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Psychological disturbance (PD) or cerebral dysfunction (CD) covers several clinical areas, and has defining features of mental retardation. Recently, we conducted a study to investigate heritable heterogeneity in Pakistani consanguineous couples with recessive autosomal intellectual abnormalities. A cohort of three consanguineous families with multiple birth defects, belonging two to district lower Dir and one to district Lodhra, were selected for molecular analysis. All the affected individuals in the cohort showed autosomal recessive non-syndromic mental disturbances. DNA was extracted and subjected to Single tagged sequence (STS) marker analyses to all known non-syndromic autosomal recessive mental retardation (NS-ARMR) genes, while autozygosity mapping was performed by advanced SNP techniques. Fragment analyses of the NS-ARMR disease genes CRBN, CC2D2A, PRSS12, GRIK2, TUSC3, and CC2D1A using polymorphic STS markers confirmed these to be contender genes for the alteration. Mapping of autozygosity in all the study subjects using genome study revealed nine novel linkage intervals, i.e. four intervals for MR4, two intervals for MR8 and three intervals for MR13. In spite of being a monogenic condition, autosomal recessive mental retardation shows genetic heterogeneity and several genes are involved in different families; hence, there is a chance for involvement of separate gene in each family.

Background Joubert syndrome and related disorders are a group of ciliopathies characterized by mid-hindbrain malformation, developmental delay, hypotonia, oculomotor apraxia, and breathing abnormalities. Molar tooth sign in brain imaging is the hallmark for diagnosis. Joubert syndrome is a clinically and genetically heterogeneous disorder involving mutations in 35 ciliopathy-related genes. We present a large cohort of 59 patients with Joubert syndrome from 55 families. Molecular analysis was performed in 35 families (trio). Methods Clinical exome analysis was performed to identify causal mutations, and genotype-phenotype correlations were evaluated. Results All of the cases were stratified into pure Joubert syndrome (62.7%), Joubert syndrome with retinal disease (22.0%), polydactyly (8.5%), and liver (1.7%) and kidney (1.7%) involvement. Joubert syndrome-related disorders include Meckel-Gruber syndrome in 5.1% cases and Leber congenital amaurosis (1.7%). Of the 35 Joubert syndrome-related genes, 11 were identified in these patients, i.e., CEP290, C5ORF, TCTN1, CC2D2A, RPGRP1L, TCTN3, AHI1, INPP5E, TCTN2, NPHP1, and TMEM237. For the first time, we identified a ciliopathy gene, CCDC28B, as a causal gene in Joubert syndrome in one family. CEP290 accounted for 37.8% cases of pure Joubert syndrome, Joubert syndrome with retinal and renal disease, and Meckel-Gruber syndrome. The p.G1890∗ allele in CEP290 is highly recurrent. Of the six families with Joubert syndrome who had a prenatal diagnosis, one fetus was normal, two were carriers, and three were affected. Conclusions This is the largest study of Joubert syndrome from India. Although a high degree of locus and allelic heterogeneity was observed, CEP290 variants were the most common among these patients.

Background Mutations in ciliary genes cause a spectrum of both overlapping and distinct clinical syndromes (ciliopathies). CEP120 and CC2D2A are paradigmatic examples for this genetic heterogeneity and pleiotropy as mutations in both cause Joubert syndrome but are also associated with skeletal ciliopathies and Meckel syndrome, respectively. The molecular basis for this phenotypical variability is not understood but basal exon skipping likely contributes to tolerance for deleterious mutations via tissue‐specific preservation of the amount of expressed functional protein. Methods We systematically reviewed and annotated genetic variants and clinical presentations reported in CEP120‐ and CC2D2A‐associated disease and we combined in silico and ex vivo approaches to study tissue‐specific transcripts and identify molecular targets for exon skipping. Results We confirmed more severe clinical presentations associated with truncating CC2D2A mutations. We identified and confirmed basal exon skipping in the kidney, with possible relevance for organ‐specific disease manifestations. Finally, we proposed a multimodal approach to classify exons amenable to exon skipping. By mapping reported variants, 14 truncating mutations in 7 CC2D2A exons were identified as potentially rescuable by targeted exon skipping, an approach that is already in clinical use for other inherited human diseases. Conclusion Genotype‐phenotype correlations for CC2D2A support the deleteriousness of null alleles and CC2D2A, but not CEP120, offers potential for therapeutic exon skipping approaches., Mutations in CEP120 and CC2D2A cause Joubert syndrome but are also associated with skeletal ciliopathies and Meckel syndrome, respectively. Here we annotate genetic variants described in CEP120‐ and CC2D2A‐associated disease and confirm more severe clinical presentations with biallelic truncating CC2D2A mutations. Combining in silico and ex vivo studies, we identify alternative basal exon skipping in the kidney, with possible relevance for organ‐specific disease manifestations and propose a multimodal approach to classify exons amenable to exon skipping.

Meckel syndrome (MKS) is a perinatally lethal, genetically heterogeneous, autosomal recessive condition caused by defective primary cilium formation leading to polydactyly, multiple cysts in kidneys and malformations of nervous system. We performed exome sequencing in six fetuses from six unrelated families with MKS. We identified seven novel variants in B9D2, TNXDC15, CC2D2A, CEP290 and TMEM67. We describe the second family with MKS due to a homozygous variant in B9D2 and fifth family with bi-allelic variant in TXNDC15. Our data validates the causation of MKS by pathogenic variation in B9D2 and TXNDC15 and also adds novel variants in CC2D2A, CEP290 and TMEM67 to the literature.

Background Joubert syndrome (JBTS) is a heterogenous disorder characterized by intellectual disability, developmental delays, molar tooth sign in brain imaging, hypotonia, ocular motor apraxia and overlapping features of ciliopathies. There are 36 clinical subtypes of JBTS, with an equal number of genes known so far for this phenotype. Methods Whole exome sequencing (WES) and Sanger sequencing were performed for the molecular diagnosis of a Pakhtun family affected with Joubert syndrome type 9 (JBTS9). Results A novel homozygous missense variant (c.4417C>G; Pro1473Ala) in exon 34 was identified in coiled-coil and C2 domains-containing the protein 2A (CC2D2A; NM_001080522) gene. The variant co-segregated in autosomal recessive fashion within the family and was not found in 200 ethnically matched unaffected individuals. In silico analyses supported the pathogenic effect of the altered CC2D2A protein. Conclusions To the best of our knowledge, this is the first report of CC2D2A alteration co-segragating with a JBTS9 phenotype in a Pakhtun family from Pakistan. Our findings broaden the pathogenic spectrum of JBTS9, adding a novel variant to CC2D2A variation pool. WES analysis is a successful molecular diagnostic tool for rare genetic disorders, especially in those populations where the marriage of cousins is more frequent. Efficient and accurate genetic testing and counselling of the affected families are helpful for patient management and for reducing the disease burden in future generations.

Joubert syndrome (JS), first described by Marie Joubert in 1968, is a congenital anomaly characterized by episodic hyperpnea, abnormal eye movement, ataxia and intellectual disability.

We present a patient with hypotonia, developmental delays, hepatomegaly, low platelet counts, and ocular colobomas, consistent with Joubert syndrome with COACH phenotype. She carries two variants in TMEM67. Reclassification in light of clinical information suggests pathogenicity of the c.517T>C (p.Cys173Arg) variant. Early MRI imaging missed a molar tooth sign. Joubert syndrome (JS) is an autosomal recessive disorder characterized by a cerebellar and brainstem malformation called the “molar tooth sign,” hypotonia with eventual ataxia and developmental delays.1 The molar tooth sign involves hypoplasia of the cerebellar vermis, thick superior cerebellar peduncles, and a particularly deep interpeduncular fossa and is mandatory in the diagnosis of Joubert syndrome.2 Joubert syndrome is one of six subgroups of phenotypes that all present with the molar tooth sign. All six are classified in Joubert syndrome and related disorders (JSRD) group: (a) pure JS, (b) JS with ocular defect, (c) JS with renal defect, (d) JS with oculorenal defects, (e) JS with hepatic defect, and (f) JS with orofaciodigital defects.3 Although the true prevalence of JSRD is unknown, estimates range from 1:80 000 to 1:100 000.3, 4 The clinical heterogeneity of JSRD is reflected in significant genetic heterogeneity with some genotype‐phenotype correlations. Biallelic pathogenic variants have been identified in approximately 50% of patients with JSRD. Thirty‐four genes have been identified in the pathogenesis; alterations in the following genes are most commonly found: AHI1, CPLANE1, CC2D2A, CEP290, CSPP1, INPP5E, KIAA0586, MKS1, NPHP1, RPGRIP1L, TCTN2, TMEM67, and TMEM216.5 JSRD are ciliopathies as these genes encode ciliary structural and regulatory proteins. Joubert syndrome with hepatic defect is also known as COACH syndrome: Cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis.6 Mutations in TMEM67 (MKS3) are responsible for the majority of COACH syndrome, although mutations in CC2D2A and RPGRIP1L can also be causative.7 TMEM67/MKS3 is found on chromosome 8 (8q22.1) and encodes a 955‐amino acid seven‐transmembrane receptor protein that is thought to regulate cilia function and mutations in TMEM67/MKS3 can also cause Meckel‐Gruber syndrome (MKS), another autosomal recessive ciliopathy that is associated with renal cystic disease.8, 9, 10, 11 Although mutations in TMEM67/MKS3 are causative for both syndromes (accounting for 9% of JSRD cases and 16% of MKS cases), a correlation between missense mutations in exons 8‐15, particularly in combination with truncating mutations, and Meckel‐Gruber syndrome has been identified.7, 12 To evaluate pathogenicity of variants, laboratories rely on the current literature, functional validation and databases, and computational (in silico) predictive programs.13 However, as these variants may have been previously unseen or insufficient data have been collected on them, the absence of information results in variants being interpreted as neither benign nor pathogenic, but of unknown significance. Clinical correlation also contributes to variant classification, which can be difficult for the testing laboratory to obtain or to interpret. As genetic testing becomes more accessible, it is likely that more variants of unknown significance (VUS) will surface, requiring classification. Here, we present a patient without a family history of Joubert syndrome who carries two heterozygous variants, classified by the testing laboratory as variants of uncertain significance, in TMEM67: c517T>C (p.Cys173Arg) and c.934T>C (p.Ser312Pro) and the following clinical findings: molar tooth sign, hypotonia, developmental delays, hepatomegaly, low platelet counts, and ocular colobomas, consistent with Joubert syndrome with the COACH phenotype.

With the increasing availability and clinical use of exome and whole-genome sequencing, reverse phenotyping is now becoming common practice in clinical genetics. Here, we report a patient identified through the Wellcome Trust Deciphering Developmental Disorders study who has homozygous pathogenic variants in CC2D2A and a de-novo heterozygous pathogenic variant in KIDINS220. He presents with developmental delay, intellectual disability, and oculomotor apraxia. Reverse phenotyping has demonstrated that he likely has a composite phenotype with contributions from both variants. The patient is much more mildly affected than those with Joubert Syndrome or Spastic paraplegia, intellectual disability, nystagmus, and obesity, the conditions associated with CC2D2A and KIDINS220 respectively, and therefore, contributes to the phenotypic variability associated with the two conditions.

Mutations in the gene Centrosomal Protein 290 kDa (CEP290) result in multiple ciliopathies ranging from the neonatal lethal disorder Meckel-Gruber Syndrome to multi-systemic disorders such as Joubert Syndrome and Bardet-Biedl Syndrome to nonsyndromic diseases like Leber Congenital Amaurosis (LCA) and retinitis pigmentosa. Results from model organisms and human genetics studies, have suggest that mutations in genes encoding protein components of the transition zone (TZ) and other cilia-associated proteins can function as genetic modifiers and be a source forCEP290pleiotropy. We investigated the zebrafishcep290fh297/fh297mutant, which encodes a nonsense mutation (p.Q1217*). This mutant is viable as adults, exhibits scoliosis, and undergoes a slow, progressive cone degeneration. Thecep290fh297/fh297mutants showed partial mislocalization of the transmembrane protein rhodopsin but not of the prenylated proteins rhodopsin kinase (GRK1) or the rod transducin subunit GNB1. Surprisingly, photoreceptor degeneration did not trigger proliferation of Müller glia, but proliferation of rod progenitors in the outer nuclear layer was significantly increased. To determine if heterozygous mutations in other cilia genes could exacerbate retinal degeneration, we bredcep290fh297/fh297mutants toarl13b, ahi1,andcc2d2amutant zebrafish lines. Whilecep290fh297/fh297mutants lacking a single allele of these genes did not exhibit accelerated photoreceptor degeneration, loss of one alleles ofarl13borahi1reduced visual performance in optokinetic response assays at 5 days post fertilization. Our results indicate that thecep290fh297/fh297mutant is a useful model to study the role of genetic modifiers on photoreceptor degeneration in zebrafish and to explore how progressive photoreceptor degeneration influences regeneration in adult zebrafish.Nonstandard abbreviationsBBSBardet-Biedl SyndromeCOScone outer segmentsDpfDays post fertilizationGNB1rod transducin β subunitGRK1rhodopsin kinaseJTBSJoubert SyndromeLCALeber Congenital AmaurosisMKSMeckel SyndromeNPHPnephronophthisisOKRoptokinetic responsePNApeanut agglutinin lectinROSrod outer segmentsRP2Retinitis Pigmentosa 2

Rationale: Ciliopathies is a group of clinically and genetically overlapping disorders due to cilia abnormalities and multiple organ systems are involved in. Patient concerns: We present a young female patient who showed renal function impairment, Caroli syndrome (CS), liver cirrhosis, polycystic ovarian syndrome, and multiple subcutaneous cysts. Diagnoses: The patient was diagnosed with ciliopathy according to the clinical manifestations and whole-genome sequencing. Interventions: She received treatment of intravenous albumin, polyene phosphatidyl choline, furosemide, and antisterone. Outcomes: The patient showed clinical improvement in her edema and liver tests, and ultrasonography revealed that the ascites had disappeared. Unfortunately, the edema relapsed a year later. The patient received the same treatment as before, and there was clinical improvement of the edema. Since the family cannot afford liver and kidney transplantation, the patient only accepted symptomatic treatment. Lessons: Polycystic ovarian syndrome and multiple subcutaneous cysts have never before been reported to be associated with ciliopathy. This finding could remind doctors to consider the possibility of ciliopathy disease for patients suffering from similar conditions. In addition, the phenotype of the patient differs from those of patients reported with the same mutations, which also reminds doctors that the clinical manifestation of a given mutation may show patient-specific differences. This case report extends the phenotypic spectrum of ciliopathy, and these findings might represent a new ciliopathy syndrome, which could facilitate the diagnosis of ciliopathies.

Background Inherited cystic kidney disorders are a common cause of end-stage renal disease. Over 50 ciliopathy genes, which encode proteins that influence the structure and function of the primary cilia, are implicated in cystic kidney disease. Methods To define the phenotype and genotype of cystic kidney disease in fetuses and neonates, we correlated antenatal ultrasound examination and postnatal renal ultrasound examination with targeted exon sequencing, using a renal gene panel. A cohort of 44 families in whom antenatal renal ultrasound scanning findings in affected cases included bilateral cystic kidney disease, echogenic kidneys or enlarged kidneys was investigated. Results In this cohort, disease phenotypes were severe with 36 cases of stillbirth or perinatal death. Extra renal malformations, including encephalocele, polydactyly and heart malformations, consistent with ciliopathy phenotypes, were frequently detected. Renal gene panel testing identified causative mutations in 21 out of 34 families (62%), where patient and parental DNA was available. In the remaining 10 families, where only parental DNA was available, 7 inferred causative mutations were found. Together, mutations were found in 12 different genes with a total of 13 novel pathogenic variants, including an inferred novel variant in NEK8 . Mutations in CC2D2A were the most common cause of an antenatal cystic kidney disease and a suspected ciliopathy in our cohort. Conclusions In families with ciliopathy phenotypes, mutational analysis using a targeted renal gene panel allows a rapid molecular diagnosis and provides important information for patients, parents and their physicians.

Genetic investigations were performed in three brothers from a consanguineous union, the two oldest diagnosed with rod-cone dystrophy (RCD), the youngest with early-onset cone-rod dystrophy and the two youngest with nephrotic-range proteinuria. Targeted next-generation sequencing did not identify homozygous pathogenic variant in the oldest brother. Whole exome sequencing (WES) applied to the family identified compound heterozygous variants in CC2D2A (c.2774G>C p.(Arg925Pro); c.4730_4731delinsTGTATA p.(Ala1577Valfs*5)) in the three brothers with a homozygous deletion in CNGA3 (c.1235_1236del p.(Glu412Valfs*6)) in the youngest correcting his diagnosis to achromatopsia plus RCD. None of the three subjects had cerebral abnormalities or learning disabilities inconsistent with Meckel-Gruber and Joubert syndromes, usually associated with CC2D2A mutations. Interestingly, an African woman with RCD shared the CC2D2A missense variant (c.2774G>C p.(Arg925Pro); with c.3182+355_3825del p.(?)). The two youngest also carried compound heterozygous variants in CUBN (c.7906C>T rs137998687 p.(Arg2636*); c.10344C>G p.(Cys3448Trp)) that may explain their nephrotic-range proteinuria. Our study identifies for the first time CC2D2A mutations in isolated RCD and underlines the power of WES to decipher complex phenotypes.

Among more than 5,000 human monogenic disorders with known causative genes, transposable element insertion of a Long Interspersed Nuclear Element 1 (LINE1, L1) is known as the mechanistic basis in only 13 genetic conditions. Meckel-Gruber syndrome is a rare ciliopathy characterized by occipital encephalocele and cystic kidney disease. Here, we document a boy with occipital encephalocele, post-axial polydactyly, and multicystic renal disease. A medical exome analysis detected a heterozygous frameshift mutation, c.4582_4583delCG p.(Arg1528Serfs*17) in CC2D2A in the maternally derived allele. The further use of a dedicated bioinformatics algorithm for detecting retrotransposon insertions led to the detection of an L1 insertion affecting exon 7 in the paternally derived allele. The complete sequencing and sequence homology analysis of the inserted L1 element showed that the L1 element was classified as L1HS (L1 human specific) and that the element had intact open reading frames in the two L1-encoded proteins. This observation ranks Meckel-Gruber syndrome as only the 14th disorder to be caused by an L1 insertion among more than 5,000 known human genetic disorders. Although a transposable element detection algorithm is not included in the current best-practice next-generation sequencing analysis, the present observation illustrates the utility of such an algorithm, which would require modest computational time and resources. Whether the seemingly infrequent recognition of L1 insertion in the pathogenesis of human genetic diseases might simply reflect a lack of appropriate detection methods remains to be seen.

Joubert syndrome (JS) is an autosomal recessive disorder, which is characterized by hypotonia, ataxia, psychomotor delay, and variable occurrences of oculomotor apraxia and neonatal breathing abnormalities. JS is clinically and genetically heterogeneous. The present study investigated a typical JS family. The 'molar tooth sign' was observed in the proband through magnetic resonance imaging. Other symptoms of JS include cerebellar vermis hypoplasia/dysplasia, oculomotor apraxia and intellectual disability. High‑throughput sequencing revealed that JS was caused by coiled‑coil and C2 domain containing 2A (CC2D2A) compound heterozygous mutations. One CC2D2A allele was affected with a missense mutation, c.2581G>A, which led to a p.Asp861Asn amino acid replacement. The other allele was affected with a c.2848C>T nonsense mutation, which resulted in a truncated CC2D2A protein (p.Arg950Ter). Both of these alterations are novel. Further investigation indicated that the proband's father was the c.2581G>A carrier, whereas the mother was the c.2848C>T carrier. These results indicated that JS in the proband was caused by novel compound heterozygous mutations in CC2D2A, which were inherited from both parents. These findings may be used to establish prenatal molecular diagnostic criteria, which may be beneficial in future pregnancies.

Joubert syndrome (JBS) is a rare neurodevelopmental disorder belonging to the group of ciliary diseases. JBS is genetically heterogeneous, with >20 causative genes identified to date. A molecular diagnosis of JBS is essential for prediction of disease progression and genetic counseling. We developed a targeted next-generation sequencing (NGS) approach for parallel sequencing of 22 known JBS genes plus 599 additional ciliary genes. This method was used to genotype a cohort of 51 well-phenotyped Northern European JBS cases (in some of the cases, Sanger sequencing of individual JBS genes had been performed previously). Altogether, 21 of the 51 cases (41%) harbored biallelic pathogenic mutations in known JBS genes, including 14 mutations not previously described. Mutations in C5orf42 (12%), TMEM67 (10%), and AHI1 (8%) were the most prevalent. C5orf42 mutations result in a purely neurological Joubert phenotype, in one case associated with postaxial polydactyly. Our study represents a population-based cohort of JBS patients not enriched for consanguinity, providing insight into the relative importance of the different JBS genes in a Northern European population. Mutations in C5orf42 are relatively frequent (possibly due to a Dutch founder mutation) and mutations in CEP290 are underrepresented compared with international cohorts. Furthermore, we report a case with heterozygous mutations in CC2D2A and B9D1, a gene associated with the more severe Meckel-Gruber syndrome that was recently published as a potential new JBS gene, and discuss the significance of this finding.

Background Joubert syndrome (JBTS) is a predominantly autosomal recessive disorder characterised by a distinctive midhindbrain malformation, oculomotor apraxia, breathing abnormalities and developmental delay. JBTS is genetically heterogeneous, involving genes required for formation and function of non-motile cilia. Here we investigate the genetic basis of JBTS in 12 French–Canadian (FC) individuals. Methods and results Exome sequencing in all subjects showed that six of them carried rare compound heterozygous mutations in CC2D2A or C5ORF42 , known JBTS genes. In addition, three individuals (two families) were compound heterozygous for the same rare mutations in TMEM231 (c.12T>A[p.Tyr4*]; c.625G>A[p.Asp209Asn]). All three subjects showed a severe neurological phenotype and variable presence of polydactyly, retinopathy and renal cysts. These mutations were not detected among 385 FC controls. TMEM231 has been previously shown to localise to the ciliary transition zone, and to interact with several JBTS gene products in a complex involved in the formation of the diffusion barrier between the cilia and plasma membrane. siRNA knockdown of TMEM231 was also shown to affect barrier integrity, resulting in a reduction of cilia formation and ciliary localisation of signalling receptors. Conclusions Our data suggest that mutations in TMEM231 cause JBTS, reinforcing the relationship between this condition and the disruption of the barrier at the ciliary transition zone.

Background Joubert syndrome (JS) is a ciliopathy characterised by a distinctive brain malformation (the ‘molar tooth sign’), developmental delay, abnormal eye movements and abnormal breathing pattern. Retinal dystrophy, cystic kidney disease, liver fibrosis and polydactyly are variably present, resulting in significant phenotypic heterogeneity and overlap with other ciliopathies. JS is also genetically heterogeneous, resulting from mutations in 13 genes. These factors render clinical/molecular diagnosis and management challenging. CC2D2A mutations are a relatively common cause of JS and also cause Meckel syndrome. The clinical consequences of CC2D2A mutations in patients with JS have been incompletely reported. Methods Subjects with JS from 209 families were evaluated to identify mutations in CC2D2A . Clinical and imaging features in subjects with CC2D2A mutations were compared with those in subjects without CC2D2A mutations and reports in the literature. Results 10 novel CC2D2A mutations in 20 subjects were identified; a summary is provided of all published CC2D2A mutations. Subjects with CC2D2A -related JS were more likely to have ventriculomegaly (p

Dobyns, William/0000-0002-7681-2844; VERLOES, Alain/0000-0003-4819-0264 WOS: 000273581000002 PubMed: 19574260 Objective To identify genetic causes of COACH syndrome Background COACH syndrome is a rare autosomal recessive disorder characterised by Cerebellar vermis hypoplasia, Oligophrenia (developmental delay/mental retardation), Ataxia, Coloboma, and Hepatic fibrosis. The vermis hypoplasia falls in a spectrum of mid-hindbrain malformation called the molar tooth sign (MTS), making COACH a Joubert syndrome related disorder (JSRD). Methods In a cohort of 251 families with JSRD, 26 subjects in 23 families met criteria for COACH syndrome, defined as JSRD plus clinically apparent liver disease. Diagnostic criteria for JSRD were clinical findings (intellectual impairment, hypotonia, ataxia) plus supportive brain imaging findings (MTS or cerebellar vermis hypoplasia). MKS3/TMEM67 was sequenced in all subjects for whom DNA was available. In COACH subjects without MKS3 mutations, CC2D2A, RPGRIP1L and CEP290 were also sequenced. Results 19/23 families (83%) with COACH syndrome carried MKS3 mutations, compared to 2/209 (1%) with JSRD but no liver disease. Two other families with COACH carried CC2D2A mutations, one family carried RPGRIP1L mutations, and one lacked mutations in MKS3, CC2D2A, RPGRIP1L and CEP290. Liver biopsies from three subjects, each with mutations in one of the three genes, revealed changes within the congenital hepatic fibrosis/ductal plate malformation spectrum. In JSRD with and without liver disease, MKS3 mutations account for 21/232 families (9%). Conclusions Mutations in MKS3 are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L; therefore, MKS3 should be the first gene tested in patients with JSRD plus liver disease and/or coloboma, followed by CC2D2A and RPGRIP1L. US National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [K23NS45832, K24HD46712, NCRR 5KL2RR025015, R01NS050375]; March of Dimes Endowment for Healthier BabiesMarch of Dimes; Ames Endowment Fund; Allan and Phyllis Treuer Endowed Chair This work was supported by the US National Institutes of Health (grants K23NS45832 to MAP, K24HD46712 to IAG, NCRR 5KL2RR025015 to DD, R01NS050375 to WBD), the March of Dimes Endowment for Healthier Babies (DD, MAP, and IAG), the Ames Endowment Fund at Seattle Children's Hospital (IAG), and the Allan and Phyllis Treuer Endowed Chair (PFC) at Seattle Children's Hospital.

Joubert syndrome (JS) is a primarily autosomal recessive condition characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation (the "molar tooth sign"). Variable features include retinal dystrophy, cystic kidney disease, liver fibrosis and polydactyly. Recently, substantial progress has been made in our understanding of the genetic basis of JS, including identification of seven causal genes (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67/MKS3, ARL13B and CC2D2A). Despite this progress, the known genes account for

Meckel syndrome (MKS) is a lethal malformation disorder characterized classically by encephalocele, polycystic kidneys, and polydactyly. MKS is also one of the major contributors to syndromic neural tube defects (NTDs). Recent findings have shown primary cilia dysfunction in the molecular background of MKS, indicating that cilia are critical for early human development. However, even though four genes behind MKS have been identified to date, they elucidate only a minor proportion of the MKS cases. In this study, instead of traditional linkage analysis, we selected 10 nonrelated affected fetuses and looked for the homozygous regions shared by them. Based on this strategy, we identified the sixth locus and the fifth gene, CC2D2A (MKS6), behind MKS. The biological function of CC2D2A is uncharacterized, but the corresponding polypeptide is predicted to be involved in ciliary functions and it has a calcium binding domain (C2). Immunofluorescence staining of patient's fibroblast cells demonstrates that the cells lack cilia, providing evidence for the critical role of CC2D2A in cilia formation. Our finding is very significant not only to understand the molecular background of MKS, but also to obtain additional information about the function of the cilia, which can help to understand their significance in normal development and also in other ciliopathies, which are an increasing group of disorders with overlapping phenotypes.

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.

Results Core JS diagnostic features (hypotonia, ataxia, cognitive dysfunction, oculo-motor apraxia) were present in >80% of individuals, while abnormal breathing pattern was reported in 60%. Frequently associated features included retinal dystrophy (31.4%), renal disease (20.9%), coloboma (17.7%), polydactyly (15.3%), liver fibrosis (15.2%) and encephalocele (8%). Liver fibrosis and coloboma were strongly associated with each other (Odds Ratio 7.0, 95% Confidence Interval = 3.0-13.2), while retinal dystrophy and renal disease were weakly associated (O.R. 2.2, 95%C. I. = 1.7-5.6). Additional clinical features included other brain abnormalities (n = 73), seizures (n = 49), cleft palate (n = 16), hearing loss (n = 14) and psychiatric problems (n = 45). The genetic cause was identified in 60% of families, with 5 genes accounting for the majority of patients (C5ORF42, CEP290, CC2D2A, AHI1, TMEM67). Bi-allelic causal mutations in B9D2 and C2CD3 were identified in 2 families each. Bi-allelic mutations in 2 different genes were identified in 4 families and heterozygous mutations (in addition to the causal mutation) were present in 62 individuals. Significant (p

Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains-containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.

Meckel-Gruber syndrome is a severe autosomal, recessively inherited disorder characterized by bilateral renal cystic dysplasia, developmental defects of the central nervous system (most commonly occipital encephalocele), hepatic ductal dysplasia and cysts and polydactyly1,2,3. MKS is genetically heterogeneous, with three loci mapped: MKS1, 17q21-24 (ref. 4); MKS2, 11q13 (ref. 5) and MKS3 (ref. 6). We have refined MKS3 mapping to a 12.67-Mb interval (8q21.13-q22.1) that is syntenic to the Wpk locus in rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus7,8. Positional cloning of the Wpk gene suggested a MKS3 candidate gene, TMEM67, for which we identified pathogenic mutations for five MKS3-linked consanguineous families. MKS3 is a previously uncharacterized, evolutionarily conserved gene that is expressed at moderate levels in fetal brain, liver and kidney but has widespread, low levels of expression. It encodes a 995–amino acid seven-transmembrane receptor protein of unknown function that we have called meckelin.

Purpose Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish.

The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a(-/-) mouse that recapitulates features of Meckel syndrome including embryonic lethality and multiorgan defects. Cilia are absent in Cc2d2a(-/-) embryonic node and other somatic tissues; disruption of cilia-dependent Shh signalling appears to underlie exencephaly in mutant embryos. The Cc2d2a(-/-) mouse embryonic fibroblasts (MEFs) lack cilia, although mother centrioles and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a(-/-) MEFs, subdistal appendages are lacking or abnormal by transmission electron microscopy. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild-type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis.

We describe a first-trimester ultrasound examination in which the finding of fetal encephalocele and the cystic appearance of the kidneys raised suspicion of Meckel-Gruber syndrome (MKS). On the basis of sonographic findings, the patient elected termination of pregnancy, and post-termination studies using next-generation sequencing of a gene panel revealed two mutations (one previously described and the other novel) in the gene CC2D2A. Mutations in CC2D2A are known to cause MKS and Joubert syndrome, thus providing molecular confirmation of the clinical suspicion of MKS and opening the possibility for future prenatal diagnosis. This case highlights the ability to detect important anomalies in the first trimester using ultrasound, even in low-risk situations. It also demonstrates the growing role of new sequencing technologies in fetal testing.

Meckel syndrome (MKS) is a severe fetal developmental disorder reported in most populations. The clinical hallmarks are occipital meningoencephalocele, cystic kidney dysplasia, fibrotic changes of the liver and polydactyly. Here we report the identification of a gene, MKS1, mutated in MKS families linked to 17q. Mks1 expression in mouse embryos, as determined by in situ hybridization, agrees well with the tissue phenotype of MKS. Comparative genomics and proteomics data implicate MKS1 in ciliary functions.

We have identified a complex of proteins that form part of the transition zone, a region at the base of the cilium. This complex includes the three members of the Tectonic family, extracytosolic glycoproteins that interact with transmembrane components of the transition zone such as Tmem67, Tmem216, and Tmem231. These transmembrane proteins connect to an intracellular transition zone complex comprised of many known Joubert- and Meckel-associated proteins including Cc2d2a, B9d1, B9d2, Mks1. Loss of components of this transition zone complex in mice compromise ciliogenesis in some tissues, and deregulate ciliary protein composition in others. In particular, the ciliary localization of Smoothened (Smo), a central component of the Hedgehog pathway, depends on this complex. As Smo functions at the cilium, many mouse transition zone mutants show deregulation of Hh signaling, resulting in ventralization of the neural tube and polydactyly. Defining the components of the transition zone has led to the identification of additional genes underlying Joubert and Meckel syndromes including Tctn1, Tctn2 and B9d2. We hypothesize that Joubert and Meckel syndromes are caused by transition zone dysfunction that disrupts intercellular signaling, leading to developmental defects.

The cerebellum plays a role not only in motor control but also in motor learning and cognition. Joubert syndrome is a rare heterogeneous inherited genetic disorder characterized by ataxia, hypotonia, developmental delay, and at least one of the following features: neonatal respiratory disturbances or abnormal eye movement. The estimated frequency of Joubert syndrome in the United States is around 1 : 100 000. The term Joubert syndrome and related disorders (JSRD) has been recently coined to describe all disorders presenting with molar tooth sign on brain neuroimaging. Joubert syndrome is believed to be a representative of a new group of disorders named ciliopathies. The identification of seven causal genes (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67/MKS3, ARL13B, CC2D2A) has led to substantial progress in the understanding of the genetic basis of Joubert syndrome. The authors focus on clinical presentation of JSRD, differential diagnosis and molecular background.

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion.

Joubert-Boltshauser syndrome; Joubertsyndrome related disorders (JSRD), including: Cerebellar vermishypoplasia/aplasia, oligophrenia, congenital ataxia, ocular coloboma,and hepatic fibrosis (COACH) syndrome; Cerebellooculorenal orcerebello-oculo-renal (COR) syndrome; Dekaban–Arima syndrome;Va´radi–Papp syndrome or orofaciodigital type VI (OFDVI) syndrome;Malta syndrome.1.2 OMIM# of the disease213300, 243910, 216360, and 277170.1.3 Name of the analysed genes or DNA/chromosome segmentsJBTS1/INPP5E, JBTS2/TMEM216, JBTS3/AHI1, JBTS4/NPHP1,JBTS5/CEP290, JBTS6/TMEM67, JBTS7/RPGRIP1L, JBTS8/ARL13B,JBTS9/CC2D2A, and JBTS10/OFD1.1.4 OMIM# of the gene(s)613037, 613277, 608894, 607100, 610142, 609884, 610937, 608922,612013, and 300170.1.5 Mutational spectrumMissense and nonsense mutations, splice site mutations, deletions,and insertions. Genomic rearrangements so far reported onlyfor NPHP1 (homozygous deletions represent 495% mutations)and CEP290 (heterozygous multiexon deletion reported in asingle patient). Marked allelic heterogeneity, with a large numberof mutations, was reported in each of the 10 genes. Gene–phenotype correlations are known for selected genes (about 50%patients with COR phenotype and about 75% patients withCOACH phenotype have mutations in CEP290 and TMEM67 genes,respectively).1.6 Analytical methodsDirect sequencing of coding genomic regions and splice site junctions;multiplex microsatellite analysis for detection of NPHP1 homozygousdeletion. Possibly, qPCR or targeted array-CGH for detection ofgenomic rearrangements in other genes.1.7 Analytical validationDirect sequencing of both DNA strands; verification of sequence andqPCR results in an independent experiment.1.8 Estimated frequency of the disease(incidence at birth (‘birth prevalence’) or population prevalence)No good population-based data on JSRD prevalence have beenpublished. A likely underestimated frequency between 1/80000 and1/100000 live births is based on unpublished data.1.9 If applicable, prevalence in the ethnic group of investigatedpersonIn Ashkenazi Jews, the estimated carrier frequency of the foundermutation p.R73L in the TMEM216 gene is about 1%.1.10 Diagnostic settingComment: The early detection of breathing abnormalities and/oroculomotor apraxia is suggestive of JS and related disorders. Definitediagnosis is made based on the identification of the characteristichindbrain malformation on brain imaging, that is, the ‘molar toothsign’ or its component features.2. TEST CHARACTERISTICS2.1 Analytical sensitivity(proportion of positive tests if the genotype is present)Nearly 100%. A test for large deletions/duplications in genes otherthan NPHP1 should be considered, especially in patients in whom asingle heterozygous mutation has been detected with conventionalsequencing. The presence of deep intronic mutations is not exploredwith current screening methods.

Meckel–Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder characterized by developmental defects of the central nervous system that comprise neural tube defects that most commonly present as occipital encephalocele. MKS is considered to be the most common syndromic form of neural tube defect. MKS is genetically heterogeneous with six known disease genes: MKS1, MKS2/TMEM216, MKS3/TMEM67, RPGRIP1L, CEP290, and CC2D2A with the encoded proteins all implicated in the correct function of primary cilia. Primary cilia are microtubule-based organelles that project from the apical surface of most epithelial cell types. Recent progress has implicated the involvement of cilia in the Wnt and Shh signaling pathways and has led to an understanding of their role in normal mammalian neurodevelopment. The aim of this review is to provide an overview of the molecular genetics of the human disorder, and to assess recent insights into the etiology and molecular cell biology of severe ciliopathies from mammalian animal models of MKS.

Ciliary disorders share typical features, such as polydactyly, renal and biliary cystic dysplasia, and retinitis pigmentosa, which often overlap across diagnostic entities. We report on two siblings of consanguineous parents and two unrelated children, both of unrelated parents, with co-occurrence of Joubert syndrome and Jeune asphyxiating thoracic dystrophy, an association that adds to the observation of common final patterns of malformations in ciliary disorders. Using homozygosity mapping in the siblings, we were able to exclude all known genes/loci for both syndromes except for INVS, AHI1, and three genes from the previously described Jeune locus at 15q13. No pathogenic variants were found in these genes by direct sequencing. In the third child reported, sequencing of RPGRIP1L, ARL13B, AHI1, TMEM67, OFD1, CC2D2A, and deletion analysis of NPHP1 showed no mutations. Although this study failed to identify a mutation in the patients tested, the co-occurrence of Joubert and Jeune syndromes is likely to represent a distinct entity caused by mutations in a yet to be discovered gene. The mechanisms by which certain organ systems are affected more than others in the spectrum of ciliary diseases remain largely unknown. © 2010 Wiley-Liss, Inc., link_to_OA_fulltext