Alessia Micalizzi

Joubert syndrome: congenital cerebellar ataxia with the molar tooth

Joubert syndrome is a congenital cerebellar ataxia with autosomal recessive or X-linked inheritance, the diagnostic hallmark of which is a unique cerebellar and brainstem malformation recognisable on brain imaging-the so-called molar tooth sign. Neurological signs are present from the neonatal period and include hypotonia progressing to ataxia, global developmental delay, ocular motor apraxia, and breathing dysregulation. These signs are variably associated with multiorgan involvement, mainly of the retina, kidneys, skeleton, and liver. 21 causative genes have been identified so far, all of which encode for proteins of the primary cilium or its apparatus. The primary cilium is a subcellular organelle that has key roles in development and in many cellular functions, making Joubert syndrome part of the expanding family of ciliopathies. Notable clinical and genetic overlap exists between distinct ciliopathies, which can co-occur even within families. Such variability is probably explained by an oligogenic model of inheritance, in which the interplay of mutations, rare variants, and polymorphisms at distinct loci modulate the expressivity of the ciliary phenotype.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice‐site mutation in PDE6D, encoding a prenyl‐binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild‐type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl‐dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP‐bound ARL3, which acts as a cargo‐release factor for PDE6D‐bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl‐binding‐dependent trafficking in ciliopathies.

Mutations in B9D1 and MKS1 cause mild Joubert syndrome: expanding the genetic overlap with the lethal ciliopathy Meckel syndrome

Joubert syndrome is a clinically and genetically heterogeneous ciliopathy characterized by a typical cerebellar and brainstem malformation (the “molar tooth sign”), and variable multiorgan involvement. To date, 24 genes have been found mutated in Joubert syndrome, of which 13 also cause Meckel syndrome, a lethal ciliopathy with kidney, liver and skeletal involvement. Here we describe four patients with mild Joubert phenotypes who carry pathogenic mutations in either MKS1 or B9D1, two genes previously implicated only in Meckel syndrome.

Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes

Background Ciliopathies are an extensive group of autosomal recessive or X-linked disorders with considerable genetic and clinical overlap, which collectively share multiple organ involvement and may result in lethal or viable phenotypes. In large numbers of cases the genetic defect remains yet to be determined. The aim of this study is to describe the mutational frequency and phenotypic spectrum of the CEP120 gene. Methods Exome sequencing was performed in 145 patients with Joubert syndrome (JS), including 15 children with oral-facial-digital syndrome type VI (OFDVI) and 21 Meckel syndrome (MKS) fetuses. Moreover, exome sequencing was performed in one fetus with tectocerebellar dysraphia with occipital encephalocele (TCDOE), molar tooth sign and additional skeletal abnormalities. As a parallel study, 346 probands with a phenotype consistent with JS or related ciliopathies underwent next-generation sequencing-based targeted sequencing of 120 previously described and candidate ciliopathy genes. Results We present six probands carrying nine distinct mutations (of which eight are novel) in the CEP120 gene, previously found mutated only in Jeune asphyxiating thoracic dystrophy (JATD). The CEP120-associated phenotype ranges from mild classical JS in four patients to more severe conditions in two fetuses, with overlapping features of distinct ciliopathies that include TCDOE, MKS, JATD and OFD syndromes. No obvious correlation is evident between the type or location of identified mutations and the ciliopathy phenotype. Conclusion Our findings broaden the spectrum of phenotypes caused by CEP120 mutations that account for nearly 1% of patients with JS as well as for more complex ciliopathy phenotypes. The lack of clear genotype–phenotype correlation highlights the relevance of comprehensive genetic analyses in the diagnostics of ciliopathies.

Infantile and childhood onset PLA2G6-associated neurodegeneration in a large North African cohort.

Mutations in the PLA2G6 gene are causative of PLA2G6‐associated neurodegeneration (PLAN), a spectrum of neurodegenerative conditions including infantile, childhood and adult onset forms.

A rare case of cerebellar agenesis: a probabilistic Constrained Spherical Deconvolution tractographic study

Aim of this study is to show the potential of probabilistic tractographic techniques, based on the Constrained Spherical Deconvolution (CSD) algorithms, in recognizing white matter fiber bundle anomalies in patients with complex cerebral malformations, such as cerebellar agenesis. The morphological and tractographic study of a 17-year-old male patient affected by cerebellar agenesis was performed by using a 3Tesla MRI scanner. Genetic and neuropsychological tests were carried out. An MRI morphological study showed the absence of both cerebellar hemispheres and the flattening of the anterior side of the pons. Moreover, it showed a severe vermian hypoplasia with a minimal vermian residual. The study recognized two thin cerebellar remnants, medially in contact with the small vermian residual, at the pontine level. The third ventricle, morphologically normal, communicated with a permagna cerebello-medullary cistern. Probabilistic CSD tractography identified some abnormal and aberrant infratentorial tracts, symmetrical on both sides. In particular, the transverse pontine fibers were absent and the following tracts with aberrant trajectories have been identified: “cerebello-thalamic” tracts; “fronto-cerebellar” tracts; and ipsilateral and contralateral “spino-cerebellar” tracts. Abnormal tracts connecting the two thin cerebellar remnants have also been detected. There were no visible alterations in the main supratentorial tracts in either side. Neuropsychiatric evaluation showed moderate cognitive-motor impairment with discrete adaptive compensation. Probabilistic CSD tractography is a promising technique that overcome reconstruction biases of other diffusion tensor-based approaches and allowed us to recognize, in a patient with cerebellar agenesis, abnormal tracts and aberrant trajectories of normally existing tracts.

Clinical, neuroradiological and molecular characterization of cerebellar dysplasia with cysts (Poretti-Boltshauser syndrome).

Cerebellar dysplasia with cysts and abnormal shape of the fourth ventricle, in the absence of significant supratentorial anomalies and of muscular involvement, defines recessively inherited Poretti–Boltshauser syndrome (PBS). Clinical features comprise non-progressive cerebellar ataxia, intellectual disability of variable degree, language impairment, ocular motor apraxia and frequent occurrence of myopia or retinopathy. Recently, loss-of-function variants in the LAMA1 gene were identified in six probands with PBS. Here we report the detailed clinical, neuroimaging and genetic characterization of 18 PBS patients from 15 unrelated families. Biallelic LAMA1 variants were identified in 14 families (93%). The only non-mutated proband presented atypical clinical and neuroimaging features, challenging the diagnosis of PBS. Sixteen distinct variants were identified, which were all novel. In particular, the frameshift variant c.[2935delA] recurred in six unrelated families on a shared haplotype, suggesting a founder effect. No LAMA1 variants could be detected in 27 probands with different cerebellar dysplasias or non-progressive cerebellar ataxia, confirming the strong correlate between LAMA1 variants and PBS.

A novel PMCA3 mutation in an ataxic patient with hypomorphic phosphomannomutase 2 (PMM2) heterozygote mutations: Biochemical characterization of the pump defect

The neuron-restricted isoform 3 of the plasma membrane Ca2+ ATPase plays a major role in the regulation of Ca2+ homeostasis in the brain, where the precise control of Ca2+ signaling is a necessity. Several function-affecting genetic mutations in the PMCA3 pump associated to X-linked congenital cerebellar ataxias have indeed been described. Interestingly, the presence of co-occurring mutations in additional genes suggest their synergistic action in generating the neurological phenotype as digenic modulators of the role of PMCA3 in the pathologies. Here we report a novel PMCA3 mutation (G733R substitution) in the catalytic P-domain of the pump in a patient affected by non-progressive ataxia, muscular hypotonia, dysmetria and nystagmus. Biochemical studies of the pump have revealed impaired ability to control cellular Ca2+ handling both under basal and under stimulated conditions. A combined analysis by homology modeling and molecular dynamics have revealed a role for the mutated residue in maintaining the correct 3D configuration of the local structure of the pump. Mutation analysis in the patient has revealed two additional function-impairing compound heterozygous missense mutations (R123Q and G214S substitution) in phosphomannomutase 2 (PMM2), a protein that catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. These mutations are known to be associated with Type Ia congenital disorder of glycosylation (PMM2-CDG), the most common group of disorders of N-glycosylation. The findings highlight the association of PMCA3 mutations to cerebellar ataxia and strengthen the possibility that PMCAs act as digenic modulators in Ca2+-linked pathologies.

Between SCA5 and SCAR14: delineation of the SPTBN2 p.R480W-associated phenotype

We read with interest the article by Elsayed et al. [1] describing a family with autosomal recessive congenital ataxia due to a homozygous 5-bp deletion in the β3-spectrin gene (SPTBN2). In-frame heterozygous variants of this gene had previously been identified as the cause of autosomal dominant adult-onset SCA5 (MIM#600224) (Suppl.Tab.1) [2–4]. The authors postulated the existence of SPTBN2 genotype-phenotype correlates, suggesting that loss of function mutations would act recessively, producing a severe congenital ataxic phenotype associated with cognitive impairment and variable additional neurological signs. This hypothesis was partially supported by three subsequent studies, reporting homozygous missense, nonsense and splicing SPTBN2 variants, all resulting in an analogous congenital ataxia syndrome, defined as SCAR14 (MIM#615386) (Suppl.Tab.1) [5–7]. Elsayed and colleagues [1] also mentioned a previously reported patient affected by a severe infantile-onset cerebellar ataxia with developmental delay, carrying the heterozygous missense variant c.1438C>T (p.R480W) in the SPTBN2 gene [8]. Based on previous literature data on heterozygous SPTBN2 mutation carriers (presenting the typical SCA5 phenotype of adult-onset, slowly progressive pure cerebellar ataxia), they speculated that either a second-site SCA5 modifier or an undetected SPTBN2 variant in trans (e.g., deep intronic or in a non-coding regulatory region) should contribute to the phenotypic manifestation. In this regard, we would like to report an additional case of congenital severe cerebellar ataxia and intellectual impairment carrying the same SPTBN2 p.R480W variant. This is a 2-year-old girl, the third child of healthy nonconsanguineous parents. She was born by cesarean section after an uncomplicated full-term pregnancy and was referred to the neuropsychiatric clinic because of generalized hypotonia, global developmental delay, and alternating esotropia. At age 12 months, she said her first words and got head control, but could not sit without support. Two months later, her developmental quotient was calculated to be 56. She progressively developed a cerebellar syndrome with gait ataxia and dysarthria. Brain MRI performed at age 1 year 10 months showed global cerebellar hypoplasia with enlarged interfolial spaces, in the absence of any brainstem or supratentorial abnormalities (Suppl.Fig.1A). Molecular analysis consisted of a next-generation sequencing (NGS) panel of 50 genes causative of different forms of nonprogressive cerebellar ataxia (with the exclusion of Joubert Syndrome genes), using TruSeq Custom Amplicon (TSCA) technology on a MiSeq platform (Illumina, San Diego, CA, USA). As in the case reported by Jacob et al. [8], this child was heterozygous for SPTBN2 p.R480W missense variant (NM_006946). This variant was not detected in either of the unaffected parents, suggesting de novo occurrence (Suppl. Fig.1B). Potential heterozygous deletions involving one or more exons were ruled out using a custom script tool aimed at detecting significant differences in read depth from NGS data. The p.R480W variant (rs397514749) is not present in population databases, affects a highly conserved amino acid, and multiple in silico tools consistently predict it as Sara Nuovo and Alessia Micalizzi contributed equally to this work.

Erratum to: Tubulin-related cerebellar dysplasia: definition of a distinct pattern of cerebellar malformation

Romina Romaniello & Filippo Arrigoni & Elena Panzeri & Andrea Poretti & Alessia Micalizzi & Andrea Citterio & Maria Francesca Bedeschi & Angela Berardinelli & Margherita Cusmai & Stefano D’Arrigo & Alessandro Ferraris & Annette Hackenberg & Alma Kuechler & Margherita Mancardi & Sara Nuovo & Barbara Oehl-Jaschkowitz & Andrea Rossi & Sabrina Signorini & Frank Tüttelmann & Dagmar Wahl & Ute Hehr & Eugen Boltshauser & Maria Teresa Bassi & Enza Maria Valente & Renato Borgatti