Rare single-gene disorders cause chronic disease. However, half of the
6,000 recessive single gene causes of disease are still unknown. Because
recessive disease genes can illuminate, at least in part, disease
pathomechanism, their identification offers direct opportunities for improved
clinical management and potentially treatment. Rare diseases comprise the
majority of chronic kidney disease (CKD) in children but are notoriously
difficult to diagnose. Whole exome resequencing facilitates identification of
recessive disease genes. However, its utility is impeded by the large number of
genetic variants detected. We here overcome this limitation by combining
homozygosity mapping with whole exome resequencing in 10 sib pairs with a
nephronophthisis-related ciliopathy, which represents the most frequent genetic
cause of CKD in the first three decades of life. In 7 of 10 sib-ships with a
histologic or ultrasonographic diagnosis of nephronophthisis-related ciliopathy
we detect the causative gene. In six sib-ships we identify mutations of known
nephronophthisis-related ciliopathy genes, while in two additional sib-ships we
found mutations in the known CKD-causing genes SLC4A1 and
AGXT as phenocopies of nephronophthisis-related ciliopathy.
Thus whole exome resequencing establishes an efficient, non-invasive approach
towards early detection and causation-based diagnosis of rare kidney diseases.
This approach can be extended to other rare recessive disorders, thereby
providing accurate diagnosis and facilitating the study of disease
Nephronophthisis (NPHP) is a recessive disorder of the kidney that is the leading genetic cause of end-stage renal failure in children. Egypt is a country with a high rate of consanguineous marriages; yet, only a few studies have investigated the clinical and molecular characteristics of NPHP and related ciliopathies in the Egyptian population. We studied 20 children, from 17 independent families, fulfilling the clinical and the ultrasonographic criteria of NPHP. Analysis for a homozygous deletion of the NPHP1 gene was performed by polymerase chain reaction on the genomic DNA of all patients. Patients were best categorized as 75% juvenile NPHP, 5% infantile NPHP, and 20% Joubert syndrome-related disorders (JSRD). The mean age at diagnosis was 87.5 + 45.4 months, which was significantly late as compared with the age at onset of symptoms, 43.8 ± 29.7 months (P <0.01). Homozygous NPHP1 deletions were detected in six patients from five of 17 (29.4%) studied families. Our study demonstrates the clinical phenotype of NPHP and related disorders in Egyptian children. Also, we report that homozygous NPHP1 deletions account for 29.4% of NPHP in the studied families in this cohort, thereby confir-ming the diagnosis of type-1 NPHP. Moreover, our findings confirm that NPHP1 deletions can indeed be responsible for JSRD.
Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, is the most frequent genetic cause for end-stage renal failure in the first 3 decades of life. Mutations in 13 genes (NPHP1-NPHP11, AHI1, and CC2D2A) cause NPHP with ubiquitous expression of the corresponding proteins consistent with the multiorgan involvement of NPHP-related diseases. The genotype-phenotype correlation in these ciliopathies can be explained by gene locus heterogeneity, allelism, and the impact of modifier genes. In some NPHP-related ciliopathies, the nature of the recessive mutations determines disease severity. In order to define the genotypephenotype correlation more clearly, we evaluated a worldwide cohort of 440 patients from 365 families with NPHP-related ciliopathies, in whom both disease-causing alleles were identified. The phenotypes were ranked in the order of severity from degenerative to degenerative/ dysplastic to dysplastic. A genotype of 2 null alleles caused a range of phenotypes with an increasing order of severity of NPHP1, NPHP3, NPHP4, NPHP5, NPHP2, NPHP10, NPHP6 to AHI1. Only NPHP6 showed allelic influences on the phenotypes; the presence of 2 null mutations caused dysplastic phenotypes, whereas at least one missense allele rescued it to a milder degenerative phenotype. We also found 9 novel mutations in the NPHP genes. Thus, our studies have important implications for genetic counseling and planning of renal replacement therapy.
cystic kidney; end-stage renal disease; genetic renal disease; human genetics; pediatric nephrology
Nephronophthisis-associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR).
In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each which were used as templates in order to PCR-amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on a Illumina Genome-Analyzer and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease-based heteroduplex screening and confirmed by Sanger sequencing.
For proof of principle we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance.
The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single-gene disorders. The lack of mutations in 75% of patients in our cohort indicates further extensive heterogeneity in NPHP-AC.
Next-generation sequencing; Ciliopathy; Nephronophthisis
Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. We have identified ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVERSIN (INVS, NPHP2) and NPHP3 to form a distinct NPHP module. ANKS6 localizes to the proximal cilium and knockdown experiments in zebrafish and Xenopus confirmed a role in renal development. Genetic screening identified six families with ANKS6 mutations and NPH, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN (FIH) hydroxylates ANKS6 and INVS, while knockdown of Hif1an in Xenopus resembled the loss of other NPHP proteins. HIF1AN altered the composition of the ANKS6/INVS/NPHP3 module. Network analyses, uncovering additional putative NPHP-associated genes, placed ANKS6 at the center of the NPHP module, explaining the overlapping disease manifestation caused by mutations of either ANKS6, NEK8, INVS or NPHP3.
Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS)
has furthered the understanding of the pathogenesis of this disease. Here, using a
combination of homozygosity mapping and whole human exome resequencing, we identified
mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15
individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3,
which has been shown to participate in coenzyme Q10 (CoQ10)
biosynthesis. Mutations in ADCK4 resulted in reduced
CoQ10 levels and reduced mitochondrial respiratory enzyme activity in
cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of
adck4 in zebrafish and Drosophila recapitulated
nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in
glomerular podocytes and partially localized to podocyte mitochondria and foot
processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4
interacted with members of the CoQ10 biosynthesis pathway, including COQ6,
which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in
decreased migration, which was reversed by CoQ10 addition. Interestingly,
a patient with SRNS with a homozygous ADCK4 frameshift mutation had
partial remission following CoQ10 treatment. These data indicate that
individuals with SRNS with mutations in ADCK4 or other genes that
participate in CoQ10 biosynthesis may be treatable with CoQ10.
Congenital abnormalities of the kidney and urinary tract (CAKUT) constitute the most frequent cause of chronic kidney disease in children, accounting for ~50% of all cases. Although many forms of CAKUT are likely caused by single-gene defects, only few causative genes have been identified. To identify new causative genes many candidate genes need to be analyzed due to the broad genetic locus heterogeneity of CAKUT. We therefore applied our newly developed approach of DNA pooling with consecutive massively parallel exon resequencing to overcome this problem. We pooled DNA of 20 individuals and amplified by PCR all 313 exons of 30 CAKUT candidate genes. PCR products were then subjected to massively parallel exon resequencing. Mutation carriers were identified using Sanger sequencing. We repeated the experiment to cover 40 patients in total (29 with unilateral renal agenesis and 11 with other CAKUT phenotypes). We detected 5 heterozygous missense mutations in 2 candidate genes that were not previously implicated in non-syndromic CAKUT in humans, 4 mutations in the FRAS1 gene and 1 in FREM2. All mutations were absent from 96 healthy control individuals and had a PolyPhen score of >1.4 (“possibly damaging”). Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice, whereas a phenotype in heterozygous carriers has not been described. We hereby identify heterozygous missense mutations in FRAS1 and FREM2 as a new cause of non-syndromic CAKUT in human.
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.
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as ‘ciliopathies’. However, disease mechanisms remain poorly understood. Here we identify by whole exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway, hitherto not implicated in ciliopathies. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164 and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents, and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
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.
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
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
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.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
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
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
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
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.
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that constitutes the most common genetic cause of renal failure in the first three decades of life. Using positional cloning, six genes (NPHP1‐6) have been identified as mutated in NPHP. In Joubert syndrome (JBTS), NPHP may be associated with cerebellar vermis aplasia/hypoplasia, retinal degeneration and mental retardation. In Senior–Løken syndrome (SLSN), NPHP is associated with retinal degeneration. Recently, mutations in NPHP6/CEP290 were identified as a new cause of JBTS.
Mutational analysis was performed on a worldwide cohort of 75 families with SLSN, 99 families with JBTS and 21 families with isolated nephronophthisis.
Six novel and six known truncating mutations, one known missense mutation and one novel 3 bp pair in‐frame deletion were identified in a total of seven families with JBTS, two families with SLSN and one family with isolated NPHP.
; Joubert syndrome; Senior–Løken syndrome; nephronophthisis; mutational analysis