Utilization of automated cilia analysis to characterize novel INPP5E variants in patients with non-syndromic retinitis pigmentosa.

INPP5E encodes inositol polyphosphate-5-phosphatase E, an enzyme involved in regulating the phosphatidylinositol (PIP) makeup of the primary cilium membrane. Pathogenic variants in INPP5E hence cause a variety of ciliopathies: genetic disorders caused by dysfunctional cilia. While the majority of these disorders are syndromic, such as the neuronal ciliopathy Joubert syndrome, in some cases patients will present with an isolated phenotype-most commonly non-syndromic retinitis pigmentosa (RP). Here, we report two novel variants in INPP5E identified in two patients with non-syndromic RP: patient 1 with compound heterozygous variants (c.1516C > T, p.(Q506*), and c.847G > A, p.(A283T)) and patient 2 with a homozygous variant (c.1073C > T, p.(P358L)). To determine whether these variants were causative for the phenotype in the patients, automated ciliary phenotyping of patient-derived dermal fibroblasts was performed for percent ciliation, cilium length, retrograde IFT trafficking, and INPP5E localization. In both patients, a decrease in ciliary length and loss of INPP5E localization in the primary cilia were seen. With these molecular findings, we can confirm functionally that the novel variants in INPP5E are causative for the RP phenotypes seen in both patients. Additionally, this study demonstrates the usefulness of utilizing ciliary phenotyping as an assistant in ciliopathy diagnosis and phenotyping.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Bilateral posterior lamellar corneal transplant surgery in an infant of 17 weeks old: Surgical challenges and the added value of intraoperative optical coherence tomography.

This study aimed to describe the surgical challenges, management, and value of intraoperative optical coherence tomography in a case of a bilateral Descemet Stripping Automated Endothelial Keratoplasty corneal transplantation at 17 weeks of age for the treatment of severe posterior polymorphous corneal dystrophy resulting from a de novo mutation of the OVOL2-gene.

Generation of a patient-derived induced pluripotent cell line (SCTCi016-A) carrying a homozygous variant in RPE65.

Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.

Healthcare recommendations for Joubert syndrome.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder defined by a characteristic cerebellar and brainstem malformation recognizable on axial brain magnetic resonance imaging as the "Molar Tooth Sign". Although defined by the neurological features, JS is associated with clinical features affecting many other organ systems, particularly progressive involvement of the retina, kidney, and liver. JS is a rare condition; therefore, many affected individuals may not have easy access to subspecialty providers familiar with JS (e.g., geneticists, neurologists, developmental pediatricians, ophthalmologists, nephrologists, hepatologists, psychiatrists, therapists, and educators). Expert recommendations can enable practitioners of all types to provide quality care to individuals with JS and know when to refer for subspecialty care. This need will only increase as precision treatments targeting specific genetic causes of JS emerge. The goal of these recommendations is to provide a resource for general practitioners, subspecialists, and families to maximize the health of individuals with JS throughout the lifespan.

Clinical Characterization of 66 Patients With Congenital Retinal Disease Due to the Deep-Intronic c.2991+1655A>G Mutation in CEP290.

Purpose: To describe the phenotypic spectrum of retinal disease caused by the c.2991+1655A>G mutation in CEP290 and to compare disease severity between homozygous and compound heterozygous patients. Methods: Medical records were reviewed for best-corrected visual acuity (BCVA), age of onset, fundoscopy descriptions. Foveal outer nuclear layer (ONL) and ellipsoid zone (EZ) presence was assessed using spectral-domain optical coherence tomography (SD-OCT). Differences between compound heterozygous and homozygous patients were analyzed based on visual performance and visual development. Results: A total of 66 patients were included. The majority of patients had either light perception or no light perception. In the remaining group of 14 patients, median BCVA was 20/195 Snellen (0.99 LogMAR; range 0.12-1.90) for the right eye, and 20/148 Snellen (0.87 LogMAR; range 0.22-1.90) for the left. Homozygous patients tended to be more likely to develop light perception compared to more severely affected compound heterozygous patients (P = 0.080) and are more likely to improve from no light perception to light perception (P = 0.022) before the age of 6 years. OCT data were available in 12 patients, 11 of whom had retained foveal ONL and EZ integrity up to 48 years (median 23 years) of age. Conclusions: Homozygous patients seem less severely affected compared to their compound-heterozygous peers. Improvement of visual function may occur in the early years of life, suggesting a time window for therapeutic intervention up to the approximate age of 17 years. This period may be extended by an intact foveal ONL and EZ on OCT.

Refining the phenotype associated with GNB1 mutations: Clinical data on 18 newly identified patients and review of the literature.

De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.

Clinical and genetic analyses of a Dutch cohort of 40 patients with a nephronophthisis-related ciliopathy.

BACKGROUND: Nephronophthisis is an autosomal recessive ciliopathy and important cause of end-stage renal disease (ESRD) in children and young adults. Diagnostic delay is frequent. This study investigates clinical characteristics, initial symptoms, and genetic defects in a cohort with nephronophthisis-related ciliopathy, to improve early detection and genetic counseling. METHODS: Forty patients from 36 families with nephronophthisis-related ciliopathy were recruited at university medical centers and online. Comprehensive clinical and genotypic data were recorded. Patients without molecular diagnosis were offered genetic analysis. RESULTS: Of 40 patients, 45% had isolated nephronophthisis, 48% syndromic diagnosis, and 7% nephronophthisis with extrarenal features not constituting a recognizable syndrome. Patients developed ESRD at median 13 years (range 5-47). Median age of symptom onset was 9 years in both isolated and syndromic forms (range 5-26 vs. 5-33). Common presenting symptoms were fatigue (42%), polydipsia/polyuria (33%), and hypertension (21%). Renal ultrasound showed small-to-normal-sized kidneys, increased echogenicity (65%), cysts (43%), and abnormal corticomedullary differentiation (32%). Renal biopsies in eight patients showed nonspecific signs of chronic kidney disease (CKD). Twenty-three patients (58%) had genetic diagnosis upon inclusion. Thirteen of those without a genetic diagnosis gave consent for genetic testing, and a cause was identified in five (38%). CONCLUSIONS: Nephronophthisis is genetically and phenotypically heterogeneous and should be considered in children and young adults presenting with persistent fatigue and polyuria, and in all patients with unexplained CKD. As symptom onset can occur into adulthood, presymptomatic monitoring of kidney function in syndromic ciliopathy patients should continue until at least age 30.

MKS1 regulates ciliary INPP5E levels in Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a recessive ciliopathy characterised by a distinctive brain malformation 'the molar tooth sign'. Mutations in >27 genes cause JS, and mutations in 12 of these genes also cause Meckel-Gruber syndrome (MKS). The goals of this work are to describe the clinical features of MKS1-related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS. METHODS: We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a three-dimensional (3D) spheroid rescue assay to test the effects of disease-related MKS1 mutations. RESULTS: We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1-related JS carry ≥ 1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids. CONCLUSIONS: MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalisation, is a key mechanism underlying JS, downstream of MKS1 and ARL13B.

Joubert syndrome: genotyping a Northern European patient cohort.

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.

Murine Joubert syndrome reveals Hedgehog signaling defects as a potential therapeutic target for nephronophthisis.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

Chromosomal abnormalities resembling Joubert syndrome: two cases illustrating the diagnostic pitfalls.

We describe two patients with severe developmental delay, hypotonia and breathing abnormalities initially diagnosed with the autosomal recessive Joubert syndrome (JBS) who at a later stage appeared to carry chromosomal abnormalities. One case was due to a 4.8 Mb terminal 1q44 deletion, and the other due to a 15.5 Mb duplication of Xq27.2-qter containing the MECP2 gene. Critical evaluation of the clinical data showed that, retrospectively, the cases did not fulfil the diagnostic criteria for JBS, and that the diagnosis of JBS was incorrectly made. We discuss the diagnostic pitfalls and recommend adhering strictly to the JBS diagnostic criteria in the case of a negative molecular diagnosis. Critical assessment of the MRI findings by a specialized neuroradiologist is imperative. As chromosomal abnormalities may give rise to symptoms resembling JBS, we recommend array-based screening for segmental aneuploidies as an initial genetic test in all cases with a JBS-like phenotype.

Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes.

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease.

Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations.

PURPOSE: To determine the genetic defect and to describe the clinical characteristics in a cohort of mainly nonconsanguineous cone-rod dystrophy (CRD) patients. METHODS: One hundred thirty-nine patients with diagnosed CRD were recruited. Ninety of them were screened for known mutations in ABCA4, and those carrying one or two mutations were excluded from further research. Genome-wide homozygosity mapping was performed in the remaining 108. Known genes associated with autosomal recessive retinal dystrophies located within a homozygous region were screened for mutations. Patients in whom a mutation was detected underwent further ophthalmic examination. RESULTS: Homozygous sequence variants were identified in eight CRD families, six of which were nonconsanguineous. The variants were detected in the following six genes: ABCA4, CABP4, CERKL, EYS, KCNV2, and PROM1. Patients carrying mutations in ABCA4, CERKL, and PROM1 had typical CRD symptoms, but a variety of retinal appearances on funduscopy, optical coherence tomography, and autofluorescence imaging. CONCLUSIONS: Homozygosity mapping led to the identification of new mutations in consanguineous and nonconsanguineous patients with retinal dystrophy. Detailed clinical characterization revealed a variety of retinal appearances, ranging from nearly normal to extensive retinal remodeling, retinal thinning, and debris accumulation. Although CRD was initially diagnosed in all patients, the molecular findings led to a reappraisal of the diagnosis in patients carrying mutations in EYS, CABP4, and KCNV2.

A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype.

PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP). METHODS. DNA samples of the affected family members were genotyped with 250 K genome-wide SNP microarrays. Genetic defects were localized by linkage analysis and homozygosity mapping, and candidate genes were analyzed by sequencing. Patients underwent a full ophthalmic examination. RESULTS. Compound heterozygous mutations in CEP290 were identified in the proband and her two cousins: the frequent c.2991+1655A>G founder mutation and a novel nonsense mutation in exon 7 (c.451C>T, p.Arg151X). The proband had nystagmus, hyperopia, a flat electroretinogram (ERG), and decreased visual acuity (20/250) from birth. The two cousins had minimal scotopic ERG responses at the age of 2. In one of these patients, visual acuity had reached a level of 20/32 at age 5, which is high for patients with CEP290 mutations. Analysis of the CEP290 mRNA in affected individuals revealed altered splice forms in which either exon 7 or exons 7 and 8 were skipped. In both mutant cDNA products, the open reading frame was not disrupted. Furthermore, homozygosity mapping and mutation analysis in the distant family member affected by RP revealed a homozygous mutation in MERTK, but no CEP290 mutations. This MERTK mutation was heterozygously present in the most severely affected (LCA) patient, but was absent in the two more mildly affected cousins. CONCLUSIONS. A novel nonsense mutation in CEP290 results in nonsense-associated altered splicing. That the remaining open reading frame is intact may explain the less severe phenotype observed in the two affected cousins. The additional heterozygous mutation in MERTK may clarify the more severe phenotype in the proband. This study extends the phenotypic spectrum of CEP290-associated diseases at the mild end.

Dandy-Walker complex in a boy with a 5 Mb deletion of region 1q44 due to a paternal t(1;20)(q44;q13.33).

A 10-year-old boy with vermis hypoplasia, dilatation of the fourth ventricle, enlarged cisterna magna and aplasia of the corpus callosum, consistent with the Dandy-Walker complex (DWC), and slight facial dysmorphisms, severe motor and mental retardation is presented. By combining data obtained by karyotyping, array-CGH, FISH, and multiplex ligation-mediated probe amplification (MLPA) we identified a 5 Mb deletion of the 1q44 --> qter region resulting from a paternal t(1;20)(q44;q13.33). This smallest 1q44 deletion reported so far, enabled us to significantly narrow down the number of candidate genes for the DWC in this region. Since the ZNF124 transcription factor is strongly expressed in the fetal brain it may represent a candidate gene for the DWC at 1q44.

Cerebral, cerebellar, and colobomatous anomalies in three related males: Sex-linked inheritance in a newly recognized syndrome with features overlapping with Joubert syndrome.

We present a so far unrecognized X-linked mental retardation syndrome with features overlapping with Joubert syndrome (JBS). Two brothers showed hypotonia, mental retardation, ocular abnormalities with impaired vision and colobomas and a breathing pattern compatible with JBS. Neuroimaging revealed cerebellar vermis hypoplasia and ventriculomegaly. A tentative diagnosis of JBS was made, and autosomal recessive inheritance considered most likely. In a subsequent pregnancy that occurred after artificial donor insemination, ultrasound in the 22nd week revealed a Dandy-Walker malformation and hydrocephaly. At autopsy at 34 weeks of gestation, the male infant showed cerebellar vermis aplasia and abnormalities of the brainstem and cerebral cortex. He was considered to have the same disorder as his two half-brothers. This renders the pedigree highly suggestive of X-linked inheritance. The clinical symptoms of this syndrome resemble JBS. However, the absence of the molar tooth sign and the X-linked inheritance do not support JBS. We propose the name X-linked cerebral-cerebellar-coloboma syndrome to distinguish the two disorders. Differentiation of the two disorders is especially important in genetic counseling, where artificial donor insemination may be considered as a means of reducing the recurrence risk, or when female relatives of the patient are concerned.