BBS4 protein has basal body/ciliary localization in sensory organs but extra-ciliary localization in oligodendrocytes during human development.

Bardet-Biedl syndrome protein 4 (BBS4) localization has been studied in human embryos/fetuses from Carnegie stage 15 to 37 gestational weeks in neurosensory organs and brain, underlying the major clinical signs of BBS. We observed a correlation between the differentiation of the neurosensory cells (hair cells, photoreceptors, olfactory neurons) and the presence of a punctate BBS4 immunostaining in their apical cytoplasm. In the brain, BBS4 was localized in oligodendrocytes and myelinated tracts. In individual myelinated fibers, BBS4 immunolabelling was discontinuous, predominantly at the periphery of the myelin sheath. BBS4 immunolabelling was confirmed in postnatal developing white matter tracts in mouse as well as in mouse oligodendrocytes cultures. In neuroblasts/neurons, BBS4 was only present in reelin-expressing Cajal-Retzius cells. Our results show that BBS4, a protein of the BBSome, has both basal body/ciliary localization in neurosensory organs but extra-ciliary localization in oligodendrocytes. The presence of BBS4 in developing oligodendrocytes and myelin described in the present paper might attribute a new role to this protein, requiring further investigation in the field of myelin formation.

Deciphering the causes of sporadic late-onset cerebellar ataxias: a prospective study with implications for diagnostic work.

The management of sporadic late-onset cerebellar ataxias represents a very heterogeneous group of patients and remains a challenge for neurologist in clinical practice. We aimed at describing the different causes of sporadic late-onset cerebellar ataxias that were diagnosed following standardized, exhaustive investigations and the population characteristics according to the aetiologies as well as at evaluating the relevance of these investigations. All patients consecutively referred to our centre due to sporadic, progressive cerebellar ataxia occurring after 40 years of age were included in the prospective, observational study. 80 patients were included over a 2 year period. A diagnosis was established for 52 patients (65%) corresponding to 18 distinct causes, the most frequent being cerebellar variant of multiple system atrophy (n = 29). The second most frequent cause was inherited diseases (including spinocerebellar ataxias, late-onset Friedreich's disease, SLC20A2 mutations, FXTAS, MELAS, and other mitochondrial diseases) (n = 9), followed by immune-mediated or other acquired causes. The group of patient without diagnosis showed a slower worsening of ataxia (p < 0.05) than patients with multiple system atrophy. Patients with later age at onset experienced faster progression of ataxia (p = 0.001) and more frequently parkinsonism (p < 0.05) than patients with earlier onset. Brain MRI, DaT scan, genetic analysis and to some extent muscle biopsy, thoracic-abdominal-pelvic tomodensitometry, and cerebrospinal fluid analysis were the most relevant investigations to explore sporadic late-onset cerebellar ataxia. Sporadic late-onset cerebellar ataxias should be exhaustively investigated to identify the underlying causes that are numerous, including inherited causes, but dominated by multiple system atrophy.

Xq28 duplication including MECP2 in six unreported affected females: what can we learn for diagnosis and genetic counselling?

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

Hereditary systemic angiopathy (HSA) with cerebral calcifications, retinopathy, progressive nephropathy, and hepatopathy.

Several hereditary conditions affecting cerebral, retinal and systemic microvessels have recently been described. They include CADASIL, CRV, and HERNS. We here report on a variant form of a hereditary systemic angiopathy (HSA) affecting two generations of a Caucasian family. Clinical symptoms of HSA appear in the mid-forties and are characterized by visual impairment, migraine-like headache, skin rash, epileptic seizures, progressive motor paresis and cognitive decline. Late symptoms include hepatic and renal failure. Retinal capillary microaneurysms and arteriolar tortuosity are associated with marked optic disc atrophy. Radiological hallmarks consist of multiple cerebral calcifications and tumor-like subcortical white matter lesions. Brain, peripheral nerve, muscle, kidney and colon biopsies have revealed a multi organ small vessel involvement with partly altered endothelium, perivascular inflammation and thrombotic microangiopathy. No curative therapeutic options are known for hereditary cerebral vasculopathies. The use of cyclophosphamide, azathioprine and methotrexate was of no benefit in our cases of HSA. Early diagnosis of hereditary systemic angiopathies is important in order to prevent patients from repetitive invasive diagnostic measures and to avoid the use of inappropriate and potentially harmful drugs.

Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study.

Oral-facial-digital syndrome type 1 (OFD1) is characterised by an X linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, tooth, and distal abnormalities, polycystic kidney disease, and central nervous system malformations. Large interfamilial and intrafamilial clinical variability has been widely reported, and 18 distinct mutations have been previously reported within OFD1. A French and Belgian collaborative study collected 25 cases from 16 families. OFD1 was analysed using direct sequencing and phenotype-genotype correlation was performed using chi2 test. X inactivation studies were performed on blood lymphocytes. In 11 families, 11 novel mutations, including nine frameshift, one nonsense, and one missense mutation were identified, which spanned nine different exons. A combination of our results with previously reported cases showed that the majority of mutations (65.5%) was located in exons 3, 8, 9, 13, and 16. There was phenotype-genotype correlation between (a) polycystic kidney disease and splice mutations; (b) mental retardation and mutations located in exons 3, 8, 9, 13, and 16; and (c) tooth abnormalities and mutations located in coiled coil domains. Comparing the phenotype of the families with a pathogenic mutation to families with absence of OFD1 mutation, polycystic kidneys and short stature were significantly more frequent in the group with no OFD1 mutation, whereas lingual hamartomas were significantly more frequent in the group with OFD1 mutation. Finally, an X inactivation study showed non-random X inactivation in a third of the samples. Differential X inactivation between mothers and daughters in two families with high intrafamilial variability was of particular interest. Slight phenotype-genotype correlations were established, and X inactivation study showed that skewed X inactivation could be partially involved in the pathogenesis of intrafamilial clinical variability.

SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types.

Accumulation of expanded polyglutamine proteins and selective pattern of neuronal loss are hallmarks of at least eight neurodegenerative disorders, including spinocerebellar ataxia type 7 (SCA7). We previously described SCA7 mice displaying neurodegeneration with progressive ataxin-7 accumulation in two cell types affected in the human pathology. We describe here a new transgenic model with a more widespread expression of mutant ataxin-7, including neuronal cell types unaffected in SCA7. In these mice a similar handling of mutant ataxin-7, including a cytoplasm to nucleus translocation and accumulation of N-terminal fragments, was observed in all neuronal populations studied. An extensive screen for chaperones, proteasomal subunits and transcription factors sequestered in nuclear inclusions (NIs) disclosed no pattern unique to neurons undergoing degeneration in SCA7. In particular, we found that the mouse TAF(II)30 subunit of the TFIID initiation complex is markedly accumulated in NIs, even though this protein does not contain a polyglutamine stretch. A striking discrepancy between mRNA and ataxin-7 levels in transgenic mice expressing the wild-type protein but not in those expressing the mutant one, indicates a selective stabilization of mutant ataxin-7, both in this model and the P7E/N model described previously. These mice therefore provide in vivo evidence that the polyglutamine expansion mutation can stabilize its target protein.

Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.

The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntington's disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.

Differential distribution of the normal and mutated forms of huntingtin in the human brain.

Huntington's disease is an inherited disorder caused by expansion of a CAG trinucleotide repeat in the IT15 gene, which leads to expansion of a polyglutamine tract within the protein called huntingtin. Despite the characterization of the IT15 gene and the mutation involved in the disease, the normal function of huntingtin and the effects of the mutation on its function and on its neuronal location remain unknown. To study whether mutated huntingtin has the same neuronal distribution and intracellular location as normal huntingtin, we analyzed immunohistochemically both forms of this protein in the brain of 5 controls and 5 patients with Huntington's disease. We show that the distribution of mutated huntingtin is, like that of the normal form, heterogeneous throughout the brain, but is not limited to vulnerable neurons in Huntington's disease, supporting the hypothesis that the presence of the mutated huntingtin in a neuron is not in itself sufficient to lead to neuronal death. Moreover, whereas normal huntingtin is detected in some neuronal perikarya, nerve fibers, and nerve endings, the mutated form is observed in some neuronal perikarya and proximal nerve processes but is not detectable in nerve endings. Our results suggest that the expression or processing of the mutated huntingtin in perikarya and nerve endings differs quantitatively or qualitatively from the expression of the normal form in the same neuronal compartments.

SH3 domain-dependent association of huntingtin with epidermal growth factor receptor signaling complexes.

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.

Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.

Two forms of the neurodegenerative disorder spinocerebellar ataxia are known to be caused by the expansion of a CAG (polyglutamine) trinucleotide repeat. By screening cDNA expression libraries, using an antibody specific for polyglutamine repeats, we identified six novel genes containing CAG stretches. One of them is mutated in patients with spinocerebellar ataxia linked to chromosome 12q (SCA2). This gene shows ubiquitous expression and encodes a protein of unknown function. Normal SCA2 alleles (17 to 29 CAG repeats) contain one to three CAAs in the repeat. Mutated alleles (37 to 50 repeats) appear particularly unstable, upon both paternal and maternal transmissions. The sequence of three of them revealed pure CAG stretches. The steep inverse correlation between age of onset and CAG number suggests a higher sensitivity to polyglutamine length than in the other polyglutamine expansion diseases.

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms.

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.

Polyglutamine expansion as a pathological epitope in Huntington's disease and four dominant cerebellar ataxias.

A polyglutamine expansion (encoded by a CAG repeat) in specific proteins causes neurodegeneration in Huntington's disease (HD) and four other disorders, by an unknown mechanism thought to involve gain of function or toxicity of the mutated protein. The pathological threshold is 37-40 glutamines in three of these diseases, whereas the corresponding normal proteins contain polymorphic repeats of up to about 35 glutamines. The age of onset of clinical manifestations is inversely correlated to the length of the polyglutamine expansion. Here we report the characterization of a monoclonal antibody that selectively recognizes polyglutamine expansion in the proteins implicated in HD and in spinocerebellar ataxia (SCA) 1 and 3. The intensity of signal depends on the length of the polyglutamine expansion, and the antibody also detects specific pathological proteins expected to contain such expansion, in SCA2 and in autosomal dominant cerebellar ataxia with retinal degeneration, whose genes have not yet been identified.

A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture.

The fragile X syndrome is an X-linked inherited disease and is the result of transcriptional inactivation of the FMR1 gene and the absence of its encoded FMR protein (FMRP). Using a specific monoclonal antibody directed against human FMRP, we have studied the steady-state levels of its murine homolog in several tissues and organs of adult and young mice. In immunoblot analyses, the antibody recognizes a heterogeneous subset of proteins with apparent molecular weights ranging from 80 to 70 kDa. These proteins are detected in all the 27 tissues tested; however, the relative proportion of each polypeptide recognized varies between tissues, and a significantly higher expression is observed in young animals. Northern blot analysis of RNA extracted from selected tissues from adult mouse shows that these tissues express the major 4.8 kb mRNA, although at different levels, and contain several additional shorter transcripts, particularly in muscular tissues. We also report that expression of the FMR1 gene is modulated in proliferating and quiescent primary mouse kidney cell cultures with an inverse relationship between levels of FMR1 mRNA and of its encoded proteins. This suggests that FMRPs are highly stable in quiescent cells and that FMR1 expression is likely post-transcriptionally controlled. Our results document the widespread expression of the FMR1 gene, and suggest that it is controlled by different mechanisms implicated in cell growth and differentiation.

Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form.

Huntington's disease (HD) results from the expansion of a polyglutamine encoding CAG repeat in a gene of unknown function. The wide expression of this transcript does not correlate with the pattern of neuropathology in HD. To study the HD gene product (huntingtin), we have developed monoclonal antibodies raised against four different regions of the protein. On western blots, these monoclonals detect the approximately 350 kD huntingtin protein in various human cell lines and in neural and non-neural rodent tissues. In cell lines from HD patients, a doublet protein is detected corresponding to the mutated and normal huntingtin. Immunohistochemical studies in the human brain using two of these antibodies detects the huntingtin in perikarya of some neurons, neuropiles, varicosities and as punctate staining likely to be nerve endings.

Rapid antibody test for fragile X syndrome.

Fragile X syndrome is the most common known cause of inherited mental retardation. Identification of patients and carriers of fragile X syndrome is usually done with a DNA test system but we have developed a rapid antibody to identify fragile X patients. This non-invasive test requires only 1 or 2 drops of blood and can be used for screening large groups of mentally retarded people and neonates for fragile X syndrome.

Quantitative comparison of FMR1 gene expression in normal and premutation alleles.

We report studies on FMR1 gene expression in cells derived from male premutation carriers. Transcription of FMR1 genes with CGG-repeat lengths within the premutation range was demonstrated to be normal. Repeat lengths are faithfully transcribed into FMR1 mRNAs, which have steady-state levels, as measured by RNase protection, similar to those of normal cells. Premutation transcripts also are shown to have normal turnover, with the FMR1 mRNA half-life estimated to be 12 h. Measurement of FMR1 protein was also found to be in similar abundance in normal and premutation cell lines. These data support the nonpenetrant status of premutation carriers of fragile X syndrome and suggest that the occasional case reports to the contrary may reflect either other causes, including low-level mosaicism for larger, methylated FMR1 alleles, or simply coincidence.