire Gubler, Pierre Merville, Guillaume Mestrallet, Maite Meunier, Karine Moreau, Jérôme
463 Harambat, Graeme Morgan, Georges Mourad, Niksic Stuber, Odile Boespflug-Tanguy, Olivier
464 Dunand, Olivier Niel, Nacera Ouali, Paolo Malvezzi, Pauline Abou Jaoude, Solenne Pelletier, Julie
465 Peltier, M. B. Petersen, Philippe Michel, Philippe Rémy, Jean-Baptiste Philit, Valérie Pichault, Thierry
466 Billette de Villemeur, Bernard Boudailliez, Bruno Leheup, Claire Dossier, Djamal-Dine Djeddi, Yves
467 Berland, Bruno Hurault de Ligny, Susan Rigden, Christophe Robino, Annick Rossi, Sabine Sarnacki,
468 Messaoud Saidani, Albane Brodin Sartorius, Elise Schäfer, Sztricha Laszlo, Marie-Christine Thouret,
469 Angélique Thuillier-Lecouf, Howard Trachtman, Claire Trivin, Michel Tsimaratos, Rita Van Damme-
470 Lombaerts, Marjolaine Willems, Michel Youssef, Ariane Zaloszyc, Alexis Zawodnik, Marie-Julia
471 Ziliotis.

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603

604
605 **Figure Titles and Legends**

606 **Figure 1**

607 **a** Composition of the primary cilium: The mother centriole with the distal appendages (DA) connects 608 to the cellular membrane and forms the basal body, which gives rise to the microtubule-based ciliary 609 axoneme. Distal, the transition zone including two distinct protein complexes, the NPH and JBTS/MKS 610 subdomain, controls protein entry and exit and joins to the Inversin compartment. The Intraflagellar 611 transport (IFT) system carries cargo along the microtubules and interacts with the cargo adapter 612 BBSome to mediate export of ciliary proteins. NPH genes/proteins localize to the functional and 613 phenotypically based ciliary compartments (except MAPKBP1, ADAMTS9, and GLIS2 without ciliary 614 localization). The portion refer to the frequencies of the causative genes/compartments analyzed in 615 the cohort. **b** Relative frequencies of disease-causing genes in the cohort of 600 NPH patients. 616

616

617 **Figure 2**

618 **a** Distribution of disease-causing genes related to the three NPH forms according to the age of onset 619 of ESKD: infantile form (<5 years), juvenile (5-15 years) and late-onset form (> 15 years). **b** Ciliary 620 module-related kidney survival. C Mutation type related kidney survival of IFT module. Genetic IFT 621 variants were classified into compound-heterozygous missense + LoF variant (violet) and 622 homozygous or compound-heterozygous missense/VUS/in-frame deletion/hypomorphic splicing 623 variant (green). 624

624

625 **Figure 3**

626 Detailed phenotypic features by ciliary gene. Only genes that were altered in five or more patients 627 are listed. Numeric data represent the percentage of patients with a biallelic variant that display a 628 given phenotypic feature. Abbreviations: BB Basal Body; BBS Bardet Biedl Syndrome; DA Distal 629 Appendages; IFT Intraflagellar Transport; INVS Inversin; TZ Transition Zone; JBTS Joubert Syndrome; 630 MKS Meckel syndrome; NPH Nephronophthisis. 631

631

632 **Figure 4**
633 a Relative fraction of clinical NPH syndromes and the causative mutations in the overall cohort, and
634 related to the age of onset of ESKD as infantile (b), juvenile (c) and late-onset NPH form (d). NPH
635 isolated nephronophthisis, SLS Senior-Løken syndrome, JBTS Joubert syndrome, MZSDS Saldino-
636 Mainzer syndrome, CED cranioectodermal dysplasia, JATD Asphyxiating thoracic dystrophy, BBS
637 Bardet-Biedl syndrome.

638

639 **Figure 5**

640 a Unsupervised projection of eleven selected extrarenal phenotypes (eye, CNS, liver, bone,
641 polydactyly, obesity, situs inversus, heart, deafness, recurrent respiratory infections, and genital
642 disorders) of 338 syndromic patients and clustering revealed five main clusters. One dot represented
643 one patient colored according the variants corresponding ciliary module. CO correlated to a noise
644 cluster containing patients who have not been clustered in any cluster. b Number of patients in each
645 cluster carrying variants in each module. c Clustering of main ciliary modules using proportion of
646 patients presenting extrarenal main organ involvement.

647

648
<figure><img src="image_2.png" /><figcaption>**Figure 1. A Composition of the primary cilium:** The mother centriole with the distal appendages (DA) connects to the cellular membrane and forms the basal body, which gives rise to the microtubule-based ciliary axoneme. Distal, the transition zone including two distinct protein complexes, the NPH and JBTS/MKS subdomain, controls protein entry and exit and joins to the Inversin compartment. The Intraflagellar transport (IFT) system carries cargo along the microtubules and interacts with the cargo adapter BBSome to mediate export of ciliary proteins. NPH genes/proteins localize to the functional and phenotypically based ciliary compartments (except *MAPKBP1*, *ADAMTS9*, and *GLIS2* without ciliary localization). The portion refer to the frequencies of the causative genes/compartments analyzed in the cohort. **B Relative frequencies of disease causing genes** in the cohort of 600 NPH patients.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** A Distribution of disease-causing genes related to the three NPH forms according to the age of onset of ESKD: infantile form (<5 years), juvenile (5-15 years) and late-onset form (> 15 years). **B** Ciliary module-related kidney survival. **C** Mutation type related kidney survival of IFT module. Genetic IFT variants were classified into compound-heterozygous missense + LoF variant (violet) and homozygous or compound-heterozygous missense/VUS/in-frame deletion/hypomorphic splicing variant (green).</figcaption></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>
<table><thead><tr><th>Ciliary Modules</th><th>Genes</th><th>number of patients</th><th>SYNDROMIC</th><th>EYE</th><th>Retinal abnormalities</th><th>Abnormal oculomotricity</th><th>Other eye</th><th>CNS</th><th>Intellectual disability</th><th>Cerebellar abnormalities</th><th>Other CNS</th><th>LIVER</th><th>BONE</th><th>Cranium</th><th>Thorax</th><th>Axial skeleton</th><th>Appendicular skeleton</th><th>Hands/feet</th><th>Other bone / Not specified</th><th>POLYDACTYLY</th><th>HEART</th><th>Valve defects</th><th>Septal defects</th><th>Vessel defects</th><th>Other heart</th><th>SITUS</th><th>INVERSUS</th><th>MISCELLANO US</th><th>Deafness</th><th>Obesity</th><th>Genital disorders</th><th>Recurrent Respiratory</th><th>Other</th></tr></thead><tbody><tr><td>BB</td><td>OFD1</td><td>6</td><td>100</td><td>83</td><td>83</td><td></td><td></td><td>17</td><td>17</td><td></td><td></td><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>17</td><td></td><td></td><td></td><td>17</td><td></td></tr><tr><td>DA</td><td>CEP83</td><td>7</td><td>71</td><td>14</td><td>14</td><td></td><td></td><td>57</td><td>57</td><td></td><td>14</td><td>29</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">IFT-A</td><td>TTC21B</td><td>36</td><td>81</td><td>39</td><td>22</td><td>11</td><td>22</td><td>11</td><td>11</td><td></td><td>3</td><td>53</td><td>31</td><td>3</td><td></td><td></td><td></td><td>6</td><td>22</td><td>6</td><td></td><td>8</td><td></td><td></td><td>8</td><td>8</td><td>6</td><td></td><td></td><td>3</td><td>3</td></tr><tr><td>WDR19</td><td>14</td><td>71</td><td>71</td><td>64</td><td>7</td><td>29</td><td></td><td></td><td></td><td></td><td>14</td><td>16</td><td>7</td><td>7</td><td></td><td></td><td>14</td><td>7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>29</td><td>7</td><td></td><td>21</td></tr><tr><td>IFT140</td><td>12</td><td>83</td><td>58</td><td>50</td><td></td><td>17</td><td></td><td></td><td></td><td></td><td>67</td><td>83</td><td>25</td><td>33</td><td></td><td></td><td>25</td><td>75</td><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>33</td><td>8</td><td></td><td>17</td><td>17</td></tr><tr><td>WDR35</td><td>7</td><td>100</td><td>29</td><td>14</td><td></td><td>29</td><td>43</td><td>43</td><td></td><td>29</td><td>86</td><td>86</td><td>57</td><td>57</td><td>14</td><td></td><td>43</td><td>29</td><td>14</td><td>14</td><td>14</td><td></td><td></td><td></td><td></td><td>14</td><td>14</td><td></td><td></td><td></td></tr><tr><td>IFT-B</td><td>TRAF3IP1</td><td>10</td><td>100</td><td>100</td><td>60</td><td>10</td><td>40</td><td>20</td><td>20</td><td></td><td></td><td>30</td><td>60</td><td></td><td></td><td></td><td></td><td>30</td><td>10</td><td>40</td><td>30</td><td></td><td></td><td></td><td></td><td></td><td></td><td>30</td><td>30</td><td>10</td><td>20</td><td></td></tr><tr><td rowspan="3">INVS</td><td>NPHP3</td><td>27</td><td>81</td><td>15</td><td>11</td><td></td><td>4</td><td>26</td><td>15</td><td>11</td><td>7</td><td>70</td><td>15</td><td></td><td></td><td></td><td>7</td><td>7</td><td>7</td><td>7</td><td>7</td><td>11</td><td>7</td><td>4</td><td></td><td>4</td><td>19</td><td></td><td>4</td><td>4</td><td>11</td></tr><tr><td>INVS</td><td>20</td><td>70</td><td></td><td></td><td></td><td></td><td>20</td><td>15</td><td>10</td><td>5</td><td>25</td><td>10</td><td></td><td></td><td></td><td>5</td><td></td><td></td><td>5</td><td></td><td></td><td></td><td></td><td></td><td>25</td><td>20</td><td>10</td><td></td><td>10</td><td>30</td><td>5</td></tr><tr><td>ANKS6</td><td>6</td><td>33</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>33</td><td>17</td><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>17</td><td>17</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">TZ-JBTS/MKS</td><td>TMEM67</td><td>11</td><td>82</td><td>45</td><td>18</td><td>36</td><td>9</td><td>55</td><td>45</td><td>45</td><td></td><td>64</td><td>9</td><td></td><td></td><td></td><td></td><td>9</td><td>9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>9</td><td></td><td></td><td></td><td>9</td></tr><tr><td>RPGRIP1L</td><td>7</td><td>100</td><td>43</td><td>14</td><td>29</td><td>29</td><td>100</td><td>86</td><td>57</td><td>14</td><td></td><td>29</td><td>14</td><td></td><td>29</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>14</td><td>14</td><td></td><td></td><td></td></tr><tr><td rowspan="4">TZ-NPH</td><td>NPHP1</td><td>317</td><td>35</td><td>24</td><td>15</td><td>11</td><td>9</td><td>14</td><td>10</td><td>7</td><td>4</td><td>2</td><td>3</td><td></td><td>1</td><td></td><td>2</td><td>1</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td><td>1</td><td></td><td>3</td><td>1</td><td>1</td></tr><tr><td>NPHP4</td><td>28</td><td>36</td><td>25</td><td>21</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td>4</td><td></td><td></td><td>4</td><td></td></tr><tr><td>IQCB1</td><td>23</td><td>100</td><td>100</td><td>22</td><td>3</td><td>5</td><td>13</td><td>3</td><td></td><td></td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td></td><td></td><td>4</td><td></td></tr><tr><td>CEP290</td><td>23</td><td>100</td><td>100</td><td>91</td><td>30</td><td>17</td><td>61</td><td>43</td><td>57</td><td>9</td><td>4</td><td>4</td><td></td><td></td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>13</td><td>4</td><td></td><td>4</td><td>4</td></tr><tr><td>others</td><td>MAPKBP1</td><td>5</td><td>40</td><td>20</td><td></td><td></td><td>20</td><td>20</td><td>20</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>40</td><td>20</td><td></td><td></td><td></td><td>20</td></tr></tbody></table>

Colors represent a gradient of % as follows:

<table><tr><td>&lt;10%</td><td>10-24%</td><td>25-49%</td><td>50-74%</td><td>75-99%</td><td>100%</td></tr></table>
<figure><img src="image_7.png" /><figcaption>**Figure 3.** A Relative fractions of clinical NPH syndromes and the causative mutations in the overall cohort, and related to the age of onset of ESKD as infantile (B), juvenile (C) and late-onset NPH form (D). NPH isolated nephronophthisis, SLS Senior-Løken syndrome, JBTS Joubert syndrome, MSS Saldino-Mainzer syndrome, CED cranioectodermal dysplasia, JATD Asphyxiating thoracic dystrophy, BBS Bardet-Biedl syndrome.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 4.** A Unsupervised projection of eleven selected extrarenal phenotypes (eye, CNS, liver, bone, polydactyly, obesity, situs inversus, heart, deafness, recurrent respiratory infections, and genital disorders) of 338 syndromic patients and clustering revealed five main clusters. One dot represented one patient colored according the variants corresponding ciliary module. C0 correlated to a noise cluster containing patients who have not been clustered in any cluster. B Number of patients in each cluster carrying variants in each module. C Clustering of main ciliary modules using proportion of patients presenting extrarenal main organ involvement.</figcaption></figure>