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Hereditary hyperekplexia (HPX) is a genetic neurodevelopmental disorder recently defined by the triad of (1) neonatal hypertonia, (2) excessive startle reflexes, and (3) generalized stiffness following the startle. Defects in GLRA1 are the most common cause of HPX, inherited both in an autosomal dominant and autosomal recessive manner. GLRA1 mutations can also cause milder phenotypes in the startle syndromes spectrum, but the prevalence is uncertain and no clear genotype-phenotype correlation has emerged yet. Moreover, the prevalence of neurodevelopmental outcomes has not been clearly defined. Here we report a new family of patients with a typical HPX phenotype, linked to a novel GLRA1 mutation, inherited with a recessive pattern. We then perform a systematic review of the literature of GLRA1-related HPX, describing the main epidemiological features of 210 patients. We found that GLRA1-related phenotypes do not necessarily fulfill the current criteria for HPX, including also milder and later-onset phenotypes. Among clinical features of the disease, neurodevelopmental issues were reported in a third of the sample; interestingly, we found that these problems, particularly when severe, were more common in homozygous than in heterozygous patients. Additional clinical and preclinical studies are needed to define predictors of adverse neurodevelopmental outcomes and underlying mechanisms.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr (NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.

Background and Aims:: Mutations in the gene DYSF encoding dysferlin are responsible for dysferlinopathies. Dysferlin is mainly expressed in cardiac and skeletal muscle and appears to play an important role in the repair of the plasmalemma. Intra- and inter-family phenotypic variability has been reported in the literature for mutations in the DYSF gene. A new Cys1678Tyr mutation responsible of LGMD2B has been identified in a family from southern Italy. Methods:: Genomic DNA was extracted from peripheral blood lymphocytes of all family's affected and unaffected subjects using standard protocol. We developed a panel comprising 40 genes involved in LGMDs. The sequencing process was conducted on Ion Torrent PGM platform, annotation of variant calling by WANNOVAR. All the variations founded were confirmed by direct resequencing ABI PRISM 3130xl Genetic Analyzer. Results:: Molecular analysis of three patients from a southern Italian family allowed the identification of a new G5036A mutation in the DYSF gene responsible for LGMD 2B. This mutation determining the replacement of a cysteine in position 1678 with a tyrosine predicted to be deleterious by PolyPhen and silico SIFT analysis. Moreover, Swiss model pipeline analysis showed that this mutation causes the breaking of a strong disulfide bridge with important consequences on the protein activity. Conclusions:: The identification of new mutations responsible for LGMD2B and the study of several affected families will contribute to the identification of additional genetic, epigenetic, environmental factors responsible for the intra and inter-family variability reported in the literature. Furthermore, this will determine the development of new therapeutic targets and personalized medicine.

Background: Friedreich's ataxia is a rare autosomal recessive neurological disorder caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene and frataxin protein deficiency. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Methods and Results: Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5' and 3' ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3' interruptions being most frequent. The patient cohort (n=101) was stratified into groups: 5' interruption, 3' interruption, both 5' and 3' interruptions or lacking interruption. Those patients with a 3' interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Conclusions and Significance: Based on this modelling, a 3' interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5' and 3' interruptions, highlightening the key role of interruptions at the 3' end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.

Autism spectrum disorders (ASDs) constitute a set of heterogeneous neurodevelopmental conditions, characterized by a wide genetic variability that has led to hypothesize a polygenic origin. The metabolic profiles of patients with ASD suggest a possible implication of mitochondrial pathways. Although different physiological and biochemical studies reported deficits in mitochondrial oxidative phosphorylation in subjects with ASD, the role of mitochondrial DNA variations has remained relatively unexplored. In this review, we report and discuss very recent evidence to demonstrate the key role of mitochondrial disorders in the development of ASD.

During a routine screening for thalassemia conducted at Annunziata Hospital (Cosenza, Italy), a Haemoglobin variant was detected, using ion-exchange HPLC. The variant elution time was superimposable to HbAlc and it was present at 50%. For Haemoglobin characterization we utilized a MALDI-TOF approach with ISD fragmentation. The spectrum of the crude hemolysate reveals the presence both of a normai beta-chain (MW 15867, 1) and of an abnormal beta-chain (MW 15881,3) with a difference, between them, of +14 Da. Moreover, the chromatographic features of the variant were not compatible with the possible aminoacidi e change giving the + 14 Da difference observed. Then a MALDI-ISD analysis of the hemolysate was performed and the same spectra showed both the normai and the + 14 Da abnormal c-ions pattern. The (z+2)-ion pattern on the contrary was normai. This results clearly indicated that the substitution concerned the N-terminal portion of the beta chain. Because the abnormal c-ions were present starting from the ISD smaller fragment useful for analysis betac7 (VHL TPEE), the amino acid substitution likely was present in this fragment. A PSD analysis ofthe ISD abnormal fragment at m/z 967.06 (953.08+14) clearly showed, based on the y- and b-ions, that the substitution was (b1 Val7 Ac-Ala). This data was also confirmed by classica] beta chain characterization using tryptic digestion of the abnormal globin and MS/MS analysis and by DNA sequencing. In conclusion the results obtained showed that the top-down MALDI-ISD approach represents a rapid and effective methods for characterization of aminoacidic substitution and post-translational modifications ofh emoglobins

Background: With the use of Next-Generation Sequencing (NGS) an increasing number of PD related genes have been discovered and this new approach can be used to genetically define unresolved PD clinical cases. Objectives: The objective of the present study was to analyze the DNA of two brothers affected by late onset PD. The first patient suffered from mood depression and panic attacks since he was young. He presented progressive slowness of movements, oro-mandibular and right upper limb tremor from the age of 68 years. The response to L-dopa treatment was good. After six years, he showed rapid deterioration of the motor condition, progressive dementia, fluctuating cognition and recurrent visual hallucinations. His brother presented resting tremor at the right upper limb at the age of 59 years. Methods: After DNA extraction, high coverage targeted NGS data were generated at the NGS-Core of IRIB, CNR, Mangone (CS) by an amplicon-based approach. We used a custom-made panel containing 42 genes PD-related. The enriched libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM) system from ThermoFisher Scientific. The primary bioinformatics analysis was carried out using Ion Torrent Suite vers.5.10. Annotation and filtering/priorization of single-nucleotide variations (SNVs) and copy number variations (CNVs) discovered was made by annovar. Results: Sequencing generated a median of 5.85 *106 reads per run, with a mean depth close to 500 reads, a mean read length of 375 called bases and a chip loading of 71%. After bioinformatics analysis and considering only the common variants identified in the two siblings, we highlighted a heterozygous variation c.2710A>G (p.Lys904Glu) in the alphasynuclein binding domain of SNCAIP gene with MAF

To date lymphoblastoid cell lines (LCLs), that constitute the source of several worldwide biobank linked to patient's clinical history, genotype and phenotype data and molecular/functional studies, represent a severely underused bioresource to obtain induced pluripotent stem cells (iPSCs). The overall reprogramming efficiency and poor success rate constitute the limit of use of this cellular resource, and only few groups have reported the successful reprogramming of LCLs. We report an nucleofector based, optimized and reliable method for LCLs reprogramming protocol using known episomal plasmids and mouse embryonic fibroblast (MEF) conditioned culture medium. Methods iPSC reprogramming was performed in accordance with the protocol by S. Kumar et al () but with some modifications. In detail: SF Cell Line 4D-Nucleofector X kit and SF-EO100 pulse programme was utilised for nucleofection; from day 3 to day 10 complete WiCell medium (added of ?MeOH 0,1mM, ?-FGF 1,25ng/?l, NaBut 0,5mM) was utilized to maintain and expand nucleofected cell; from day 11 to day 21 Wi Cell complete medium was gradually replaced with WiCell medium without NaBut. Complete WiCell was obtained conditioning base WiCell (DMEM/F12, KO sierum 20%, MEM NEAA 0,1mM, L-glutamine 2mM, L-ascorbic acid 50ng/?l) with MEF. Reprogrammed iPSC lines were confirmed by immunochemistry. Results The utilized Nucleofection protocol allowed to obtain 40-50% transfection efficiency as shown by the GFP positive cell number detected. Adherent cells appeared already from day 3 after transfection while iPSC-like colonies appeared 2-3 weeks after transfection. In the same time lapse non adherent colonies like to the embryoid bodies also appeared. The iPSC-like colonies detached by an EDTA based, enzyme free method, showed the expression of the specific stem cell proteins such as the OCT-3/4 and SOX-2 when examined by immunofluorescence. The number of colonies obtained from 1.0*106 cells was of 30-60. Using this protocol, we achieved 100% success rate and high reprogramming efficiency. Conclusions To encourage the use of LCLs to generate iPSCs it is crucial to improve and/or to develop new effective reprogramming protocols. Our optimized method, goes in this direction. The availability of iPSC deriving from LCLs of well characterized patients may become crucial in discovering molecular mechanism and new therapeutics strategy for Mendelian or complex human disease.

La Next-generation sequencing (NGS) ha dato un forte impulso alla diagnosi genetica delle distrofie muscolari. Le distrofie muscolari dei cingoli (LGMDs) sono la quarta forma più comune, includono un ampio numero di disordini muscolari con eziologia genetica, severità, fenotipo ed età di esordio variabile. Molti geni associati ad LGMDs sono tra i più grandi del genoma rendendo impossibile fare una diagnosi molecolare con le tecniche di sequenziamento classico. NGS permette di risolvere geneticamente molti casi clinici irrisolti con un approccio rapido ed economico. Scopo dello studio è stato quello di identificare i difetti genetici responsabili di LGMD associata a cardiomiopatia dilatativa in un soggetto Calabrese le cui indagini cliniche, strumentali e bioptiche hanno mostrato una sofferenza muscolare di possibile natura primitiva. Il sequenziamento multigenico del pannello ha permesso di identificare nel paziente affetto da LGMD e cardiomiopatia dilatativa le seguenti varianti geniche in eterozigosi: G56S, R143S, Q50R, M327V and R408C nei geni CAV3, FKRP, PSN2 and TRIM32 rispettivamente. L'ereditarietà di tali varianti è stata studiata nell'intera famiglia. Lo studio suggerisce che più varianti geniche possono avere un effetto additivo nel determinare le caratteristiche fenotipiche del paziente. Varianti su più geni potrebbero spiegare la grande variabilità fenotipica osservata nelle LGMDs. Inoltre, l'identificazione dei pathways in cui tali proteine partecipano permetterà l'identificazione di target terapeutici nel trattamento di tali patologie.

The mechanisms responsible for the onset of iDCM and its evolution to overt heart failure and death are currently unknown. Literature data indicate a genetic involvement both familial and idiopathic dilated cardiomyopathies. Recently, two mutations in presenilin1 and 2 genes, have been found in 0.9% of tested DCM families. The presenilins are membrane proteins required for ?-secretase activity and for Ca2+ homeostasis. They are also expressed in the heart2. Mutations in CAV3 gene are responsible of a broad spectrum of clinical phenotypes as Limb Girdle Muscular Dystrophy, rippling, distal myopathy, hyperCKemia and cardiomyopathy. Caveolin3 is mainly expressed in striated muscle but also in cardiomyocytes. It is essential for the formation of caveolae involved in cellular metabolic regulation through calcium signaling. Moreover, TRIM32 expression is decreased in human hearts with dilated cardiomyopathy3. We reported a patient from southern Italy in which the additive effect of mutations on different genes (CAV3, PSN2, TRIM32) is responsible of the characteristics of his phenotype. In fact, the loss of the protective role played by TRIM32 in the cardiac hypertrophy and heart failure, the alteration of calcium signaling and in the formation of caveolae induced by TRIM32, presenilin2 and caveolin3 mutations respectively, seem to increase in our case the susceptibility to dilatated cardiomyopathy manifested at 20 years. Exploring the pathways in which these proteins take part means adding new elements to the identification of potential therapeutic targets in the prevention of DCM.

During a routine screening for thalassemia conducted at Annunziata Hospital (Cosenza, italy), a haemoglobin variant was detected, using ion-exchange HPLC. The variant elution time was superimposable to HbA1c and it was present at 50%. For hemoglobin characterization we utilized a MALDI-TOF approach with ISD fragmentation. The spectrum of the crude hemolysate reveals the presence both of a normal ?-chain (15867 Da) and of an abnormal ?-chain (15881 Da) with a difference, between them, of +14 Da(m/z). Moreover the chromatographic characteristic of the variant were not compatible with the possibles aminoacidic change giving the +14Da difference observed . Then a MALDI-ISD analysis of the hemolysate was performed and the (same) spectra showed both the normal and a +14 abnormal c-ions pattern. The z+2 ion pattern on the contrary was normal. This results clearly indicated that the substitution concerned the N-terminal portion of the b chain. In particular because the abnormal C-ions were present starting from the ISD smaller fragment useful for analysis (C-7) (VHLTPEE), the aminoacid substitution likely was present in this fragment. A PSD analysis of the ISD abnormal fragment at m/z 967.06 (953.08+14) clearly showed on the basis of the y and b ions, that the substitution was (?1Val?Ac-Ala). This data were also confirmed by classical beta chain characterization using tryptic digestion of the abnormal globin and MS/MS analysis and by DNA sequencing. In conclusion the results obtained showed that the MALDI-ISD approach represents a rapid and effective methods for characterization of aminoacidic substitution and post-traslational modifications of Hemoglobins.

Background: Neurological disorders are a highly heterogeneous group of pathological conditions that affect both the peripheral and the central nervous system. These pathologies are characterized by a complex and multifactorial etiology involving numerous environmental agents and genetic susceptibility factors. For this reason, the investigation of their pathogenetic basis by means of traditional methodological approaches is rather arduous. High-throughput genotyping technologies, including the microarray-based comparative genomic hybridization (aCGH), are currently replacing classical detection methods, providing powerful molecular tools to identify genomic unbalanced structural rearrangements and explore their role in the pathogenesis of many complex human diseases. Methods: In this report, we comprehensively describe the design method, the procedures, validation, and implementation of an exon-centric customized aCGH (NeuroArray 1.0), tailored to detect both single and multi-exon deletions or duplications in a large set of multi- and monogenic neurological diseases. This focused platform enables a targeted measurement of structural imbalances across the human genome, targeting the clinically relevant genes at exon-level resolution. Conclusion: An increasing use of the NeuroArray platform may offer new insights in investigating potential overlapping gene signatures among neurological conditions and defining genotype-phenotype relationships.

Histone H4K16 hypoacetylation in a PKAN lymphoblastoid cell line from a patients compound heterozygous for the PANK2 gene: a MALDI-ISD study.

Multiple sclerosis (MS) might share clinical features with muscular diseases (MD); however, only sporadic association between MS and MD has been reported [1–3]. A 44-year-old woman presented with a family history of MS and MD. Her mother and her uncle were wheelchair bound due to a progressive muscle weakness and wasting in the four limbs; two sisters were affected by MS. Patient clinical history revealed severe obesity and autoimmune thyroiditis. Neurological symptoms started at 39 years of age with progressive muscle weakness and dysesthesias in the left limbs. She developed ataxia and urinary urgency at 41 years of age. Brain, spinal MRI showed several T2-hyperintense lesions involving frontal, parietal, temporoparietal white matter, corpus callosum, semioval centers and cervical spinal cord (Fig. 1). Visualand sensorial-evoked potential showed, respectively, a bilateral delay of P100 and a delay in right N13 wave; brainstem-evoked potentials were normal. Cerebrospinal fluid analysis showed intrathecal oligoclonal IgG synthesis. MS was diagnosed and interferon beta-1a was initiated (but early suspended due to liver toxicity). Then azathioprine was begun but the development of herpetic stomatitis required interruption. On our observation, the patient showed ataxic/spastic gate, possible only for 300 m, with nystagmus. The clinical assessment of the upper limbs showed diffuse muscle weakness, wasting of the scapular girdle, and brisk tendon reflexes. In lower limbs muscle weakness, wasting of the pelvic girdle, calf pseudohypertrophy and normal deep tendon reflexes were observed. Left hemisomatic tactile, pain hypoesthesia, moderate hypopallesthesia, and urinary urgency were also observed. EDSS score was 4.5. Laboratory assessment showed a slight CK increase (350 U/L), a decrease in D-vitamin content (10 ng/mL). A careful neuromuscular evaluation was performed to better elucidate the possible cause of the predominant pelvic and scapular girdles muscle weakness and wasting, which is not usual in patients with relapsing remitting MS. Electromyography was myopathic. Muscular biopsy of the right deltoid showed caliber variability of the muscle fibers, a subsarcolemmal increased mitochondrial proliferation was present in some fibers on SDH. Histoenzymatic analysis revealed the absence of myophosphorylase enzymatic activity confirmed in scheletric muscle homogenate (0.02 nmol/min/mg protein; normal: 0.4 ± 0.12). Molecular analysis of the 20 exons and flanking intronic boundaries of myophosphorylase (PGYM) gene, conducted by polymerase chain reaction and direct sequencing, revealed a transition G[A at codon 220 with no aminoacid change (Q220Q) in compound heterozygosity with IVS14?1G[A. None of the family members gave the informed consent to the genetic counseling. Treatment with glatiramer acetate was started. In the following 3 years the patient developed an optic neuritis, recovering after methylprednisolone treatment, and a worsening of gait imbalance and urinary urgency, only partially improving after steroids. She presented also partial complex seizures fully controlled with levetiracetam. & Stefano Zoccolella stefzoc@hotmail.it

Introduction.The post-translational (PTMs) modifications of nuclear histones, as well known, play an essential role both in the DNA packaging mechanism in chromosomes that in the regulation of gene expression in different cellular processes, especially in response to molecular agents of environmental origin (epigenetic regulation). Aim.The characterization of histone PTMs is traditionally performed by the use of antibodies. However, these methods are often not sufficiently specific and today systems that use mass spectrometry are increasingly used. In this context, our research aims to improve the methodologies based on mass spectrometry mainly in relation to the comprehensivity, simplicity and rapidity of the characterization. Methods.For this purpose, a top-down analysis of MALDI- In Source Decay (MALDI-ISD) analysis, on HPLC fraction of nuclear extracts obtained from immortalized EBV lymphocytic lines, was applied. The cells grown in RPMI 1640 medium were lysed and the collected nuclear fraction was subjected to acid extraction with 0.8M HCl. A preliminary MALDI-TOF profiling analysis was performed on the whole crude extract, and successively this was subjected to HPLC separation from which homogenous fractions of histones (H2A, H2B, H3.1, H3.2, H3.3 and H4) were obtained. Fractions of interest were analyzed using MALDI-ISD using 1.4 DAN as matrix, in order to obtain sequencing and PTMs data. In some cases, a PSD analysis was also performed on fragments generated by the ISD (MS3) to confirm data. Results.The MALDI-TOF profiling of the crude nuclear extract revealed the presence of the principal types of histones, allowing also a relative quantitative evaluation. Subsequently, the MALDI-ISD analysis of the HPLC fractions provided information on both the amino acid sequence and the PTMs present. In particular, we were able to define, in the same experiment the presence in H4 of a dimethylation (above 90%) in the lysine at position 20 (H4K20Me2) Conclusions.In conclusion, the MALDI-ISD methodology allows to obtain global information on histone PTMs with specificity, rapidity and simplicity, effectively contributing to reveal the influence of environmental molecular factors

Tay Sachs disease (TSD) is an autosomal recessive neurological disorder characterized by significant deficiency of lysosomal enzyme ?-Hexosaminidase A and subsequent intralysosomal accumulation of GM2 ganglioside. Tay Sachs disease has been extensively studied in the Ashkenazy Jewish population because of an elevated incidence of cases.We have described in a recent work a young woman of 30 years old with early onset depression at around age 9, inbalance with tendency to fall and cerebellar ataxia developed at 19 years old. She was no longer able to walk without assistance at age of 25. This patient originated from an isolated village of Calabria, South Italy. Genetic tests excluded recessive spinocerebellar ataxias. In vitro determination of the ?-Hexosaminidase A activity showed a low value (< 10%) suggesting a diagnosis of TSD. Direct sequencing of HEXA gene revealed Gly269Ser mutation in compound heterozygosity with Leu127Arg. The identification of two different mutations in HEXA gene in the young woman reported by us originating from an isolated village of southern Italy, and the finding of consanguinity in the family (proband's parents were second-degree cousins), suggested the presence in this country of a founder effect of Ashkenazi Jews origin. In support of this hypothesis, historical sources indicated the presence of Jews in Calabria since eleventh century.

γ-Secretase proteins complex cleaves the amyloid precursor protein (APP) to generate amyloid-β (Aβ) peptides. Considerable evidence suggests that alterations in genes encoding these proteins exert their influence on the pathogenesis of familial Alzheimer's disease (FAD). Presenilin enhancer-2 gene ( PEN-2 ) is a necessary component of the γ-Secretase complex. Recently, it has been shown that PEN-2 mutations could be involved in Alzheimer's disease (AD). We performed a mutational screening of all PEN-2 coding and promoter regions in a FAD cohort derived from Southern Italy. Four hundred and fifty-two subjects (FAD: 97; Controls: 355) were recruited for this study. We identified for the first time in a key region necessary for the promoter activity a novel 3 bp deletion in a subject with early-FAD. Our genetic data demonstrate that the mutant allele may influence the transcriptional activity of the PEN-2 gene. Although the effective role of the PEN-2 promoter deletion in AD is not entirely clear, these findings might lead to more studies on its functional and genetic role.

COL4A1 mutations have been associated with cerebral small-vessel disease, including perinatal intracerebral hemorrhage with consequent porencephaly, microbleeds, and lacunar strokes. Moreover, involvement of multiple organs and tissues like kidney, muscle, and large vessels have been reported. Three related patients with porencephaly bearing the G749S mutation in the COL4A1 gene and one healthy control belonging to the same family underwent skin biopsy. Tissue was examined by means of immunofluorescence microscopy and immunoreactivity for collagen type IV in skin basement membranes was tested. In subjects with COL4A1 mutation, we did not detect significant alterations of immunofluorescence patterns in basal membranes of different skin structures. Heterozygous COL4A1 G749S mutation is associated with a normal immunofluorescence pattern of skin basement membranes. Further studies are needed to clarify the role of possible functional abnormalities of the basement membranes in patients with this mutation.

We report on 2 brothers (aged 19 and 12 years) with Marinesco-Sjogren syndrome who also had very low serum vitamin E concentrations with an absence of postprandial chylomicrons. The molecular study ruled out ataxia with isolated vitamin E deficiency, abetalipoproteinemia, and hypobetalipoproteinemia. The electron microscopy of the intestinal mucosa was consistent with a chylomicron retention disease. We speculate that both chylomicron retention disease and Marinesco-Sjogren syndrome are related to defects in a gene crucial for the assembly or secretion of the chylomicron particles, leading to very low serum levels of vitamin E.

The dopamine D2 receptor (DRD2) gene has been proposed as a candidate gene underlying several psychiatric and neurologic disorders. The aim of the present study was to examine if selected polymorphisms in the DRD2 gene are associated with Parkinson's disease (PD). We determined the allelic frequencies for four polymorphisms located in the DRD2 gene in a sample of 135 patients with PD and 202 normal control subjects. No significant difference was observed in the allelic frequencies between patients with PD and control subjects with regard to the -141C Ins/Del and the Ser311/Cys311 variants. On the contrary, the A1 allele of the TaqIA polymorphism and the B1 allele of the TaqIB polymorphism were more frequent in patients with PD than in control subjects (control subjects: TaqIA A1 = 14.6%, TaqIB B1 = 10.6%; patients with PD: TaqIA A1 = 20.7%, TaqIB B1 = 17.4%). Patients carrying the A1 allele or the B1 allele had an increased risk of developing PD (TaqIA, odds ratio: 1.71, 95% confidence intervals: 1.08–2.73; TaqIB, odds ratio: 1.83, 95% confidence intervals: 1.12–3.02). The TaqIA and TaqIB polymorphisms were in strong linkage disequilibrium, suggesting that these two polymorphisms convey the same information about the risk of presenting with PD. Genetic variation in the DRD2 gene may influence the risk of developing PD, thus confirming that the DRD2 gene is a susceptibility locus for PD.

Spinocerebellar ataxia type 2 (SCA2) is the most common form of autosomal dominant cerebellar ataxia in southern Italy (1). The expansion of a CAG trinucleotide repeat in exon 1 of the SCA2 gene, located on chromosome 12q23-24.1, is responsible for the disease. Normal alleles have 14 to 31 CAG repeats interrupted by one to three CAA trinucleotides, whereas expanded alleles have 33 to over 200 pure repeats, as reported in neonatal cases (2). Clinically, SCA2 is characterized by gait and limb ataxia, dysarthria, slow saccadic eye movements, supranuclear ophthalmoplegia and peripheral neuropathy. Recently, several patients have been described with expansion in the SCA2 gene and an L-dopa responsive parkinsonism

We report the first case of a heterozygous T78M mutation in the caveolin-3 gene (CAV3) associated with rippling muscle disease and proximal myopathy. The patient displayed also bilateral winged scapula with limited abduction of upper arms and marked asymmetric atrophy of leg muscles shown by magnetic resonance imaging. Immunohistochemistry on the patient’s muscle biopsy demonstrated a reduction of caveolin-3 staining, compatible with the diagnosis of caveolinopathy. Interestingly, consistent with the possible diagnosis of FSHD, the patient carried a 35 kb D4Z4 allele on chromosome 4q35. We discuss the hypothesis that the two genetic mutations may exert a synergistic effect in determining the phenotype observed in this patient.

Autosomal Dominant Optic Atrophy (ADOA or Kier's disease) is one of the most frequent forms of inherited optic atrophy, often presenting in the first decade of life with progressive impairment of visual acuity, variably combined with dyschromatopsia and optic nerve pallor. More than 90 mutations spanning throughout the Optic Atrophy 1 (OPA1) gene were disease-associated with most cases of ADOA. Genotype-phenotype comparisons have been inconclusive except for ADOA complicated with a rare sensorineural deafness (ADOAD). Here we present a family with an unusual phenotype of ADOAD and peripheral polineuropathy associated with a novel OPA1 mutation.

Alterations in the leucine-rich kinase 2 gene (LRRK2; MIM *609007) have been shown to cause an autosomal dominant form of PARK8-linked parkinsonism (1, 2). Within this gene, the G6055A mutation (exon 41; Gly2019Ser) represents the most common mutation described to date both in familial and in sporadic forms of Parkinson’s disease (PD).

Lymphocyte and monocyte brain infiltration determines inflammation in multiple sclerosis. The trafficking of these cells into the CNS results from the VLA-4 binding with its ligand on brain endothelial cells. MS patients treated with an antibody against the alpha-4 subunit, which inhibits this interaction, prevents brain lesion development. We investigated the association between VLA-4 gene polymorphisms and MS in a study on 275 patients and 255 controls. No differences were detected, thus suggesting that these polymorphisms are not a significant genetic risk factor for susceptibility to MS in Italy.

Tumor Necrosis Factor Receptor Super Family 6 gene (TNFRSF6), also known as FAS, encodes the Fas antigen, a cell surface receptor mediating cell apoptosis, situated on chromosome 10q located near the region of linkage to sporadic Alzheimer’s disease (sAD). FAS levels have been reported elevated in the brain of AD patients. Due to both positional and pathobiological criteria, the association of the FAS antigen with this pathology is of great interest. We have tested two SNPs in the FAS gene in 223 Italian patients with non-familial AD from Southern Italy (Calabria region) and 211 healthy control subjects. No significant differences in allelic and genotypic distributions were found between cases and controls, or late and early-onset AD patients, thus suggesting that these polymorphisms do not represent an AD risk factor in our population.

We performed a detailed molecular study in two unrelated families with pantothenate kinase-associated neurodegeneration (PKAN) and the specific magnetic resonance imaging (MRI) eye-of-the-tiger pattern. In the first family with classic PKAN, linkage analysis using polymorphic markers from the PANK2 region ruled out linkage with this locus, and no mutation of the PANK2 gene was found. In the second family with atypical PKAN, we identified a novel homozygous C-to-T transition at nucleotide 1069 of the PANK2 gene, which resulted in an arginine to tryptophane substitution at codon 357. As far as we are aware, this is the first case of classic PKAN with the specific MRI eye-of-the-tiger pattern not carrying a PANK2 mutation. Therefore, the present observation reinforces the notion of the phenotypic and genetic heterogeneity in PKAN. Copyright (c) 2005 Movement Disorder Society.

Background Several factors, both clinical and genetic, may account for the risk of developing levodopa-induced peak-dose dyskinesias (PDD) in patients with Parkinson disease, but it is unclear how these factors interact for modulating the individual susceptibility for PDD. Objective To examine clinical and genetic risk factors for determining individual susceptibility of PDD in patients with Parkinson disease. Design Cohort study. Setting Referral center for Parkinson disease in Calabria, southern Italy. Patients Two hundred fifty patients with Parkinson disease were screened for the presence or absence of PDD following a short-term levodopa administration, and 215 subjects were available for further evaluations, including genotypic analysis of the CA dinucleotide short tandem repeat (CA n -STR) polymorphism located in the dopamine receptor D2 gene ( DRD2 ). Results One hundred five patients (48.8%) exhibited PDD following short-term levodopa administration, and 110 patients (51.2%) did not. Multivariate logistic regression analysis showed that independent predictors for the occurrence of PDD were female sex, earlier age at onset of Parkinson disease, longer duration of treatment, and higher dose of levodopa. Genetic factors related to the DRD2 CA n -STR polymorphism were not independent predictors for PDD in the total population, but they had a strong protective effect on the appearance of PDD when the multivariate analysis was performed in men (odds ratio, 0.34 [95% confidence interval, 0.14-0.84]). In women, a genetic protective effect on PDD was not evident. Conclusions Risk factors for PDD, both clinical and genetic, act in different ways for men and women. Genetic factors related to the DRD2 polymorphic status have a protective effect on PDD development in men but not in women. A female sex–related effect for the risk of PDD may be so strong that it overcomes any protective effect due to genetic factors.

There is evidence that male subjects with a clinical picture of action tremor, Parkinsonism, and cerebellar ataxia may have Fragile X premutations (FRAXA). We analyzed FRAXA and FRAXE triplet repeats in 203 male subjects with Parkinson's disease (PD) and 370 healthy controls. No full mutations or premutations at the FRAXA and FRAXE loci were found in the subjects with PD or in the controls. FRAXA allele distribution was similar in patients and controls. FRAXE intermediate alleles (31-60 repeats CCG) were found in 13 of 203 (6.4%) subjects with PD and in only one of the 370 (0.27%) healthy controls (P < 0.001), thus indicating that these relatively large alleles may be associated with PD.

Background: The genes encoding myeloperoxiclase (MPO) and alpha(2),-macroglobulin (A2M) are involved in molecular pathways leading to beta-amyloid deposition. Two polymorphic sites in these genes (MPO-G/A and A2M-Ile/Val) have been associated with Alzheimer disease (AD), but conflicting findings have been reported in populations with different ethnic backgrounds. Objectives: To study the association of MPO-G/A and A2M-Ile/Val polymorphisms, with sporadic AD and to investigate the interactions among the MPO, A2M, and apolipoprotein E (APOE) gene polymorphisms in determining the risk of the development of AD. Design: Case-control study. Setting: Referral center for AD in Calabria, southern Italy Participants: One hundred forty-eight patients with sporadic AD and 158 healthy control subjects. Results: The MPO-G and A2M-Val alleles were found more frequently in cases than in controls, as were the MPO-G/G and A2M-Val/Val genotypes. The odds ratio (OR) for the MPO-G/G genotype was 1.78 (95% confidence interval [CI], 1.13-2.80); for the A2M-Val/Val genotype, 3.81 (95% Cl, 1.66-8.75). The presence of MPO-G/G and A2M-Val/Val genotypes synergistically increased the risk of AD (OR, 25.5; 95% Cl, 4.65-139.75). Stratification of cases by sex, age at onset of AD, and APOE-epsilon4 status did not show significant differences in the distribution of MPO or A2M polymorphisms. Conclusions: The MPO and A2M polymorphisms are associated with sporadic AD in southern Italy. Moreover, a genomic interaction between these polymorphisms increases the risk of the development of AD.

became evident, and it was associated with behavioral difficulties and emotional lability. At age 11 years, versive and staring seizures developed. His interictal EEG showed normal background activity and bilateral occipital and generalized spike‐ wave discharges associated with a photoparoxysmal response. Brain computed tomography and magnetic resonance imaging study were normal. Treatment with vigabatrin (VGB; 2,000 mg/day) was started, and a good control of seizures was achieved. After 3 years, at age 14 years, partial visual seizures and staring episodes developed, associated with jerks of both arms and legs. His interictal EEG showed a slow background activity and diffuse spike‐ and polyspike‐wave discharges. Moreover, axilla skin biopsy revealed pathognomonic polyglucosan

Contradictory evidence has been reported on the role of the polymorphic mixed dinucleotide repeat (NACP-REP1) of the α-synuclein gene as a risk factor for sporadic Parkinson's disease (PD). In the present study we genotyped the NACP-REP1 polymorphism in 189 PD patients from southern Italy and 182 healthy control subjects. We failed to demonstrate an association of any NACP-REP1 allele with PD.

The effects of serotonin (5-HT) in the CNS are mediated through receptor subtypes distinguished by seven major families. The 5-HT6 receptor gene maps to the human chromosome region 1p35-p36 and a polymorphism (C267T) of the 5-HT6 gene has been associated with the risk of developing schizophrenia.1 Unlike most 5-HT receptors, the 5-HT6 receptor is highly expressed in the striatum,2 suggesting a possible involvement of this receptor in the extrapyramidal function. In this study, we investigated whether the allelic variant C267T of the 5-HT6 gene could contribute to the risk for developing PD. Patients diagnosed with sporadic PD (n = 243) and control subjects (n = 234) were included in our study. Patients with PD were consecutively selected starting in March 1999 until December 2000 from the outpatients attending the Institute of Neurology of the University of Catanzaro, Italy. PD was defined by the presence of at least two of four cardinal signs (bradykinesia, rigidity, rest tremor, and postural instability). Only cases with idiopathic PD responsive to levodopa were included. No patient had …

Alpha-2 macroglobulin (A2M) is a component of Lewy bodies, a hallmark of Parkinson's disease (PD). In 159 PD patients and 190 normal controls, we studied two A2M polymorphisms by the polymerase chain reaction-restriction fragment length polymorphism method: a five-nucleotide deletion at the 5' splice site of exon 18; and a valine to isoleucine exchange in amino acid position 1000 near the thiolester active site. No significant differences in allelic and genotypic distribution were found between cases and controls or between early and late-onset PD patients. The present data suggest that these polymorphisms do not represent a risk factor for PD and do not modulate the age at onset of PD.

Mutations in the parkin gene have been reported in patients with early onset PD. The authors investigated the parkin gene in 118 patients who had an onset of PD after age 45 years: 95 subjects were sporadic patients and 23 subjects were from 18 families with a probable autosomal recessive inheritance. No pathogenetic mutations in the parkin gene were detected either in familial or in sporadic patients. Moreover, no differences were found between patients and 100 age-matched normal controls in the allele and genotype frequencies of four exonic parkin polymorphisms.

We report on the clinical, neuropsychological, neurophysiological, computerized eye movement, magnetic resonance imaging (MRI) and molecular findings from 17 individuals affected with spinocerebellar ataxia type 2 (SCA2) belonging to three families. The average age at onset of the symptoms was 35.6, 11.9 (mean, SD) years. The mean age at onset of the symptoms in the parents was 44.8, 8.2 years, and in the offspring it was 28.7, 7.2 years. In 12 parent-child pairs, the mean anticipation was -15.75, 9.1 years (range -8.1 to -23.3 years, t = -4.9, P =0.002). The mutated SCA2 alleles ranged from 38 to 42 CAG repeats, while the normal alleles ranged from 22 to 24 repeats, with 97% of the alleles having 22 repeats. Small differences in the number of CAG repeats influenced the age at onset and rate of progression of the disease considerably. Indeed, patients presenting with their first symptom at an age of 35 years or later with a slower course of the disease harboured between 38 and 39 repeats. In contrast, patients carryingor = 40 CAG repeats manifested the disease prior to 30 years of age and had a faster disease progression toward incapacity. The presenting symptom was always gait ataxia. Slow saccades occured from the beginning of the disease despite normal delay, accuracy and smooth pursuit eye movements. The neuropsychological study showed early and selective impairment of conceptual reasoning ability, as detected by the Wisconsin Card Sorting Test (WCST). It is noteworthy that a significant mutual relationship was observed between performance on the WCST and saccade velocity. All of these findings favour the hypothesis that the disease process of SCA2 in regions other than the cerebellum and brain stem affects severely and early those cortical structures involved in the control of both visually guided saccades and WCST performance.

) for muta-tions in SCN1A, SCN1B, and GABRG2 genes (1–3).Probands were ascertained from the clinical practice inthree epilepsy centers in southern Italy. Detailed familypedigrees were constructed, including maternal and pa-ternal lines extending as far back as possible. In the ninefamilies, we investigated 110 members of whom 37 indi-viduals were determined to be affected. Most patients hadfebrile seizures (FSs) or FS plus (FS

Objective: To investigate whether polymorphisms in the genes for dopamine receptors D 1 and D 2 are associated with the risk of developing peak-dose dyskinesias in PD. Background: Peak-dose dyskinesias are the most common side effects of levodopa therapy for PD. The identified predictors may only partially account for the risk of developing peak-dose dyskinesias because a substantial proportion of patients never develop peak-dose dyskinesias. Genetic factors could play a role in determining the occurrence of peak-dose dyskinesias. Methods: A case-control study of 136 subjects with sporadic PD and 224 population control subjects. We studied three polymorphisms involving the dopamine receptor D 1 gene and one intronic short tandem repeat polymorphism of the dopamine receptor D 2 gene. Results: The polymorphisms of the dopamine receptor D 1 gene were not associated with the risk of developing PD or peak-dose dyskinesias. The 15 allele of the polymorphism of the dopamine receptor D 2 gene was more frequent in parkinsonian subjects than in control subjects. More important, the frequency of both the 13 allele and the 14 allele of the dopamine receptor D 2 gene polymorphism was higher in nondyskinetic than in the dyskinetic PD subjects. The risk reduction of developing peak-dose dyskinesias for PD subjects carrying at least 1 of the 13 or 14 alleles was 72% with respect to the PD subjects who did not carry these alleles. Conclusions: Certain alleles of the short tandem repeat polymorphism of the dopamine receptor D 2 gene reduce the risk of developing peak-dose dyskinesias and could contribute to varying susceptibility to develop peak-dose dyskinesias during levodopa therapy.

Background: Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is caused by mutations in the α4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) gene, mapping on chromosome 20q13.2. A second ADNFLE locus was mapped on chromosome 15q24. Objective: To report a new third ADNFLE locus on chromosome 1 in a large Italian family. Methods: The authors performed a clinical and genetic study in a large, three-generation ADNFLE family from southern Italy, including eight affected individuals and three obligate carriers. Results: The age at onset of seizures was around 9 years of age and all affected individuals manifested nocturnal partial seizures of frontal lobe origin. Interictal awake and sleep EEG recordings showed no definite epileptiform abnormalities in most patients. Ictal video-EEG showed that the attacks were partial seizures with a frontal lobe semiology. Intellectual and neurologic examinations, and brain CT or MRI results were always normal. Carbamazepine was effective in all treated patients. Exclusion mapping of the known loci linked to ADNFLE—ENFL1, and ENFL2, on chromosomes 20q13.2 and 15q24—was performed on the pedigree before starting the genome-wide linkage analysis. The whole genome scan mapping allowed the identification of a new ADNFLE locus spanning the pericentromeric region of chromosome 1. Conclusions: The authors provided evidence for a third locus associated to autosomal dominant nocturnal frontal lobe epilepsy on chromosome 1. Among the known genes mapping within this critical region, the β2 subunit of the nicotinic receptor (CHRNB2) represents the most obvious candidate.