Clinical and neurogenetic characterisation of autosomal recessive RBL2-associated progressive neurodevelopmental disorder.

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.

Autoimmunity to heterogeneous nuclear ribonucleoprotein A1 in psoriatic patients and correlation with disease severity.

BACKGROUND AND OBJECTIVES: The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been postulated as an autoantigen of psoriasis, but correlation between serum levels of anti-hnRNP-A1 autoantibodies and the severity of disease has not been investigated. We aimed to assess the frequency of anti-hnRNP-A1 autoimmunity in patients with moderate to severe psoriasis and in healthy controls, and to determine the correlation between serum levels of anti-hnRNP-A1 autoantibodies and disease severity. PATIENTS AND METHODS: We performed a case-control study on 40 adult psoriatic patients with a PASI (Psoriasis Area and Severity Index) of > 10 and 40 healthy controls matched for age and gender. Immunoblotting was used to assess serum levels of anti-hnRNP-A1 autoantibodies. RESULTS: Anti-hnRNP-A1 autoantibodies were found in 9/40 psoriatic patients (22.5 %) but in no healthy controls. The PASI was significantly higher in anti-hnRNP-A1-positive patients than in anti-hnRNP-A1-negative patients (40.33 ± 3.24 vs 26.06 ± 9.28, p = 0.0001). In patients positive for anti-hnRNP-A1, serum levels of such autoanti-bodies were correlated with the PASI (R = 0.89, p = 0.001). CONCLUSIONS: Consistent with reports in the literature, our results suggest a role of anti-hnRNP-A1 autoimmunity in psoriasis, although probably not as the primary cause or initial/fundamental event. Unlike previously published reports, our results also suggest that anti-hnRNP-A1 autoimmunity is particularly frequent among psoriatic patients with more severe disease. Further studies are necessary with a larger number of patients.

Autoimmunität gegen heterogenes nukleäres Ribonukleoprotein A1 bei Psoriasispatienten und Korrelation mit dem Schweregrad der Erkrankung.

HINTERGRUND UND ZIELE: Das heterogene nukleäre Ribonukleoprotein A1 (hnRNP-A1) wurde als Autoantigen bei Psoriasis vorgeschlagen; eine mögliche Korrelation zwischen dem Serumspiegel von Anti-hnRNP-A1-Antikörpern und dem Schweregrad der Erkrankung wurde bislang nicht untersucht. Unser Ziel war es, die Häufigkeit der Anti-hnRNP-A1-Autoimmunität bei Patienten mit mäßig schwerer bis schwerer Psoriasis und gesunden Kontrollpersonen zu bestimmen und festzustellen, ob eine Korrelation zwischen dem Anti-hnRNP-A1-Autoantikörper-Serumspiegel und dem Schweregrad der Erkrankung besteht. PATIENTEN UND METHODEN: Wir führten eine Fallkontrollstudie an 40 erwachsenen Psoriasispatienten mit einem Psoriasis Area and Severity Index (PASI) von > 10 und 40 gesunden Kontrollpersonen mit ähnlicher Alters- und Geschlechtsverteilung durch. Die Bestimmung des Serumspiegels von hnRNP-A1-Autoantikörpern erfolgte mit Immunoblots. ERGEBNISSE: Anti-hnRNP-A1-Autoantikörper wurden bei 9 von 40 Psoriasispatienten (22,5 %) nachgewiesen, jedoch bei keiner der gesunden Kontrollpersonen gefunden. Der PASI-Wert war bei Anti-hnRNP-A1-positiven Patienten signifikant höher als bei Anti-hnRNP-A1-negativen Patienten (40,33 ± 3,24 vs. 26,06 ± 9,28, p  =  0,0001). Bei Anti-hnRNP-A1-positiven Patienten korrelierte der Serumspiegel der Autoantikörper mit dem PASI-Wert (R = 0,89, p = 0,001). SCHLUSSFOLGERUNGEN: Übereinstimmend mit Berichten aus der Literatur legen unsere Ergebnisse nahe, dass Anti-hnRNP-A1-Autoimmunität bei Psoriasis eine Rolle spielt, wenn auch möglicherweise nicht als primäre Ursache oder als initiales oder grundlegendes Ereignis. Anders als bei früheren Publikationen weisen unsere Daten auch darauf hin, dass Autoimmunität gegen Anti-hnRNP-A1 unter Patienten mit schwererer Erkrankung besonders häufig ist. Weitere Studien mit einer größeren Anzahl von Patienten sind erforderlich.

Meningiomas and Proteomics: Focus on New Potential Biomarkers and Molecular Pathways.

Meningiomas are one of the most common tumors affecting the central nervous system, exhibiting a great heterogeneity in grading, treatment and molecular background. This article provides an overview of the current literature regarding the molecular aspect of meningiomas. Analysis of potential biomarkers in serum, cerebrospinal fluid (CSF) and pathological tissues was reported. Applying bioinformatic methods and matching the common proteic profile, arising from different biological samples, we highlighted the role of nine proteins, particularly related to tumorigenesis and grading of meningiomas: serpin peptidase inhibitor alpha 1, ceruloplasmin, hemopexin, albumin, C3, apolipoprotein, haptoglobin, amyloid-P-component serum and alpha-1-beta-glycoprotein. These proteins and their associated pathways, including complement and coagulation cascades, plasma lipoprotein particle remodeling and lipid metabolism could be considered possible diagnostic, prognostic biomarkers, and eventually therapeutic targets. Further investigations are needed to better characterize the role of these proteins and pathways in meningiomas. The role of new therapeutic strategies are also discussed.

Prognostic value of HMGB1 and oxidative stress markers in multiple trauma patients: A single-centre prospective study.

Serious multiple traumatic injuries may rapidly become fatal or be complicated by a life-threatening sequelae leading to a significant increase of the mortality rate. Trauma scoring systems are used to evaluate the critical status of the patient and recently many different biomarkers have been taken into account to better estimate the potential clinical outcome. The aim of the present study is to analyse the expression pattern of high-mobility group box-1 (HMGB1), oxidative stress markers and nuclear factor erythroid 2-related (Nrf2) in critically ill traumatic patients (at hospital admittance and after 6 and 24 h), in order to find out their potential role as early post-traumatic predictors markers. Forty-seven patients admitted for multiple trauma and 15 healthy participants were prospectively recruited. Eight patients (17%) died within 92 h of admission; this subgroup of patients presented the highest severity scores and their HMGB1 expression levels were significantly correlated with ISS, whereas patients with higher ISS exhibited higher levels of HMGB1 (P <0.001). Our study suggests the role of HMGB1 as a predictive biomarker of outcome in injured patients and hypothesizes the protective role of Nrf2 in bringing down the oxidative stress and HMGB1 release; measuring HMGB1 in combination with Nrf2 might represent a potentially useful tool in the early detection of post-trauma complications.

Type I hyperprolinemia and proline dehydrogenase (PRODH) mutations in four Italian children with epilepsy and mental retardation.

Type I hyperprolinemia (HPI) is an autosomal recessive disorder caused by proline oxidase deficiency. This enzyme is encoded by the proline dehydrogenase (PRODH) gene on 22q11. The functional consequences of different PRODH mutations on proline oxidase activity have been characterized in vitro. Few patients with HPI with epilepsy and cognitive/behavioral disturbances have been described so far. We screened four Italian children with HPI presenting epilepsy, mental retardation, and behavioral disorders for PRODH gene mutations, and attempted a genotype-phenotype correlation.

Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients.

Limb girdle muscular dystrophies (LGMD) are characterized by genetic and clinical heterogeneity: seven autosomal dominant and 12 autosomal recessive loci have so far been identified. Aims of this study were to evaluate the relative proportion of the different types of LGMD in 181 predominantly Italian LGMD patients (representing 155 independent families), to describe the clinical pattern of the different forms, and to identify possible correlations between genotype, phenotype, and protein expression levels, as prognostic factors. Based on protein data, the majority of probands (n=72) presented calpain-3 deficiency; other defects were as follows: dysferlin (n=31), sarcoglycans (n=32), alpha-dystroglycan (n=4), and caveolin-3 (n=2). Genetic analysis identified 111 different mutations, including 47 novel ones. LGMD relative frequency was as follows: LGMD1C (caveolin-3) 1.3%; LGMD2A (calpain-3) 28.4%; LGMD2B (dysferlin) 18.7%; LGMD2C (gamma-sarcoglycan) 4.5%; LGMD2D (alpha-sarcoglycan) 8.4%; LGMD2E (beta-sarcoglycan) 4.5%; LGMD2F (delta-sarcoglycan) 0.7%; LGMD2I (Fukutin-related protein) 6.4%; and undetermined 27.1%. Compared to Northern European populations, Italian patients are less likely to be affected with LGMD2I. The order of decreasing clinical severity was: sarcoglycanopathy, calpainopathy, dysferlinopathy, and caveolinopathy. LGMD2I patients showed both infantile noncongenital and mild late-onset presentations. Age at disease onset correlated with variability of genotype and protein levels in LGMD2B. Truncating mutations determined earlier onset than missense substitutions (20+/-5.1 years vs. 36.7+/-11.1 years; P=0.0037). Similarly, dysferlin absence was associated with an earlier onset when compared to partial deficiency (20.2+/-standard deviation [SD] 5.2 years vs. 28.4+/-SD 11.2 years; P=0.014).

Identification of the infant-type R631C mutation in patients with the benign muscular form of CPT2 deficiency.

Carnitine palmitoyltransferase 2 (CPT2) deficiency is the most common defect of mitochondrial fatty acid oxidation; three different clinical phenotypes have been described but the adult form, involving exclusively the skeletal muscle, is the most frequent. We describe herein 3 families where 4 individuals manifested with the adult form of CPT2 deficiency. CPT2 gene molecular analysis identified the homozygous R631C mutation, so far only reported in severe infantile cases. Our data evidenced that R631C mutation is not exclusively detected in the infantile form but it may be present in a wider spectrum of CPT2 phenotypes. These findings indirectly suggest that other modulators may influence clinical severity of CPT2 deficiency.

Immunohistochemical analysis of human skeletal muscle AMP deaminase deficiency. Evidence of a correlation between the muscle HPRG content and the level of the residual AMP deaminase activity.

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.

Nuclear factor-kappab activation is associated with glutamate-evoked tissue transglutaminase up-regulation in primary astrocyte cultures.

We have previously demonstrated that alterations of cell redox state, evoked by glutamate, are associated with tissue transglutaminase increases in primary astrocyte cultures. Furthermore, glutamate exposure activated the nuclear factor (NF)-kappaB pathway, and its effects were significantly reduced by antioxidants. Here, we investigated the possible involvement of activated NF-kappaB pathway in glutamate-evoked tissue transglutaminase up-regulation in primary astrocytes. The presence of DNA binding activity by NF-kappaB in nuclear extracts of astrocytes, treated for 24 hr with glutamate (500 microM) or untreated, was assessed by EMSA, using an oligonucleotide probe containing the NF-kappaB consensus sequence present in the tissue transglutaminase promoter. Supershifting with monoclonal antibodies revealed that activated NF-kappaB dimer complexes were composed of p50 and p65 subunits. Interestingly, the specific NF-kappaB inhibitor SN50 (but not its inactive analogue SN50M), when added to cell cultures 30 min prior to glutamate treatment, was able gradually to reduce glutamate-induced NF-kappaB activation. Western blot analysis confirmed the reduction of the p50 amount in nuclear extracts. Notably, the preincubation with SN50 also diminished glutamate-increased tissue transglutaminase expression, as showed by both RT-PCR and Western blotting. Competition experiments, carried out with an excess of a probe containing the NF-kappaB consensus sequence present in the kappa-light-chain promoter, demonstrated a preferential binding of the tissue transglutaminase specific NF-kappaB probe in the nuclear extracts of glutamate-treated astrocytes compared with untreated astrocytes. These preliminary data suggest that NF-kappaB activation, which has been demonstrated to be involved in astrocyte response to glutamate, could also be associated with the molecular pathway leading to glutamate-evoked tissue transglutaminase up-regulation.

Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies.

Mutations in the DYSF gene underlie two main muscle diseases: Limb Girdle Muscular Dystrophy (LGMD) 2B and Miyoshi myopathy (MM). Dysferlin is involved in muscle membrane-repair and is thought to interact with other dysferlin molecules and annexins A1 and A2 at the sarcolemma. We performed genotype/phenotype correlations in a large cohort of dysferlinopathic patients and explored the possible role of annexins as modifier factors in LGMD-2B and MM. In particular, clinical examination, expression of sarcolemmal proteins and genetic analysis were performed on 27 dysferlinopathic subjects. Expression of A1 and A2 annexins was investigated in LGMD-2B/MM subjects and in patients with other muscle disorders. We identified 24 different DYSF mutations, 10 of them being novel. We observed no clear correlation between mutation type and clinical phenotype, but MM patients were found to display muscle symptoms significantly earlier in life than LGMD subjects. Remarkably, dysferlinopathic patients and subjects suffering from other muscular disorders expressed higher levels of both annexins compared to controls; a significant correlation was observed between annexin expression levels and clinical severity scores. Also, annexin amounts paralleled the degree of muscle histopathologic changes. In conclusion, our data indicate that the pathogenesis of different inherited and acquired muscle disorders involves annexin overexpression, probably because these proteins actively participate in the plasmalemma repair process. The positive correlation between annexin A1 and A2 and clinical severity, as well as muscle histopathology, suggests that their level may be a prognostic indicator of disease.

Asymptomatic hyperCKemia in a case of Danon disease due to a missense mutation in Lamp-2 gene.

Primary lysosome-associated membrane protein-2 (LAMP-2) deficiency is an X-linked disease, characterized by the clinical triad of cardiomyopathy, vacuolar myopathy and mental retardation, previously known as Danon disease. Mutations of lamp-2 gene have been reported so far in about 20 patients, one of whom was Italian. We describe a new Italian case with persistent hyperCKemia, exercise intolerance and hypertrophic cardiomyopathy but with no muscle weakness or mental impairment. Muscle biopsy revealed a vacuolar myopathy with mild glycogen storage, and immunohistochemical studies detected LAMP-2 deficiency. A new nucleotide substitution (T961C) on exon 8 of lamp-2 gene was identified as responsible for the protein deficiency. This is the first missense mutation so far described. LAMP-2 deficiency should be considered as a cause of recurrent hyperCKemia and hypertrophic cardiomyopathy.

Immunolocalization and activation of transcription factor nuclear factor kappa B in dysimmune neuropathies and familial amyloidotic polyneuropathy.

BACKGROUND: Recently, immunoreactivity of transcription factor nuclear factor kappaB (NF-kappaB) was found in peripheral nerves from patients with Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), and familial amyloidotic polyneuropathy (FAP), suggesting a role in their pathogenesis. OBJECTIVE: To investigate expression and activation of NF-kappaB in nerve biopsy specimens from patients with peripheral neuropathies of different origins. PATIENTS: Nerve biopsies from 17 patients (5 with CIDP, 3 with vasculitis, 4 with Charcot-Marie-Tooth disease, and 5 with FAP) and 3 normal sural nerves were studied by immunocytochemistry and Western blot of nuclear extracts for the activated form of NF-kappaB. Nuclear factor kappaB DNA-binding activity was studied by electrophoretic mobility shift assay. RESULTS: Immunobinding for the activated form p65 of NF-kappaB was found in 2% to 5% of endoneurial vessel walls, in the external myelin of 5% to 10% of fibers, and in a few axons in CIDP specimens. It was also found in 5% to 15% of epineurial and endoneurial vessels in vasculitis specimens and at the level of amyloid deposits in FAP nerves. Nuclear factor kappaB immunoreactivity was not correlated to type of inflammatory cells, but it often corresponded to the deposition of the terminal complement complex C5b9. Western blot analysis of nuclear extracts showed a single band corresponding to 65 kDa in all affected nerves. Nuclear factor kappaB DNA-binding activity was revealed by electrophoretic mobility shift assay in specimens from patients with CIDP, vasculitis, and FAP. CONCLUSION: Our novel findings suggest a crucial role of NF-kappaB in inflammatory neuropathies and FAP.