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Nephronophthisis (NPHP) is an autosomal recessive kidney disorder characterized by chronic tubulointerstitial nephritis and leading to end-stage renal failure. NPHP as a renal entity is often part of a multisystem disorder and has been associated with many syndromes including Joubert syndrome (and related disorders) and Senior–Loken syndrome. Recent molecular genetic advances have allowed identification of several genes underlying NPHP. Most of these genes express their protein products, named nephrocystins, in primary cilial/basal body structures. Some nephrocystins are part of adherens junction and focal adhesion kinase protein complexes. This shared localization suggests that common pathogenic mechanisms within the kidney underlie this disease. Functional studies implicate nephrocystins in planar cell polarity pathways, which may be crucial for renal development and maintenance of tubular architecture.
Nephronophthisis (NPHP) is an autosomal recessively inherited renal disorder, which leads to progressive renal failure, usually within the first 3 decades of life.1 Nephronophthisis literally means ‘disappearance of nephrons'. Typical ultrasound features include normal or reduced renal size, loss of corticomedullary differentiation and corticomedullary cysts (Figure 1). Renal biopsy findings include tubular atrophy, interstitial fibrosis and tubular basement membrane defects, including abrupt transition between thickening and attenuation or disintegration.2, 3 A rare form of NPHP may lead to end-stage renal failure (ESRF) within 5 years of age and is termed infantile NPHP.4 This differs from typical NPHP in that there is moderate renal enlargement, histological changes that include cortical microcysts, cystic dilatation of Bowman's spaces and lack of tubular basement membrane disruption.
NPHP is often part of a spectrum of multisystem disease and may not be detected unless appropriate investigations on relevant systems are performed. These disease associations form a very heterogeneous group (Table 1). The most commonly associated syndrome is retinal dystrophy and retinal degeneration leading to blindness (Senior–Loken syndrome).1 Other associations include Joubert syndrome and related diseases (JSRD, reviewed in reference5), which often involves a cerebellar, retinal and renal phenotype referred to as CORS (cerebello-oculo-renal syndrome). Apart from these, a whole variety of syndromes have been reported in association with NPHP (Table 1).
NPHP has been reported worldwide, yet the incidence varies. A Canadian study reported an incidence of 1 in 50000 live births,6 whereas the incidence in the United States of America was estimated to be 9 per 8.3 million.7 A more recent European study reported an incidence of NPHP as 1 in 61800 live births.8 However, as NPHP may present in adults with late enuresis and renal failure,9 these figures may be an underestimate.
NPHP is genetically and clinically heterogeneous. Traditionally, NPHP has been subdivided into infantile, juvenile and adolescent forms, based on the age of onset of renal failure. It remains useful to distinguish the much rarer infantile NPHP from the more typical (non-infantile) forms of NPHP, to allow a targeted approach to diagnosis and molecular testing (Figure 2).
Many disorders have been described in which NPHP is a clinical feature. Such multisystem features and pleiotropy are typical of ‘ciliopathies' such as NPHP. Extrarenal manifestations are seen in 10–20% of cases of NPHP.12
Here retinal dysplasia and degeneration (also known as tapetoretinal degeneration or retinitis pigmentosa) may lead to early and severe visual loss (within 2 years of age), resembling Leber's congenital amaurosis (LCA). Later onset forms present initially with night blindness, which progresses to visual loss by the age of 10 years. Diagnosis is made by performing an electroretinogram, which may show abnormalities before the physical signs of retinitis pigmentosa and visual loss. Molecular mechanisms of blindness are secondary to photoreceptor cell defects (reviewed in reference13).
Joubert syndrome and related disorders (JSRD) are characterized by cerebellar vermis hypoplasia and brainstem abnormalities.5 Brain imaging (MRI) reveals a characteristic appearance of the brain stem known as the ‘molar tooth sign'. Typically an affected child will have an irregular breathing pattern in the newborn period and often abnormal eye movements. During infancy hypotonia develops, with ataxia developing late in childhood. Other conditions associated with JSRD include CNS anomalies, ocular coloboma, retinal dystrophy, skeletal defects such as polydactyly, hepatic fibrosis and cystic dysplastic kidneys or NPHP.
This is characterized by abnormal eye movements, which include nystagmus and difficulty with saccades (smooth visual pursuits). The transient inability to perform horizontal gaze eye movements in the first years of life is referred to as oculomotor apraxia (OMA) type Cogan and is associated with NPHP gene mutations.14 Indeed, OMA may be a mild form of JSRD, as cerebellar vermis aplasia has been described in this condition.15
A variety of associated skeletal defects have been reported, the most frequent are cone-shaped epiphyses.16, 17 Scoliosis due to poor muscle tone (as part of a JSRD syndrome) and polydactyly (postaxial, most commonly) may also occur.
Other syndromes that include NPHP have been described. These include Ellis van Creveld syndrome,20 RHYNS (retinitis pigmentosa, hypopituitarism and skeletal dysplasia),21 Alstrom syndrome, COACH syndrome, Jeune syndrome and Arima syndrome (Table 1).
The association of occipital encephalocoele, polydactyly and ductal proliferation in the portal area of the liver and cystic kidney dysplasia is known as Meckel–Gruber syndrome (MKS). Recently, mutations in some of the genes implicated in NPHP/JSRD have been found in patients with MKS.18, 22, 23, 24 This broadens the phenotypic spectrum of diseases associated with NPHP gene defects and implies a common pathogenesis.
Diagnosis relies on a clinical suspicion of the disorder. NPHP should initially be investigated non-invasively.
Key features would include:
Following appropriate genetic counselling, homozygous or heterozygous NPHP1 deletion (found in around 25% of cases) can be screened easily by PCR. Other NPHP genes may be tested by direct sequencing (see http://www.orpha.net for a list of laboratories). A renal biopsy should not be necessary if a molecular genetic diagnosis can be made. If a molecular diagnosis is not available, a renal biopsy may be required to confirm or exclude NPHP (Figure 2).
Preparation for ESRF (renal replacement therapy) and consideration for renal transplantation should be undertaken during subsequent reviews of the patient, once a diagnosis has been made. NPHP does not recur in transplanted kidneys. Living-related kidney donation from unaffected family members, including heterozygous carriers (eg parents), is possible following clinical evaluation. Referral to the Joubert Syndrome Foundation (http://www.joubertsyndrome.org/) and other support organizations for families of children with disabilities (eg http://www.cafamily.org.uk/services.html or http://www.orpha.net) may be appropriate.
NPHP should not be confused with autosomal dominant polycystic kidney disease (ADPKD) which is characterized by bilateral, multiple renal cysts resulting in kidney enlargement over time, with extrarenal manifestations which include simple liver cysts, which arise from the biliary epithelium.
NPHP should be distinguished from medullary cystic kidney disease (MCKD), which shares pathological appearances at the macroscopic and microscopic level. However, unlike NPHP, MCKD is inherited in an autosomal dominant pattern, and the age of ESRF is usually later. Two different variants of MCKD are known, MCKD1 (gene remains unidentified) and MCKD2 (secondary to UMOD mutations), with a median onset of ESRD at 62 and 32 years,25 respectively. In contrast to NPHP, the only extra-renal manifestation of MCKD is the occurrence of hyperuricaemia and gout.25
Given the antenatal/early childhood onset of renal disease in infantile NPHP, care must be taken to exclude autosomal recessive polycystic kidney disease (ARPKD; Figure 2). Like NPHP, ARPKD may present at a wide age distribution, from antenatally to adulthood. Antenatal ultrasound scanning may reveal markedly enlarged kidneys with increased echogencity. Kidney microcysts and fusiform dilation of collecting ducts are typical of ARPKD. Liver involvement is always present in ARPKD and may be the predominant clinical feature, with dilated intrahepatic bile ducts, liver fibrosis and portal hypertension. The gene defect is in the PKHD1 gene, encoding its protein product fibrocystin (or polyductin).26
Finally, Bardet–Biedl syndrome (BBS) must be considered in the differential diagnosis of NPHP (Figure 2). BBS is another ciliopathy affecting multiple organ systems.27 Clinical features may include obesity, learning difficulties, genitourinary tract malformations and limb deformities.28 Renal lesions may include renal cysts, dysplasia, concentrating defects and progressive renal failure.28 Histologically, cystic dilatation of the renal collecting ducts have been described,29 reminiscent of infantile NPHP.
There are a growing number of genes implicated in NPHP. These will be briefly reviewed in terms of their phenotype, frequency and most common disease associations. NPHP is largely inherited as an autosomal recessive disease with homozygous single gene mutations/deletions or compound heterozygous mutations occurring in a single NPHP gene. This usually allows a molecular diagnosis and accurate genetic counselling to be performed. However oligogenicity, where allelic variants at multiple loci contribute to disease, has been documented for NPHP.30 Likewise, additional NPHP gene mutations may modulate the phenotype in an epistatic way.31 Thus a wide spectrum of clinical variants with any mutant gene(s) is possible (Table 3). The encoded NPHP proteins, called nephrocystins, typically posses multiple domains (Figure 3).
NPHP1 was the first NPHP gene identified, using positional cloning strategies in consanguineous families.32, 33 Homozygous deletions of ~250kb DNA in the region 2q13 are the most frequent genetic abnormality found.34 Other mutations include compound heterozygosity for the NPHP1 gene deletion combined with a single point mutation in the NPHP1 gene. NPHP1 mutations account for about 25% of cases of NPHP. NPHP1 mutations may be associated with congenital OMA type Cogan14 and Senior–Loken syndrome35 and also give rise to JSRD phenotypes.31, 36
NPHP1 encodes a protein product named nephrocystin-1. Nephrocystin-1 has been localized to the primary renal cilium19 and to epithelia cell adherens junctions.37, 38 More recently, the primary cilial localization has been refined to the transition zone (at the ciliary base) in renal and respiratory epithelia and to the connecting cilia in photoreceptor cells.39 Targeting of nephrocystin-1 to the transition zone of the cilia is dependent on casein kinase 2 phosphorylation and an interaction with PACS-1.40 Nephrocystin-1 also interacts with other nephrocystins (Nephrocystin-2, -3, -4 and Jouberin16, 41, 42, 43, 44) and there is evidence that this complex of proteins may function in multiple intracellular locations including the cilium, cell–cell adherens junctions and at focal adhesions.19, 37, 38, 44, 45 Within the human kidney nephrocystin-1 is expressed in renal collecting ducts.44
Mutations in INVS/NPHP2 give rise to infantile NPHP.19 These mutations are rare and account for <1% of all cases of NPHP worldwide. The gene encodes the protein named inversin, which has a dynamic distribution during cell cycle46 and is expressed in renal cilia.19, 46, 47 INVS mutations may cause situs inversus in affected patients, and knockout animals mimic the human disease, with large cystic kidneys at an early age, situs inversus and hepatobiliary malformations.48 Retinitis pigmentosa is an uncommon but reported association with INVS mutations.49 Inversin seems to play a crucial role in Wnt signalling, acting as a switch between canonical and non-canonical Wnt signalling pathways50, 51 and is required for convergent extension movements.50 This suggests that inversin plays a role in the developing nephron and in maintenance of the tubular architecture. This coordinated ability of epithelial cells to divide and reorganize themselves to form and maintain tubular structures relies on planar cell polarity (PCP) signalling. PCP signalling is mediated via proteins associated with the primary cilia/basal body complex, such as inversin50 and its disruption may underlie the pathophysiology of cyst development.51
Mutations in NPHP3 can produce diverse phenotypes. Mutations were originally identified in a large Venezuelan kindred who exhibited NPHP.16 Mutations in NPHP3 were associated with hepatic fibrosis and retinal degeneration in some affected individuals.16 Recently the phenotype of NPHP3 mutations has been expanded to include Meckel–Gruber like syndrome.18
NPHP3 encodes nephrocystin-3, which interacts with nephrocystin-116 and inversin,18 and can inhibit canonical Wnt signalling. A mouse model of NPHP type 3, named pcy, displays cystic kidney disease which responds to treatment with the aquaretic agents/vasopressin-2-receptor antagonists.52
NPHP4 encodes nephrocystin-4 (alias nephroretinin), a highly conserved protein which interacts with nephrocystin-1.42 Nephrocytsin-4 complexes with α-tubulin and localizes to the primary cilium and basal bodies.41 NPHP4 mutations may cause isolated NPHP, NPHP with RP and NPHP with OMA.53 Recently, nephrocystin-4 has been reported to interact with RPGRIP1L.24, 54
The NPHP5/IQCB1 gene encodes nephrocystin-5. This protein contains two IQ calmodulin binding sites, which surround a coiled-coil domain. Similar to inversin, nephrocystin-5 interacts directly with calmodulin via its IQ domains, with which it colocalizes to the primary cilium, and forms a complex with RPGR.55 The clinical phenotype of NPHP5 mutations is always associated with severe retinal degeneration (early onset Senior–Loken syndrome).
The NPHP6 (alias CEP290) gene encodes the nephrocystin-6 protein. Mutations in NPHP6 account for a growing spectrum of clinical phenotypes which include isolated NPHP, Senior–Loken syndrome, JSRD,56, 57 MKS22, 23 and BBS.58 Mutations in NPHP6 have also been described in 21% patients with isolated LCA, making this the most common gene defect for isolated LCA.59 A mouse model, named rd16 has an in-frame deletion in Nphp6/Cep290 and mimics this phenotype, with early onset retinal degeneration, but no kidney or brain disease. Nephrocystin-6 directly interacts with and activates the cAMP related transcription factor, CREB2 (alias ATF4).56 Interestingly, single heterozygous mutations in NPHP6 have been described in individuals with NPHP who have NPHP1 homozygous deletions.31 Similarly, a heterozygous nonsense mutation in NPHP6 was described together with a heterozygous NPHP4 missense mutation in an individual affected with Senior–Loken syndrome.57 This tendency towards digenic and oligogenicity has recently been reported for other NPHP genes.30, 60
The NPHP7/GLIS2 gene encodes the Kruppel-like zinc-finger transcription factor GLIS2 that localizes to both the primary cilia and the nucleus.61 Mutations were reported in a consanguineous Oji-Cree Canadian family with affected members having isolated NPHP and early onset renal failure (by 8 years of age) but remains a rare genetic cause of NPHP.61 A mouse model of targeted Glis2 disruption within the kidney reveals increased rates of apoptosis, with tubular atrophy and fibrosis.
The RPGRIP1L gene encodes a protein named retinitis pigmentosa GTPase regulator interacting protein 1-like protein (RPGRIP1L). Mutations were initially reported in fetuses affected with MKS and patients with JSRD.24, 62 Additional features in some patients included scoliosis, polydactyly, pituitary agenesis and partial growth hormone deficiency, reminiscent of RHYNS syndrome.62 Regarding RPGRIP1L mutations, some phenotype–genotype correlations can be drawn as homozygous truncating mutations seem to cause MKS24, 62 whereas a heterozygous truncating mutation or a homozygous missense mutation causes JSRD. RPGRIP1L is a centrosomal protein, which interacts with nephrocystin-4. JSRD causing mutations in RPGRIP1L confer loss off interaction with nephrocystin-4.24
A mouse model Ftm−/− (Fantom or fused-toe mouse) represents inactivation of the mouse ortholog Rpgrip1l (Ftm) and recapitulates the cerebral, renal and hepatic defects of JSRD and MKS.
The NEK8 gene encodes the NEK8 protein (never in mitosis A-related kinase 8). Mutations have been described in two families with NPHP and one consanguineous family with infantile NPHP. In one NPHP family with a homozygous NPHP5 mutation, which accounts for the disease phenotype, a single heterozygous NEK8 mutation was found.60 These findings demonstrate firstly, the rarity of NEK8 mutations and secondly, that NEK8 mutations may contribute to oligogenicity in patients with NPHP. The jck mouse model of cystic kidney disease contains a missense mutation (G448V) in Nek8. Nek8 and polycystin-2 form a protein complex together, which adds weight to the argument that there are common mechanisms underlying NPHP and ADPKD.63, 64
The AHI1 (Abelson helper integration site 1) gene encodes the AHI1 protein, which is also known as Jouberin. Mutations in AHI1 were initially described in individuals with a JSRD phenotype, with no renal disease.65, 66 Subsequently, AHI1 mutations were found in individuals with NPHP67 and with retinal degeneration.68 Jouberin is localized to adherens junctions, basal bodies and primary cilia.69 Jouberin interacts with nephrocystin-1, and has been localized to the renal collecting duct.69
NPHP1 gene mutations account for around 25% of all cases of NPHP. The remaining nine genes are each found in 0.05–3% of cases, and collectively probably only account for another 25% of cases of NPHP, meaning that many cases remain ‘unsolved'. For JSRD, at least two additional loci have been reported. These are JBTS1 on chromosome 9q3470 and JBTS2 (CORS2) on chromosome 11 (a large pericentromeric region).71 Patients linked to the JBTS2 locus often have renal disease as part of their disease spectrum. Very recently, mutations in ARL13B, which encodes a cilial protein, were found in patients with classical JS, with no renal phenotype.72
The identification of genetic causes of NPHP has highlighted the paradigm, that all protein products of cystic kidney diseases are expressed in the primary renal cilium/basal body complex.73 The primary cilium is present on nearly every cell in the human body and is a cell surface projection which acts as an ‘antenna'. This organelle extends from the basal body and consists of an axoneme comprising nine microtubular doublets. Assembly of the axoneme occurs via a process called IFT where proteins are moved up and down the cilium.73 Nephrocystins are located within this cilial subcellular domain, where they form complexes with themselves and other related proteins, probably to facilitate signalling cascades. Primary cilia are thought to sense tubular luminal flow (of urine) and regulate calcium entry (mediated by polycystin-2 channels).74 Nephrocystins are expressed in the connecting cilium of the photoreceptor cell of the retina and defects here correlate with retinal defects and degeneration, often associated with NPHP gene mutations. Related syndromes such as Jeune syndrome and Ellis van Creveld syndrome (EVC) have held true to the cilial paradigm. Jeune syndrome is secondary to mutations in the IFT protein IFT8075 and EVC (together with EVC2) mutations underlie EVC, and encodes a cilial/basal body protein.76
Given that the renal disease NPHP is often being managed in the context of extra-renal manifestations, ongoing surveillance of affected patients by appropriate specialists is important.
Patients with NPHP will invariably progress to end-stage renal failure. Management in a ‘low clearance' setting is appropriate to allow time for consideration of renal replacement therapies. USS scans may detect renal cystic changes as the disease progresses. Growth, endocrine and sexual maturation and neurological evaluations should be regularly performed. Retinal disease may become progressive. Annual eye examinations commencing at the time of diagnosis is recommended.5 Liver function tests should be performed regularly and liver ultrasound scan should be performed if suspicion of liver disease.
NPHP is a genetically heterogeneous disorder, however testing for the most common gene defect, a homozygous deletion of NPHP1 (Figure 2), is readily available (see http://www.ukgtn.nhs.uk/gtn/Home; http://www.orpha.net and http://www.genetests.org/). Direct sequencing of other NPHP genes may also be performed (see http://autozygosity.org/diagnostic; http://www.renalgenes.org/ and http://www.orpha.net). Technologies are however changing rapidly and given that the genomic regions covered by all known NPHP genes is less than 1mb (Table 4) a gene capture service followed by use of high throughput sequencing platforms may allow an efficient way of screening patients with NPHP in the near future. Indeed, with the recent descriptions of oligogenicity30 and epistasis31 in NPHP, testing of all NPHP associated genes may be important to understand this complex disorder.
Genetic testing should not be performed before appropriate consent and genetic counselling. NPHP is inherited in an autosomal recessive manner, however in some affected individuals more than one associated gene may contribute to disease.30 Such oligogenicity has also been reported in BBS.77 In general, NPHP is a moderately severe disorder with major impacts on renal function and other aspects of health and development. The variable severity of the disorder in different families and even between individuals within families makes predicting outcome difficult.
For families with a genetic diagnosis of NPHP, prenatal testing is possible. Prenatal imaging may reveal cystic kidney disease and other abnormalities (such as structural CNS lesions and polydactyly) in at-risk pregnancies. INVS/NPHP2 mutations typically produce a prenatal cystic phenotype, as may the genes which have been reported to give a MKS-like phenotype (Table 3).
At present there are no proven treatments for NPHP. Treatment must centre on the progressive renal failure, which leads to ESRF and the need for dialysis and transplantation. Potential treatments, targeted towards the collecting duct may be available for future use. These include vasopressin V2 receptor antagonists, which may alter cystogenesis and progression of disease. The pcy mouse model of NPHP type 3 responded to treatment with OPC31260.52 Evidence is also growing for use of rapamycin, an mTOR inhibitor, for reducing renal cystogenesis.78, 79
During the past decade significant insight has been made in the molecular genetics of NPHP. This disease has moved from being a pathological description to an inherited ciliopathy, whereby the most common form may be readily detected by gene testing, without the need for a renal biopsy. Additional genes will no doubt be found, and high throughput technologies show promise for providing a screen of all currently known genes implicated in ciliopathies. The major challenge remains to understand the biological function of nephrocystin proteins, the molecular mechanisms that lead to renal failure and the potential treatments, which may prevent or reverse these changes. In practical terms, NPHP must be considered among the differential diagnosis of any cause of renal failure of unknown origin. The recognition that NPHP is part of a ciliopathy, with a wide clinical spectrum of disease will allow earlier diagnosis to be made, allowing for time for genetic counselling, appropriate genetic testing and improved treatment planning for ESRF.
RJS is funded by a Mason Medical Research Fellowship. JAS is funded by GlaxoSmithKline (Clinician Scientist Award).