Walcott-Rallison syndrome (WRS) and the recently delineated microcephaly with simplified gyration, epilepsy and permanent neonatal diabetes syndrome (MEDS) are clinically overlapping autosomal recessive disorders characterized by early-onset diabetes, skeletal defects, and growth retardation. While liver and renal symptoms are more severe in WRS, neurodevelopmental characteristics are more pronounced in MEDS patients, in which microcephaly and uncontrolled epilepsy are uniformly present. Mutations in the EIF2AK3 gene were described in patients with WRS and defects in this gene lead to increased susceptibility to apoptotic cell death. Mutations in IER3IP1 have been reported in patients with MEDS and similarly, loss of activity results in apoptosis of neurons and pancreatic beta cells in patients. Here we report on a homozygous mutation of the IER3IP1 gene in four patients from two unrelated consanguineous Egyptian families presenting with MEDS who display burst suppression patterns on EEG. All patients presented with mildly elevated liver enzymes, microalbuminuria, and skeletal changes such as scoliosis and osteopenia, leading to repeated bone fractures. We expand the phenotypic spectrum of MEDS caused by IER3IP1 gene mutations and propose that WRS and MEDS are overlapping clinical syndromes, displaying significant gene-dependent clinical variability.
microcephaly; epilepsy; autosomal recessive; infantile diabetes mellitus; burst suppression; Walcott-Rallison syndrome
We describe six cases from three unrelated consanguineous Egyptian families with a novel characteristic brain malformation at the level of the diencephalic–mesencephalic junction. Brain magnetic resonance imaging demonstrated a dysplasia of the diencephalic–mesencephalic junction with a characteristic ‘butterfly’-like contour of the midbrain on axial sections. Additional imaging features included variable degrees of supratentorial ventricular dilatation and hypoplasia to complete agenesis of the corpus callosum. Diffusion tensor imaging showed diffuse hypomyelination and lack of an identifiable corticospinal tract. All patients displayed severe cognitive impairment, post-natal progressive microcephaly, axial hypotonia, spastic quadriparesis and seizures. Autistic features were noted in older cases. Talipes equinovarus, non-obstructive cardiomyopathy and persistent hyperplastic primary vitreous were additional findings in two families. One of the patients required shunting for hydrocephalus; however, this yielded no change in ventricular size suggestive of dysplasia rather than obstruction. We propose the term ‘diencephalic–mesencephalic junction dysplasia’ to characterize this autosomal recessive malformation.
diencephalon; mesencephalon; mental retardation; brainstem malformation; brain wiring
Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.
•miCLIP detects NSun2-mediated cytosine-5 methylation in RNA•Vault noncoding RNA is methylated by NSun2•Cytosine-5 in Vault RNA determines its processing into small RNA (svRNA)•svRNAs bind to Argonaute proteins and exhibit microRNA-like functions
Comprehensive analyses of cytosine-5 methylation in the RNA transcriptome have previously been hampered by the lack of sensitive and reliable molecular techniques. In this work, Ule, Frye, and colleagues describe the methylation-iCLIP (miCLIP) method that they used to identify target sites of the RNA methyltransferase, NSun2. Among the targeted noncoding RNAs were vault RNAs, previously associated with resistance to chemotherapy in cancer. They further show how NSun2-mediated methylation of vault ncRNAs influences their processing into small microRNA-like molecules.
Wnt signaling encompasses a variety of signaling cascades that can be activated by secreted Wnt ligands. Two such pathways, the canonical or β-catenin pathway and the planar cell polarity pathway, have recently received attention for their roles in multiple cellular processes within the kidney. Both of these pathways are important for kidney development as well as homeostasis and injury repair. Disruption of either pathway can lead to cystic kidney disease, a class of genetic diseases that includes the most common hereditary life-threatening syndrome polycystic kidney disease. Recent evidence implicates canonical and noncanonical Wnt pathways in cyst formation and points to a remarkable role for developmental processes in the adult kidney.
cyst; Wnt; cilia; ciliopathy; signaling; planar cell polarity; kidney; injury; repair; development
Genetic diseases known as ciliopathies have recently entered the limelight, placing new importance on a previously mysterious organelle: the primary cilium. Mutations affecting the primary cilium in both humans and animal models can lead to a plethora of distinct phenotypes including retinal degeneration, kidney cysts, and brain malformations. New findings are quickly lending insight into the functions of this cellular extension that seems to be especially important in modulation of subcellular signaling cascades at various stages of development and adult homeostasis.
We review some of the key recent findings in the field of human cortical development. This development is divided into three major time-dependent phases: neural proliferation, migration, and maturation. The cells that constitute the cerebral cortex, including both inhibitory and excitatory neurons, proliferate in spatially distinct regions. These cells then migrate through multiple cellular boundaries, one of which is known as the subplate, before achieving final positioning within the 6-layered cerebral cortex. Following this migration, neurons undergo morphological changes that result in elaboration of dendrites and axons, and establish the multitude of cellular contacts that underlie neuronal processing. Many of the neurocognitive disorders that we treat in the clinic can trace their origin to a disorder in one or more of these key steps. Along with this update, we also highlight work that offers a glimpse at the future of therapy for developmental brain disorders that can result from disorders of these cellular events.
'Ciliopathies' are an emerging class of genetic multisystemic human disorders that are caused by a multitude of largely unrelated genes that affect ciliary structure/function. They are unified by shared clinical features, such as mental retardation, cystic kidney, retinal defects and polydactyly, and by the common localization of the protein products of these genes at or near the primary cilium of cells. With the realization that many previously disparate conditions are a part of this spectrum of disorders, there has been tremendous interest in the function of cilia in developmental signaling and homeostasis. Ciliopathies are mostly inherited as simple recessive traits, but phenotypic expressivity is under the control of numerous genetic modifiers, putting these conditions at the interface of simple and complex genetics. In this review, we discuss the ever-expanding ciliopathy field, which has three interrelated goals: developing a comprehensive understanding of genes mutated in the ciliopathies and required for ciliogenesis; understanding how the encoded proteins work together in complexes and networks to modulate activity and structure-function relationships; and uncovering signaling pathways and modifier relationships.
Dysequilibrium syndrome is a genetically heterogeneous condition that combines autosomal recessive, nonprogressive cerebellar ataxia with mental retardation. The condition has been classified into cerebellar ataxia, mental retardation and disequilibrium syndrome types 1 (CAMRQ1), 2 (CAMRQ2) and 3 (CAMRQ3) and attributed to mutations in VLDLR, CA8 and WDR81 genes, respectively. Quadrupedal locomotion in this syndrome has been reported in association with mutations in all three genes.
SNP mapping and candidate gene sequencing in one consanguineous Omani family from the United Arab Emirates with cerebellar hypoplasia, moderate mental retardation, delayed ambulation and truncal ataxia was used to identify the mutation. In a second unrelated consanguineous Omani family, massively parallel exonic sequencing was used.
We identified a homozygous missense mutation (c.2117 G > T, p.C706F) in the VLDLR gene in both families on a shared affected haplotype block.This is the first reported homozygous missense mutation in VLDLR and it occurs in a highly conserved residue and predicted to be damaging to protein function.
We have delineated the phenotype associated with dysequilibrium syndrome in two Omani families and identified the first homozygous missense pathogenic mutation in VLDLR gene with likely founder effect in the southeastern part of the Arabian Peninsula.
Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/− neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear “cage” to the centrosome. Lis1+/− neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus–centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.
migration; Lis1; doublecortin; centrosome; nucleus
Disorders within the “ciliopathy” spectrum include Joubert (JS), Bardet-Biedl syndromes (BBS) and nephronophthisis (NPHP). Although mutations in single ciliopathy genes can lead to these different syndromes between families, there have been no reports of phenotypic discordance within a single family. We report on two consanguineous families with discordant ciliopathies in sibling. In Ciliopathy-672, the older child displayed dialysis-dependent NPHP whereas the younger displayed the pathognomonic molar tooth MRI sign (MTS) of JS. A second branch displayed two additional children with NPHP. In Ciliopathy-1491, the oldest child displayed classical features of BBS whereas the two younger children displayed the MTS. Importantly, the children with BBS and NPHP lacked MTS, whereas children with JS lacked obesity or NPHP, and the child with BBS lacked MTS and NPHP. Features common to all three disorders included intellectual disability, postaxial polydactyly, and visual reduction. The variable phenotypic expressivity in this family suggests that genetic modifiers may determine specific clinical features within the ciliopathy spectrum.
Molar-tooth; polydactyly; intellectual disability; retinal blindness; obesity; nephronophthisis
We identified a two-branch consanguineous family in which four affected members (three females and one male) presenting with constitutive growth delay, severe psychomotor retardation, microcephaly, cerebellar hypoplasia, and second degree heart block. They also shared distinct facial features and similar appearance of their hands and feet. Childhood-onset insulin-dependent diabetes mellitus developed in one affected child around the age of 9 years. Molecular analysis excluded mutations in potentially related genes such as PTF1A, EIF2AK3, EOMES and WDR62. This condition appears to be unique of other known conditions, suggesting a unique clinical entity of autosomal recessive mode of inheritance.
Microcephaly; insulin-dependent diabetes; cerebellar hypoplasia; mental retardation; heart block
We previously reported the existence of a unique autosomal recessive syndrome consisting of macrocephaly, multiple epiphyseal dysplasia and distinctive facial appearance mapping to chromosome 15q26.
In this manuscript, we have used whole exome sequencing on two affected members of a consanguineous family with this condition and carried out detailed bioinformatics analysis to elucidate the causative mutation.
Our analysis resulted in the identification of a homozygous p.N1060S missense mutation in a highly conserved residue in KIF7, a regulator of Hedgehog signaling that has been recently found to be causing Joubert syndrome, fetal hydrolethalus and acrocallosal syndromes. The phenotype in our patients partially overlaps with the phenotypes associated with those syndromes but they also exhibit some distinctive features including multiple epiphyseal dysplasia.
We report the first missense homozygous disease-causing mutation in KIF7 and expand the clinical spectrum associated with mutations in this gene to include multiple epiphyseal dysplasia. The missense nature of the mutation might account for the unique presentation in our patients.
KIF7; Acrocallosal; Joubert; Sonic hedgehog; Dysmorphism; Multiple epiphyseal dysplasia; Fetal hydrolethalus
Mechanisms of signal transduction regulation remain a fundamental question in a variety of biological processes and diseases. Previous evidence suggests the primary cilium can act as a signaling hub1, but its exact role in many of its described pathways has remained elusive. Here, we investigate the mechanism of cilia regulation of the canonical Wnt pathway through systematic knock-down and knock-out approaches. We found that the primary cilium dampens canonical Wnt signaling through a unique spatial mechanism involving compartmentalization of signaling components. The cilium, through regulated intraflagellar transport (IFT), diverts Jouberin (Jbn), a ciliopathy protein and context specific Wnt pathway regulator2, away from the nucleus and limits β-catenin nuclear entry. This repressive regulation does not silence the pathway, but instead maintains a discrete range of Wnt responsiveness; cells without cilia have potentiated Wnt responses whereas cells with more than one cilium display inhibited responses. Furthermore, we show that this regulation occurs during embryonic development and is disrupted in cancer cell proliferation. Together these data explain a unique spatial mechanism of regulation of Wnt signaling which may provide insight into ciliary regulation of other signaling pathways.
Signaling; cilia; Wnt; β-catenin; Jouberin; Ahi1; Intraflagellar transport; Kif3A; Dnchc2; ciliopathy
The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear1–3. In order to investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome gene4,5. These mice exhibit cerebellar hypoplasia with a vermis/midline fusion defect early in development. This defect is concomitant with expansion of the roof plate and is also evident in a mouse mutant for another Joubert syndrome gene, Cep2906,7. Further, fetal magnetic resonance imaging (MRI) from human subjects with Joubert syndrome reveals a similar midline cleft suggesting parallel pathogenic mechanisms. Previous evidence has suggested a role for Jouberin (Jbn), the protein encoded by Ahi1, in canonical Wnt signaling8. Consistent with this, we found decreased Wnt reporter activity at the site of hemisphere fusion in the developing cerebellum of Ahi1 mutant mice. This decrease was accompanied by reduced proliferation at the site of fusion. Finally, treatment with lithium, a Wnt pathway agonist9, partially rescued this phenotype. Our findings implicate a defect in Wnt signaling in the cerebellar midline phenotype seen in Joubert syndrome, which can be overcome with Wnt stimulation.
Neuroscience; signaling; Wnt; ataxia; cerebellum; cilia; Joubert syndrome; Jouberin; Ahi1
Hereditary spastic paraplegia (HSP) represents a large group of neurological disorders characterized by progressive spasticity of the lower limbs. One subtype of HSP shows an autosomal recessive form of inheritance with this corpus callosum (ARHSP-TCC), and displays genetic heterogeneity with four known loci. We identified a consanguineous Egyptian family with five affected individuals with ARHSP-TCC. We found linkage to the SPG11 locus and identified a novel homozygous p.Q498X stop codon mutation in exon 7 in the SPG11 gene encoding Spatacsin. Cognitive impairment and polyneuropathy, reported as frequent in SPG11, were not evident. This family supports the importance of SPG11 as a frequent cause for ARHSP-TCC, and expands the clinic SPG11 spectrum.
Hereditary spastic paraparesis; Thin corpus callosum; SPG11; Spatacsin; Linkage analysis; Autosomal recessive
Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.
Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.
Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.
The “ciliopathies” are a newly defined group of disorders characterized by defects in the structure or function of the cellular primary cilium. Patients with these disorders display variably expressive fibrocystic renal disease, retinal blindness, polydactyly, obesity, and brain dysgenesis as well as neurocognitive impairments. Joubert syndrome is a ciliopathy defined by cerebellar vermis hypoplasia, oculomotor apraxia, intermittent hyperventilation, and mental retardation. Recent evidence suggests important roles for the primary cilium in mediating a host of extracellular signaling events such as morphogen, mitogen, homeostatic and polarity signals. Based upon the clinical features of ciliopathies and cilia mediated signaling pathways, the data support a role for the primary cilium in modulating neurogenesis, cell polarity, axonal guidance and possibly adult neuronal function.
Ciliopathy; Joubert syndrome; Neuronal function; Primary cilia
Primary cilia are evolutionarily conserved cellular organelles that organize diverse signaling pathways1,2. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities3. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance4. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. PPC was labeled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provide potential target molecules for future study.
Cystic kidney disease represents a major cause of end-stage renal disease, yet the molecular mechanisms of pathogenesis remain largely unclear. Recent emphasis has been placed on a potential role for canonical Wnt signaling, but investigation of this pathway in adult renal homeostasis is lacking. Here we provide evidence of a previously unidentified canonical Wnt activity in adult mammalian kidney homeostasis, the loss of which leads to cystic kidney disease. Loss of the Jouberin (Jbn) protein in mouse leads to the cystic kidney disease nephronophthisis, owing to an unexpected decrease in endogenous Wnt activity. Jbn interacts with and facilitates β-catenin nuclear accumulation, resulting in positive modulation of downstream transcription. Finally, we show that Jbn is required in vivo for a Wnt response to injury and renal tubule repair, the absence of which triggers cystogenesis.
Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease.
Purine; pyrimidine; deaminase; salvage; translation; GTP; de novo synthesis; neurodegeneration
It has been suggested that autism, like other complex genetic disorders, may benefit from the study of rare or Mendelian variants associated with syndromic or non-syndromic forms of the disease. However, there are few examples in which common variation in genes causing a Mendelian neuropsychiatric disorder has been shown to contribute to disease susceptibility in an allied common condition. Joubert syndrome (JS) is a rare recessively inherited disorder, with mutations reported at several loci including the gene Abelson’s Helper Integration 1 (AHI1). A significant proportion of patients with JS, in some studies up to 40%, have been diagnosed with autism spectrum disorder (ASD) and several linkage studies in ASD have nominally implicated the region on 6q where AHI1 resides. To evaluate AHI1 in ASD, we performed a three-stage analysis of AHI1 as an a priori candidate gene for autism. Re-sequencing was first used to screen AHI1, followed by two subsequent association studies, one limited and one covering the gene more completely, in Autism Genetic Resource Exchange (AGRE) families. In stage 3, we found evidence of an associated haplotype in AHI1 with ASD after correction for multiple comparisons, in a region of the gene that had been previously associated with schizophrenia. These data suggest a role for AHI1 in common disorders affecting human cognition and behavior.
Joubert syndrome (JS) is a developmental brain disorder characterized by cerebellar vermis hypoplasia, abnormal eye movement, ataxia and mental retardation. Mutations in CEP290 mutations are responsible for the cerebello–oculo–renal subtype of JS that includes kidney cysts and retinal degeneration, two phenotypes commonly linked to ciliopathies. CEP290 mutations are also associated with Meckel–Gruber syndrome and Bardet–Biedl syndrome (BBS). Here we demonstrate that CEP290 interacts with a centriolar satellite protein PCM-1, which is implicated in BBS4 function. CEP290 binds to PCM-1 and localizes to centriolar satellites in a PCM-1- and microtubule-dependent manner. The depletion of CEP290 disrupts subcellular distribution and protein complex formation of PCM-1. In accord with PCM-1’s role in microtubule organization, CEP290 knockdown causes the disorganization of the cytoplasmic microtubule network. Moreover, we show that both CEP290 and PCM-1 are required for ciliogenesis and are involved in the ciliary targeting of Rab8, a small GTPase shown to collaborate with BBS protein complex to promote ciliogenesis. Our results suggest that PCM-1 is a potential mediator that may link CEP290 with BBS proteins in common molecular pathways.
Joubert Syndrome Related Disorders (JSRDs) are autosomal recessive pleiotropic conditions sharing a peculiar cerebellar and brainstem malformation known as the “molar tooth sign” (MTS). Recently, mutations in a novel ciliary gene, RPGRIP1L, have been shown to cause both JSRDs and Meckel-Gruber syndrome. We searched for RPGRIP1L mutations in 120 patients with proven MTS and phenotypes representative of all JSRD clinical subgroups. Two homozygous mutations, the previously reported p.T615P in exon 15 and the novel c.2268_2269delA in exon 16, were detected in two out of 16 families with cerebello-renal presentation (~12%). Conversely, no pathogenic changes were found in patients with other JSRD phenotypes, suggesting that RPGRIP1L mutations are largely confined to the cerebello-renal subgroup, while they overall represent a rare cause of JSRD (<2%).
RPGRIP1L; Joubert Syndrome Related Disorders; molar tooth sign; nephronophthisis
Subcortical band heterotopia (SBH) is a neuron migration disorder characterized by an aberrant ‘band-like’ accumulation of neurons within the neocortical white matter, frequently leading to mental retardation and epilepsy. SBH can now be regressed by reactivating neuronal migration.