Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, leads to chronic renal failure in children. The genes mutated in NPHP1 and NPHP4 have been identified, and a gene locus associated with infantile nephronophthisis (NPHP2) was mapped. The kidney phenotype of NPHP2 combines clinical features of NPHP and polycystic kidney disease (PKD). Here, we identify inversin (INVS) as the gene mutated in NPHP2 with and without situs inversus. We show molecular interaction of inversin with nephrocystin, the product of the gene mutated in NPHP1 and interaction of nephrocystin with β-tubulin, a main component of primary cilia. We show that nephrocystin, inversin and β-tubulin colocalize to primary cilia of renal tubular cells. Furthermore, we produce a PKD-like renal cystic phenotype and randomization of heart looping by knockdown of invs expression in zebrafish. The interaction and colocalization in cilia of inversin, nephrocystin and β-tubulin connect pathogenetic aspects of NPHP to PKD, to primary cilia function and to left-right axis determination.
Nephrotic syndrome (NS) is divided into steroid-sensitive (SSNS) and -resistant (SRNS) variants. SRNS causes end-stage kidney disease, which cannot be cured. While the disease mechanisms of NS are not well understood, genetic mapping studies suggest a multitude of unknown single-gene causes. We combined homozygosity mapping with whole-exome resequencing and identified an ARHGDIA mutation that causes SRNS. We demonstrated that ARHGDIA is in a complex with RHO GTPases and is prominently expressed in podocytes of rat glomeruli. ARHGDIA mutations (R120X and G173V) from individuals with SRNS abrogated interaction with RHO GTPases and increased active GTP-bound RAC1 and CDC42, but not RHOA, indicating that RAC1 and CDC42 are more relevant to the pathogenesis of this SRNS variant than RHOA. Moreover, the mutations enhanced migration of cultured human podocytes; however, enhanced migration was reversed by treatment with RAC1 inhibitors. The nephrotic phenotype was recapitulated in arhgdia-deficient zebrafish. RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS.
Focal segmental glomerulosclerosis (FSGS) is a kidney disease that presents with nephrotic syndrome and is often resistant to glucocorticosteroids and progresses to end-stage kidney disease in 50–70% of patients. Genetic studies in familial FSGS indicate that it is a disease of the podocytes, major components of the glomerular filtration barrier. However the molecular cause of over half of primary FSGS is unknown, and effective treatments have been elusive.
We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive linkage area in a family with autosomal recessive FSGS and sequenced a newly discovered gene in 52 unrelated FSGS patients. Immunohistochemistry was performed in human kidney biopsies and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified.
Two mutations (A159P and Y695X) in MYO1E, encoding the non-muscle class I myosin, myosin 1E (Myo1E), which segregated with FSGS in two independent pedigrees were identified. Patients were homozygous for the mutations and were resistant to glucocorticosteroids. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney biopsies in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and the tail domains of Myo1E.
MYO1E mutations lead to childhood onset steroid-resistant FSGS. These data support a role of Myo1E in podocyte function and the consequent integrity of the glomerular permselectivity barrier.
Neighboring genes are often coordinately expressed within cis-regulatory modules, but evidence that nonparalogous genes share functions in mammals is lacking. Here, we report that mutation of either TMEM138 or TMEM216 causes a phenotypically indistinguishable human ciliopathy, Joubert syndrome. Despite a lack of sequence homology, the genes are aligned in a head-to-tail configuration and joined by chromosomal rearrangement at the amphibian-to-reptile evolutionary transition. Expression of the two genes is mediated by a conserved regulatory element in the noncoding intergenic region. Coordinated expression is important for their interdependent cellular role in vesicular transport to primary cilia. Hence, during vertebrate evolution of genes involved in ciliogenesis, nonparalogous genes were arranged to a functional gene cluster with shared regulatory elements.
Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.
Homozygous or compound heterozygous Renin (REN) mutations cause renal tubular dysgenesis (RTD), which is characterized by death in utero due to renal failure and pulmonary hypoplasia. The phenotype resembles the fetopathy caused by angiotensin-converting enzyme inhibitor or angiotensin receptor blocker intake during pregnancy. Recently, heterozygous REN mutations were shown to result in early-onset hyperuricemia, anemia and chronic renal failure. So far, only three different heterozygous REN mutations were reported.
We performed mutation analysis of the REN gene in 39 kindreds with hyperuricemia and chronic kidney disease (CKD) previously tested negative for mutations in the UMOD and HNF1β genes. We identified one kindred with a novel c.28T>C (p.W10R) REN mutation in the signal sequence, concluding that REN mutations are rare events in CKD patients. Affected individuals over four generations were identified carrying the novel REN mutation and were characterized by significant anemia, hyperuricemia and CKD. Anemia was severe and disproportional to the degree of renal impairment. Moreover all heterozygous REN mutations are localized in the signal sequence. Therefore, screening of the REN gene for CKD patients with hyperuricemia and anemia may be focusing on exon 1 sequencing, which encodes the signal peptide.
Integrin α3 is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α3 gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis.
We describe a child of Middle Eastern descent by first-cousin mating with idiopathic neurogenic bladder and high grade vesicoureteral reflux at 1 year of age, whose characteristic facial grimace led to the diagnosis of Ochoa (Urofacial) syndrome at age 5 years. We used homozygosity mapping, exome capture and paired end sequencing to identify the disease causing mutation in the proband. We reviewed the literature with respect to the urologic manifestations of Ochoa syndrome. A large region of marker homozygosity was observed at 10q24, consistent with known autosomal recessive inheritance, family consanguinity and previous genetic mapping in other families with Ochoa syndrome. A homozygous mutation was identified in the proband in HPSE2: c.1374_1378delTGTGC, a deletion of 5 nucleotides in exon 10 that is predicted to lead to a frameshift followed by replacement of 132 C-terminal amino acids with 153 novel amino acids (p.Ala458Alafsdel132ins153). This mutation is novel relative to very recently published mutations in HPSE2 in other families. Early intervention and recognition of Ochoa syndrome with control of risk factors and close surveillance will decrease complications and renal failure.
Ochoa syndrome; Urofacial syndrome; HPSE2 mutation; Neurogenic bladder
The ciliopathies are a category of diseases caused by disruption of the physiological functions of cilia. Ciliary dysfunction results in a broad range of phenotypes, including renal, hepatic, and pancreatic cyst formation; situs abnormalities; retinal degeneration; anosmia; cerebellar or other brain anomalies; postaxial polydactyly; bronchiectasis; and infertility. The specific clinical features are dictated by the subtype, structure, distribution, and function of the affected cilia. This review highlights the clinical variability caused by dysfunction of motile and nonmotile primary cilia and emphasizes the genetic heterogeneity and phenotypic overlap that are characteristics of these disorders. There is a need for additional research to understand the shared and unique functions of motile and nonmotile cilia and the pathophysiology resulting from mutations in cilia, basal bodies, or centrosomes. Increased understanding of ciliary biology will improve the diagnosis and management of primary ciliary dyskinesia, syndromic ciliopathies, and cilia-related cystic diseases.
primary cilia; ciliopathy; heterotaxy; nephronophthisis; cyst
Tubulin glutamylation is a post-translational modification (PTM) occurring predominantly on ciliary axonemal tubulin and has been suggested to be important for ciliary function 1,2. However, its relationship to disorders of the primary cilium, termed ‘ciliopathies’, has not been explored. Here, in Joubert syndrome (JBTS) 3, we identify the JBTS15 locus and the responsible gene as CEP41, encoding a centrosomal protein of 41 KDa 4. We show that CEP41 is localized to the basal body/primary cilium, and regulates the ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme 5. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mouse, and induces cilia axonemal glutamylation defects. Our data identify loss of CEP41 as a cause of JBTS ciliopathy and highlight involvement of tubulin PTM in pathogenesis of the ciliopathy spectrum.
Centrioles are microtubule-derived structures that are essential to form centrosomes, cilia and flagella. The centrosome is the major microtubule organiser in animal cells, participating in a variety of processes from cell polarization to cell division, while cilia and flagella contribute to several mechanisms in eukaryotic cells from motility to sensing. Although it was suggested more than a century ago that these microtubule-derived structures are involved in human disease, the molecular bases of this association have only recently been discovered. Surprisingly, there is very little overlap between the genes affected in the different diseases, suggesting there are tissue-specific requirements for these microtubule-derived structures. Knowledge of these requirements and disease mechanisms has opened new avenues for therapeutical strategies. Here, we give an overview of recent developments in this field focusing on cancer, diseases of brain development and ciliopathies.
Nephronophthisis (NPHP), Joubert (JBTS) and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins, and discovered three connected modules: “NPHP1-4-8” functioning at the apical surface; “NPHP5-6” at centrosomes; and “MKS” linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
Nephronophthisis (NPHP) is an autosomal recessive kidney disease characterized by tubular basement membrane disruption, interstitial infiltration, and tubular cysts. NPHP leads to end-stage renal failure in the first two decades of life and is the most frequent genetic cause of chronic renal failure in children and young adults. Mutations in eleven genes (NPHP1-11) have been identified. Extrarenal manifestations are known, such as retinitis pigmentosa (Senior-Løken syndrome, SLS), brainstem and cerebellar anomalies (Joubert syndrome), liver fibrosis, and ocular motor apraxia type Cogan.
We report on a Turkish family with clinical signs of nephronophthisis. The phenotype occurred in two generations and therefore seemed to be inherited in an autosomal dominant pattern. Nevertheless, a deletion analysis of the NPHP1 gene on chromosome 2 was performed and showed a homozygous deletion. Analysis of the family pedigree indicated no obvious consanguinity in the last three generations. However, haplotype analysis demonstrated homozygosity on chromosome 2 indicating a common ancestor to the parents of all affected individuals. NPHP1 deletion analysis should always be considered in patients with apparently dominant nephronophthisis, especially from likely consanguineous families.
Nephronophthisis; NPHP1; cystic kidney disease
Homozygosity mapping has played an important role in detecting recessive mutations using families of consanguineous marriages. However, detection of homozygous regions identity by descent (HBD) when family data is not available, or when relationship is hidden, is still a challenge. Making use of population data from high-density SNP genotyping may allow detection of regions HBD from recent common founders in singleton patients without genealogy information. We report a novel algorithm that detects such regions by estimating the population haplotype frequencies (HF) for an entire homozygous region. We also developed a simulation method to evaluate the probability of HBD for a homozygous region by examining the best regions in unaffected controls from the host population. The method can be applied to diseases of Mendelian inheritance and can be further extended to complex diseases to detect rare founder mutations using multiplex families or sporadic cases. Testing of the method on both real cases (singleton affected) and simulated data demonstrated its superb sensitivity and great resistance to genetic heterogeneity.
homozygosity mapping; recessive mutation; founder mutation; runs of homozygosity; hidden relationship
Mutations in genes encoding ciliary components cause ciliopathies, but how many of these mutations disrupt ciliary function is unclear. We investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel (MKS) and Joubert (JBTS) syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2, and Cc2d2a. Components of the Tectonic ciliopathy complex colocalize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of complex components Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes JBTS. Thus, a transition zone complex of MKS and JBTS proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.
Ciliopathies represent a growing group of human genetic diseases whose etiology lies in defects in ciliogenesis or ciliary function. Given the established entity of renal–retinal ciliopathies, we have been examining the role of cilia-localized proteins mutated in retinitis pigmentosa (RP) in regulating renal ciliogenesis or cilia-dependent signaling cascades. Specifically, this study examines the role of the RP2 gene product with an emphasis on renal and vertebrate development. We demonstrate that in renal epithelia, RP2 localizes to the primary cilium through dual acylation of the amino-terminus. We also show that RP2 forms a calcium-sensitive complex with the autosomal dominant polycystic kidney disease protein polycystin 2. Ablation of RP2 by shRNA promotes swelling of the cilia tip that may be a result of aberrant trafficking of polycystin 2 and other ciliary proteins. Morpholino-mediated repression of RP2 expression in zebrafish results in multiple developmental defects that have been previously associated with ciliary dysfunction, such as hydrocephalus, kidney cysts and situs inversus. Finally, we demonstrate that, in addition to our observed physical interaction between RP2 and polycystin 2, dual morpholino-mediated knockdown of polycystin 2 and RP2 results in enhanced situs inversus, indicating that these two genes also regulate a common developmental process. This work suggests that RP2 may be an important regulator of ciliary function through its association with polycystin 2 and provides evidence of a further link between retinal and renal cilia function.
An emerging group of human genetic diseases termed ‘ciliopathies’ are caused by dysfunction of two functionally and physically associated organelles, the centrosome and cilium. These organelles are central to perception of the physical environment through detection of a diverse variety of extracellular signals such as growth factors, chemicals, light and fluid flow. Many of the described ciliopathies display multi-organ involvement, with renal and retina being the most commonly affected. Nephronophthisis is a recessive disorder of the kidney that is the leading cause of end-stage renal failure in children. Through positional cloning, many of the causative mutations have been mapped to genes involved in centrosome and cilia function. In this review, we discuss the identified causative mutations that give rise to nephronophthisis and how these are related to the disease etiology in both the kidney and other organs.
Nephronophthisis; NPHP genes; Joubert syndrome; Meckel-Gruber syndrome; Senior-Løken syndrome
Background. Recessive mutations in the NPHS1 gene encoding nephrin account for ∼40% of infants with congenital nephrotic syndrome (CNS). CNS is defined as steroid-resistant nephrotic syndrome (SRNS) within the first 90 days of life. Currently, more than 119 different mutations of NPHS1 have been published affecting most exons.
Methods. We here performed mutational analysis of NPHS1 in a worldwide cohort of 67 children from 62 different families with CNS.
Results. We found bi-allelic mutations in 36 of the 62 families (58%) confirming in a worldwide cohort that about one-half of CNS is caused by NPHS1 mutations. In 26 families, mutations were homozygous, and in 10, they were compound heterozygous. In an additional nine patients from eight families, only one heterozygous mutation was detected. We detected 37 different mutations. Nineteen of the 37 were novel mutations (∼51.4%), including 11 missense mutations, 4 splice-site mutations, 3 nonsense mutations and 1 small deletion. In an additional patient with later manifestation, we discovered two further novel mutations, including the first one affecting a glycosylation site of nephrin.
Conclusions. Our data hereby expand the spectrum of known mutations by 17.6%. Surprisingly, out of the two siblings with the homozygous novel mutation L587R in NPHS1, only one developed nephrotic syndrome before the age of 90 days, while the other one did not manifest until the age of 2 years. Both siblings also unexpectedly experienced an episode of partial remission upon steroid treatment.
mutation analysis; nephrotic syndrome; NPHS1
Uromodulin (UMOD) mutations were described in patients with medullary cystic kidney disease (MCKD2), familial juvenile hyperuricemic nephropathy (FJHN), and glomerulocystic kidney disease (GCKD). UMOD transcription is activated by the transcription factor HNF1B. Mutations in HNF1B cause a phenotype similar to FJHN/GCKD but also congenital anomalies of the kidney and the urinary tract (CAKUT). Moreover, we recently detected UMOD mutations in 2 patients with CAKUT. As HNF1B and UMOD act in the same pathway and cause similar phenotypes we here examined, whether UMOD mutations would be found in patients with CAKUT.
Mutation analysis of UMOD was performed in 96 individuals with CAKUT by direct sequencing of exons 4 and 5 and by heteroduplex analysis following CEL I digestion assay of the exons 3 and 6–12.
The mean age of patients was 11.4 years and in 36.4% of patients the family history was positive for CAKUT. In the CEL I assay 12 aberrant bands were detected in 103 of 960 PCR products and were sequenced. Two previously known and eight new SNPs were detected. As no UMOD mutations were identified in these 96 patients with CAKUT, UMOD mutations do not seem to be a significant cause of CAKUT in this cohort.
Uromodulin; Tamm-Horsfall protein; urinary tract malformation; CAKUT; mutation analysis
Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the ‘nephronophthisis–MCKD complex’, a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.
Background. Congenital anomalies of the kidney and urinary tract (CAKUT) account for the majority of end-stage renal disease in children (50%). Previous studies have mapped autosomal dominant loci for CAKUT. We here report a genome-wide search for linkage in a large pedigree of Somalian descent containing eight affected individuals with a non-syndromic form of CAKUT.
Methods. Clinical data and blood samples were obtained from a Somalian family with eight individuals with CAKUT including high-grade vesicoureteral reflux and unilateral renal agenesis. Total genome search for linkage was performed using a 50K SNP Affymetric DNA microarray. As neither parent is affected, the results of the SNP array were analysed under recessive models of inheritance, with and without the assumption of consanguinity.
Results. Using the non-consanguineous recessive model, a new gene locus (CAKUT1) for CAKUT was mapped to chromosome 8q24 with a significant maximum parametric Logarithm of the ODDs (LOD) score (LODmax) of 4.2. Recombinations were observed in two patients defining a critical genetic interval of 2.5 Mb physical distance flanked by markers SNP_A-1740062 and SNP_A-1653225.
Conclusion. We have thus identified a new non-syndromic recessive gene locus for CAKUT (CAKUT1) on chromosome 8q24. The identification of the disease-causing gene will provide further insights into the pathogenesis of urinary tract malformations and mechanisms of renal development.
congenital anomalies of the kidney and urinary tract (CAKUT); kidney development; total genome search for linkage; ureteropelvic junction obstruction; vesicoureteral reflux
Mutations of LAMB2 typically cause autosomal recessive Pierson syndrome, a disorder characterized by congenital nephrotic syndrome, ocular and neurologic abnormalities, but may occasionally be associated with milder or oligosymptomatic disease variants. LAMB2 encodes the basement membrane protein laminin β2 which is incorporated in specific heterotrimeric laminin isoforms and has an expression pattern corresponding to the pattern of organ manifestations in Pierson syndrome. Herein we review all previously reported and several novel LAMB2 mutations in relation to the associated phenotype in patients from 39 unrelated families. The majority of disease-causing LAMB2 mutations are truncating, consistent with the hypothesis that loss of laminin β2 function is the molecular basis of Pierson syndrome. While truncating mutations are distributed across the entire gene, missense mutations are clearly clustered in the N-terminal LN domain, which is important for intermolecular interactions. There is an association of missense mutations and small in frame deletions with a higher mean age at onset of renal disease and with absence of neurologic abnormalities, thus suggesting that at least some of these may represent hypomorphic alleles. Nevertheless, genotype alone does not appear to explain the full range of clinical variability, and therefore hitherto unidentified modifiers are likely to exist.
LAMB2; Pierson syndrome; nephrotic syndrome; autosomal recessive; podocyte; laminin; ocular malformation
Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive disorder characterized by the five cardinal features retinitis pigmentosa, postaxial polydactyly, mental retardation, obesity and hypogenitalism. In addition, renal cysts and other anomalies of the kidney and urinary tract can be present. To date, mutations in 12 BBS genes as well as in MKS1 and CEP290 have been identified as causing BBS. The vast genetic heterogeneity of BBS renders molecular genetic diagnosis difficult in terms of both the time and cost required to screen all 204 coding exons. Here, we report the use of genome-wide homozygosity mapping as a tool to identify homozygous segments at known BBS loci in BBS individuals from inbred and outbred background. In a worldwide cohort of 45 families, we identified, via direct exon sequencing, causative homozygous mutations in 20 families. Eleven of these mutations were novel, thereby increasing the number of known BBS mutations by 5% (11/218). Thus, in the presence of extreme genetic locus heterogeneity, homozygosity mapping provides a valuable approach to the molecular genetic diagnosis of BBS and will facilitate the discovery of novel pathogenic mutations.
Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.