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Caratteristica comune di molti tumori solidi è l’aneuploidia, conseguenza dell’instabilità cromosomica (CIN). Tuttavia non sono ancora chiari i meccanismi alla base dell’aneuploidia e i percorsi che permettono la sua tolleranza. Lo Spindle Assembly Checkpoint (SAC) è un meccanismo di sorveglianza cellulare che controlla la stabilità genomica durante la mitosi. Alterazioni nei membri del SAC generano aneuploidia, ma non è ancora chiaro se questi difetti sono sufficienti per promuovere la tumori-genesi. In questo processo, infatti, il contesto genetico della cellula gioca un ruolo importante. È risaputo che i difetti di p53 aiutano le cellule a proliferare velocemente tollerando la CIN. Al contrario, l’attivazione di una p53-wt contrasta l’aneuploidia. Poco si sa, invece, circa il ruolo del tumor suppressor p14ARF nel contrastare l’aneuploidia. In questa tesi ho voluto valutare la relazione tra alcuni geni del SAC, la cui deplezione induce aneuploidia, e i geni tumor suppressor. Inizialmente, per indagare sul ruolo di p14ARF nel contrastare l’aneuploidia, l’ho espresso ectopicamente in cellule HCT116 (quasi diploidi) dopo aver ridotto, in contemporanea, l’espressione del gene MAD2, elemento cruciale del SAC. Il silenziamento post-trascrizionale di MAD2 ha indotto alti livelli di aneuploidia e di mitosi anomale, che sono diminuiti con l’espressione contemporanea di p14ARF. Inoltre, l’espressione ectopica di p14ARF in cellule MAD2-deplete ha attivato un percorso apoptotico, associato a un incremento della proteina p53. Questa risposta non è stata rilevata in cellule HCT116 p53KO, suggerendo che p14ARF contrasta l’aneuploidia attivando un’apoptosi p53-dipendente. Poi, ho voluto approfondire la relazione tra la proteina motrice Cenp-E, che funziona solo nell’attivazione del SAC, e l’aneuploidia in cellule umane. A tal fine, ho utilizzato due diversi tipi cellulari, fibroblasti primari umani IMR90 e cellule HCT116 che non esprimono la proteina p14ARF, analizzando gli effetti della deplezione di Cenp-E fino a quattro settimane. I due tipi cellulari hanno risposto in maniera differente poiché l’aneuploidia è più tollerata dalle cellule che non esprimono p14ARF piuttosto che dai fibroblasti primari. Inoltre, l’osservazione che la riduzione dell’aneuploidia nelle IMR90 coincide con l’incremento di p14ARF e che la sua espressione ectopica riduce l’aneuploidia nelle HCT116 conferma l’abilità del gene di contrastare l’aneuploidia. Infine, per approfondire questi risultati, ho generato cellule HCT116 che esprimono una proteina p14ARF funzionale, in cui ho valutato gli effetti della deplezione di Cenp-E. In conclusione, questi risultati suggeriscono che p14ARF potrebbe avere un ruolo importante nel contrastare l’aneuploidia attivando un’apoptosi p53-dipendente e che, in maniera più generale, è coinvolto nell’ostacolare l’aneuploidia indotta da differenti stimoli. The majority of solid tumors are characterized by aneuploidy that is believed to be the consequence of chromosomal instability (CIN). The mechanisms leading to aneuploidy and the pathway (s) that allows its tolerance are not completely understood. The Spindle Assembly Checkpoint (SAC) is a cellular surveillance mechanism that works to maintain the genomic balance in mitosis. Alterations of SAC components can induce aneuploidy but it is not clear if these defects are sufficient for tumorigenesis. In this process the genetic background of the cell plays an important role. It is known that p53 defects allow cells to quickly proliferate tolerating CIN. On the contrary, activation of wt-p53 counteracts aneuploidy. Less is known about the role of the p14ARF tumor suppressor to counteract aneuploidy. In this thesis, I investigate the relationship between some of the SAC genes that if depleted induce aneuploidy and tumor suppressor genes. First, to investigate the role of p14ARF to counteract aneuploidy it was ectopically expressed in HCT116 cells (near diploid) after MAD2 depletion a crucial component of the SAC. MAD2 posttranscriptional silencing induced high levels of aneuploid cells and aberrant mitosis that decreased when p14ARF was simultaneously expressed. In addition, p14ARF ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. This response was not detected in HCT116 p53KO cells suggesting that p14ARF counteracts aneuploidy activating apoptosis p53-dependent. Second, I wanted to probe the relationship between the motor protein CENP-E, which works only in the SAC signaling, and aneuploidy in human cells. To this aim I used two types of cells, human primary fibroblasts (IMR90) and near diploid cells (HCT116) lacking p14ARF, and analyzed the effects of CENP-E depletion up to four weeks. These experiments showed a different response for the two cell types. Aneuploidy was tolerated for longer times in cells lacking p14ARF expression rather than in primary cells. In addition the observations that the reduction of aneuploidy in IMR90 cells was proportional to the increase of p14ARF gene expression, and that its ectopic expression in HCT116 cells reduced aneuploidy confirm the ability of p14ARF to counteract aneuploidy. Third, to improve these results I generated HCT116 cells expressing a functional p14ARF to assess the effects of CENP-E depletion. Collectively, these results suggest that the tumor suppressor p14ARF may have an important role to contrast aneuploidy activating a p53-dependent apoptosis pathway and that it is generally involved to counterbalance aneuploidy induced by different stimuli.

Contains fulltext : 167700.pdf (Publisher’s version ) (Open Access) Chorea is a hyperkinetic movement disorder resulting from dysfunction of striatal medium spiny neurons (MSNs), which form the main output projections from the basal ganglia. Here, we used whole-exome sequencing to unravel the underlying genetic cause in three unrelated individuals with a very similar and unique clinical presentation of childhood-onset chorea and characteristic brain MRI showing symmetrical bilateral striatal lesions. All individuals were identified to carry a de novo heterozygous mutation in PDE10A (c.898T>C [p.Phe300Leu] in two individuals and c.1000T>C [p.Phe334Leu] in one individual), encoding a phosphodiesterase highly and selectively present in MSNs. PDE10A contributes to the regulation of the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both substitutions affect highly conserved amino acids located in the regulatory GAF-B domain, which, by binding to cAMP, stimulates the activity of the PDE10A catalytic domain. In silico modeling showed that the altered residues are located deep in the binding pocket, where they are likely to alter cAMP binding properties. In vitro functional studies showed that neither substitution affects the basal PDE10A activity, but they severely disrupt the stimulatory effect mediated by cAMP binding to the GAF-B domain. The identification of PDE10A mutations as a cause of chorea further motivates the study of cAMP signaling in MSNs and highlights the crucial role of striatal cAMP signaling in the regulation of basal ganglia circuitry. Pharmacological modulation of this pathway could offer promising etiologically targeted treatments for chorea and other hyperkinetic movement disorders.

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helixes. The resulting channel's loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.

Introduction ADCY5 mutations have been recently identified as an important cause of early-onset hyperkinetic movement disorders. The phenotypic spectrum associated with mutations in this gene is expanding. However, the ADCY5 mutational frequency in cohorts of paediatric patients with hyperkinetic movement disorders has not been evaluated. Methods We performed a screening of the entire ADCY5 coding sequence in 44 unrelated subjects with genetically undiagnosed childhood-onset hyperkinetic movement disorders, featuring chorea alone or in combination with myoclonus and dystonia. All patients had normal CSF analysis and brain imaging and were regularly followed-up in tertiary centers for paediatric movement disorders. Results We identified five unrelated subjects with ADCY5 mutations (11% of the cohort). Three carried the p. R418W mutation, one the p. R418Q and one the p. R418G mutation. Mutations arose de novo in four cases, while one patient inherited the mutation from his similarly affected father. All patients had delayed motor and/or language milestones with or without axial hypotonia and showed generalized chorea and dystonia, with prominent myoclonic jerks in one case. Episodic exacerbations of the baseline movement disorder were observed in most cases, being the first disease manifestation in two patients. The disease course was variable, from stability to spontaneous improvement during adolescence. Conclusion Mutations in ADCY5 are responsible for a hyperkinetic movement disorder that can be preceded by episodic attacks before the movement disorder becomes persistent and is frequently misdiagnosed as dyskinetic cerebral palsy. A residual degree of neck hypotonia and a myopathy-like facial appearance are frequently observed in patients with ADCY5 mutations. © 2017 The Authors

BACKGROUND: Increased body mass index and weight gain are risk factors for psoriasis, and the prevalence of obesity in patients with psoriasis is higher than in the general population. Limited data exist regarding the role of diet in psoriasis. Here we assessed the impact of a dietary intervention combined with physical exercise for weight loss on improving psoriasis in overweight or obese patients. METHODS: This study included 303 overweight or obese patients with moderate-to-severe chronic plaque psoriasis not achieving clearance after 4 weeks of continuous systemic treatment. They were randomized to receive a 20-week quantitative and qualitative dietary plan associated with physical exercise for weight loss, or simple informative counseling at baseline about the utility of weight loss for clinical control of psoriatic disease. The main outcome was any reduction of the Psoriasis Area and Severity Index (PASI) from baseline to week 20. RESULTS: Intention-to-treat analysis showed a median PASI reduction of 48% (95% confidence interval, 33.3 to 58.3%) in the dietary intervention arm and 25.5% (95% confidence interval, 18.2 to 33.3%) in the information-only arm (P=0.02). Among secondary outcomes, PASI50 significantly differed between study arms (49.7% with dietary intervention vs. 34.2% with information only, P=0.006). The weight-loss target (a ≥5% reduction from baseline) was reached by 29.8% of patients in the dietary intervention arm compared to 14.5% in the information-only arm (P=0.001). CONCLUSIONS: A 20-week dietetic intervention associated with increased physical exercise reduced psoriasis severity in systemically treated overweight or obese patients with active psoriasis. This article is protected by copyright. All rights reserved.

Introduction: Cerebral small vessel diseases (CSVDs) are an important cause of stroke, cognitive impairment, and mood disorders among the elderly. Usually CSVD is sporadic, but early-onset monogenic forms of CSVD have been reported. CADASIL is one of the most common hereditary CSVD caused by autosomal dominant NOTCH3 gene mutations, while CARASIL is a rare autosomal recessive CSVD caused by HTRA1 gene mutations. More than 200 mutations are known in NOTCH3 gene and only 12 HTRA1 mutated CARASIL families have been reported so far. Recently, heterozygous HTRA1 mutations have been described associated with an autosomal dominant form of CSVD with reduced penetrance, showing clinical features differing from those of CARASIL. Patients and methods: We report a family with three affected individuals, referred for stroke and cognitive impairment segregating as an autosomal dominant disorder. Mutations in NOTCH3 and HTRA1 genes have been screened by Sanger sequencing. In silico prediction tools PolyPhen and SIFT have been used to predict the pathogenicity of the variants found. Skin biopsy of one patient was examined with transmission electron microscope. Results: A heterozygous HTRA1 missense mutation segregating with the disease and predicted to be deleterious has been found. The mutation affects a highly conserved aminoacid, which, if mutated, strongly reduce the HTRA1 proteolytic activity. The biopsy showed deposits of osmophilic material in the arterial wall, very similar to those characterizing CADASIL. Conclusions: We confirm the dominant inheritance of some HTRA1 mutations, overlapping clinical, MRI and ultramicroscopic features of CADASIL, in absence of the typical CARASIL extra-neurological symptoms.

Chorea is a hyperkinetic movement disorder resulting from dysfunction of striatal medium spiny neurons (MSNs), which form the main output projections from the basal ganglia Here, we used whole-exome sequencing to unravel the underlying genetic cause in three unrelated individuals with a very similar and unique clinical presentation of childhood-onset chorea and characteristic brain MRI showing symmetrical bilateral striatal lesions. All individuals were identified to carry a de novo heterozygous mutation in PDE10A (c.898T>C [p.Phe300Leu] in two individuals and c.1000T>C [p.Phe334Leu] in one individual), encoding a phosphodiesterase highly and selectively present in MSNs. PDE10A contributes to the regulation of the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both substitutions affect highly conserved amino acids located in the regulatory GAF-B domain, which, by binding to cAMP, stimulates the activity of the PDE10A catalytic domain. In silico modeling showed that the altered residues are located deep in the binding pocket, where they are likely to alter cAMP binding properties. In vitro functional studies showed that neither substitution affects the basal PDE10A activity, but they severely disrupt the stimulatory effect mediated by cAMP binding to the GAF-B domain. The identification of PDE10A mutations as a cause of chorea further motivates the study of cAMP signaling in MSNs and highlights the crucial role of striatal cAMP signaling in the regulation of basal ganglia circuitry. Pharmacological modulation of this pathway could offer promising etiologically targeted treatments for chorea and other hyperkinetic movement disorders.

Small-diameter melanocytic lesions represent a diagnostic challenge for clinicians, as they do not follow the ABCD rule for diagnosis and do not always display reliable histopathological criteria.To analyse the confocal features of small-diameter lesions (naevi and melanomas with diameter ≤ 5 mm) to determine whether they show specific morphological criteria.Twenty-four melanomas and 72 naevi were subjected to dermoscopic and confocal evaluation along with histopathology. Significant dermoscopic and confocal differences between melanomas and naevi were evaluated by means of the Pearson χ(2) test. Odds ratios and 95% confidence intervals were calculated for each parameter. Binary logistic regression was performed to identify the reflectance confocal microscopy (RCM) independently significant features for melanoma diagnosis.The seven-point checklist dermoscopic score was ≥ 3 in 22 melanomas and in 33 naevi. The combination of cells' pleomorphism and architectural disorder (i.e. nonspecific pattern or irregular junctional nests upon confocal examination) are the most striking criteria for consistent diagnosis of small melanoma. The presence of atypical cells, more than five atypical cells per mm(2) , and roundish atypical cells at the dermoepidermal junction showed the highest odds ratios. From logistic regression, the presence of at least five pagetoid cells per mm(2) , tangled lines within the epidermis, and atypical roundish cells at the dermoepidermal junction resulted in the three independent confocal parameters that characterized small melanomas.Small melanomas frequently reveal specific dermoscopic and confocal features. Moreover, the combination of dermoscopy and RCM can lead to a correct diagnosis of a number of naevi that share some morphological aspects with melanomas.

Spinocerebellar Ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease characterized by late onset, slowly progressive, mostly pure cerebellar ataxia. It is one of three allelic disorders associated to CACNA1A gene, coding for the Alpha1 A subunit of P/Q type calcium channel Cav2.1 expressed in the brain, particularly in the cerebellum. The other two disorders are Episodic Ataxia type 2 (EA2), and Familial Hemiplegic Migraine type 1 (FHM1). These disorders show distinct phenotypes that often overlap but have different pathogenic mechanisms. EA2 and FHM1 are due to mutations causing, respectively, a loss and a gain of channel function. SCA6, instead, is associated with short expansions of a polyglutamine stretch located in the cytoplasmic C-terminal tail of the protein. This domain has a relevant role in channel regulation, as well as in transcription regulation of other neuronal genes; thus the SCA6 CAG repeat expansion results in complex pathogenic molecular mechanisms reflecting the complex Cav2.1 C-terminus activity. We will provide a short review for an update on the SCA6 molecular mechanism.

Benign hereditary chorea (BHC) is a rare autosomal dominant condition characterized by early onset, non-progressive chorea, usually caused by mutations in the thyroid transcription factor-1 gene (TITF1). We describe a novel mutation arising de novo in a proband presenting in infancy with delayed walking and ataxia. She later developed chorea, then hypothyroidism and a large cystic pituitary mass. Her daughter presented in infancy with delayed walking and ataxia and went on to develop non-progressive chorea and a hormonally inactive cystic pituitary mass. Mutational analysis of the whole coding region of the TITF1 gene was undertaken and compared with a population study of 160 control subjects. This showed that both affected subjects have a heterozygous A > T substitution at nucleotide 727 of the TITF1 gene changing lysine to a stop codon at residue 211. Genetic analysis of parents and siblings of the proband confirmed that the mutation arose de novo in the proband. The mutated lysine is an evolutionarily highly conserved amino acid in the protein homoeodomain (HD) where most point mutations associated with BHC are located. The range of mutations in BHC is reviewed with particular emphasis on pituitary abnormalities. Cystic pituitary masses and abnormalities of the sella turcica are reported in just 6.4 % of published cases. This is a new nonsense mutation associated with ataxia, benign chorea and pituitary abnormalities which further extends the phenotype of this condition. Mutational screening of TITF1 is important in cases of sporadic or dominant juvenile-onset ataxia, with mild chorea where no other cause is found, particularly if pituitary abnormalities are seen on imaging. © 2014 The Author(s).

The CACNA1A gene on chromosome 19p13 encodes the alpha1A subunit of P/Q type voltage-gated Ca2+ channels (Cav2.1) expressed mainly in the cerebellum (in Purkinje and granule cells) and at the neuromuscular junction. Mutations in this gene are responsible for three rare autosomal dominant neurological disorders: Episodic Ataxia 2 (EA2), Familial Hemiplegic Migraine 1 (FHM1) and Spinocerebellar Ataxia 6 (SCA6) due to the expansion of a CAG repeat at the 3'end of the gene. The Cav2.1 alpha1A subunit is a four domain transmembrane protein of about 280KDa with cytoplasmic N- and C-terminal tails. The cytoplasmic C-terminus plays regulatory roles in the gating and trafficking of many ion channels. The C-terminus of the Cav2.1 alpha1A subunit, which represent the 25% of the entire protein (about 700 aminoacids) and contains the CAG repeat expanded in SCA6, plays a regulatory role in gating and trafficking and contains residues involved in channel inactivation and modulation induced by intracellular signalling proteins. Recently, the C-terminal tail was shown to be cleaved in vivo and conveyed into the nucleus, suggesting its involvement in regulating some transcriptional processes. In order to elucidate the role of the Cav2.1 C-terminal tail as possible transcription factor, gene expression arrays were carried out in HEK293 cell line wild type and over-expressing Cav2.1 C-terminal tail. Total RNA, extracted from these cells, was analyzed using Agilent 44K whole human genome oligo microarray system. Gene expression in HEK293 cells wild type and over-expressing Cav2.1 C-terminal tail was compared and for genes resulted up or down-regulated more than 5 folds, the expression profile was checked by using standard qRT-PCR analysis. These results, for the first time, provide evidence that Cav2.1 C-terminus, beside modulating channel activity, regulates the expression of genes important for the neuronal excitability such as gap junctions, ion channels, signalling proteins.

The CACNA1A gene codes for the alpha(1A) pore-forming subunit of Ca(2+) voltage-gated Cav2.1 channels. CACNA1A mutations are responsible for Familial Hemiplegic Migraine (FHM) type 1, Episodic Ataxia (EA) type 2 and Spinocerebellar Ataxia type 6. The structure of the human gene includes, at present, 49 exons; however almost nothing is known about the 5' regulatory region, and there is now evidence suggesting the presence of additional exons at the 3' of the gene. The 892 bp fragment upstream of exon 1 and its deletion mutants were characterised for their transcriptional activity by using luciferase as a reporter gene. The 3' region was analysed by Rapid Amplification of the cDNA 3' End. Both regions were screened for mutations in a series of FHM and EA patients by SSCP and sequencing. At the 5' end of the gene a minimal promoter region was identified within the first 497 bp from ATG. By screening a larger fragment for mutations, the 5 bp deletion (g.-757_-753delCTTTC) was identified in a FHM patient. The deletion significantly increased the transcriptional activity, most likely due to the removal of half a turn of the DNA helix, changing the orientation of downstream binding sites for transcriptional factors. At the 3' end of the gene a new exon 48, followed by a strong poly-A signal, was identified as well as a new splice variant. The 5 bp insertion (g.38429_38430insCTTTT) in this exon was found in an EA patient. The two new regions can open the way for the study of human CACNA1A gene expression regulation and can be sites of mutations associated with FHM or EA phenotypes.

Benignhereditarychorea (BHC) is an autosomal dominant disorder of early onset characterised by non progressive choreic movements with normal cognitive function occasionally associated with hypothyroidism and respiratory problems. Numerous pieces of evidence link BHC with TITF-1/NKX2.1 gene mutations. We studied apatient with a familial benignhereditarychorea and normal thyroid and respiratory function. Sequence analysis of TITF-1 revealed the presence of a heterozygous C > T substitution at nucleotide 532, predicted to change an arginine (CGA) with a stop codon (TGA) at position 178 (R178X). Afunctional analysis shows that the mutated TTF-1 is not binding DNA, nor activating the canonical thyroid target gene promoter or interfering with the ability of wild type TTF-1 to activate transcription. In addition, the mutated protein is predominantly cytoplasmic, rather than nuclear as in the case of the wild type TTF-1. Thus, we have identified a new mutation in the TTF-1 coding gene in apatient with benignhereditarychorea. The results show that the mutation leads to a haploinsufficiency of TITF-1 and opens the question of genotype/phenotype correlation.

The CACNA1A gene, coding for the 1A-subunit (Cav2.1) of voltage-gated calcium channel type P/Q is responsible for Episodic Ataxia type 2 (EA2), Familial Hemiplegic Migraine (FHM) and Spinocerebellar Ataxia type 6 (SCA6). Several mutations causing these diseases have been described. In addition, families segregating for the disease with the CACNA1A, but not showing any mutations in the coding region, have also been found.Thirthy-four patients with typical EA2, and 8 with cerebellar ataxia of unknown genetic type, are screened for CACNA1A gene mutations. Four new Cav2.1 missense or non-truncating mutations and 1 truncating mutation have been detected. From an analysis of the localization of the non-truncating mutations together with those reported in literature it is further confirmed that: 1) they tend to be preferentially located in specific protein regions, namely S5-S6 linkers and their borders; 2) their associated clinical phenotype suggests a higher age at onset and a lower frequency of mental retardation. Families segregating for the disease with the CACNA1A markers, but not showing mutations of the coding region, suggest a further characterization of the gene. A new 3terminal exon, namely 48B, has been characterized. An alternative splice site about 300 bp upstream from 5end of exon 48 creates a so far unknown isoform similar to BI-1(V2.V3) but with a longer exon 48. Results from bioinformatic analysis show that the two isoforms with the alternatively spliced exon 48 have at least two different polyadenilation signal candidates. Since preliminary results suggest that mRNAs of this gene have dendritic localization and mRNA transport studies show a clear implication of the 3 tail in subcellular localization, these exons are being characterized both for the alternative polyadenilation signal usage and transport and for mutations causing disease in EA2 and cerebellar ataxia patients. Supported by grants MIUR cofin 2003 to CJ and FIRB2001, RBNE01XMP4-008, FISR2000 to MF.

The CACNA1A gene, coding for the 1A-subunit (Cav2.1) of voltage-gated calcium channel type P/Q is responsible for Episodic Ataxia type 2 (EA2), Familial Hemiplegic Migraine (FHM) and Spinocerebellar Ataxia type 6 (SCA6). Several mutations causing these diseases have been described. In addition, families segregating for the disease with the CACNA1A, but not showing any mutations in the coding region, have also been found.Thirthy-four patients with typical EA2, and 8 with cerebellar ataxia of unknown genetic type, are screened for CACNA1A gene mutations. Four new Cav2.1 missense or non-truncating mutations and 1 truncating mutation have been detected. From an analysis of the localization of the non-truncating mutations together with those reported in literature it is further confirmed that: 1) they tend to be preferentially located in specific protein regions, namely S5-S6 linkers and their borders; 2) their associated clinical phenotype suggests a higher age at onset and a lower frequency of mental retardation. Families segregating for the disease with the CACNA1A markers, but not showing mutations of the coding region, suggest a further characterization of the gene. A new 3terminal exon, namely 48B, has been characterized. An alternative splice site about 300 bp upstream from 5end of exon 48 creates a so far unknown isoform similar to BI-1(V2.V3) but with a longer exon 48. Results from bioinformatic analysis show that the two isoforms with the alternatively spliced exon 48 have at least two different polyadenilation signal candidates. Since preliminary results suggest that mRNAs of this gene have dendritic localization and mRNA transport studies show a clear implication of the 3 tail in subcellular localization, these exons are being characterized both for the alternative polyadenilation signal usage and transport and for mutations causing disease in EA2 and cerebellar ataxia patients. Supported by grants MIUR cofin 2003 to CJ and FIRB2001, RBNE01XMP4-008, FISR2000 to MF.

Spinocerebellar ataxia type 6 (SCA6) is one of three allelic disorders caused by mutations of CACNA1A gene, coding for the pore-forming subunit of calcium channel type P/Q. SCA6 is associated with small expansions of a CAG repeat at the 3' end of the gene, while point mutations are responsible for its two allelic disorders (Episodic Ataxia type 2 and Familial Hemiplegic Migraine). Genetic, clinical, pathological and pathophysiological data of SCA6 patients are reviewed and compared to those of other SCAs with expanded CAG repeats as well as to those of its allelic channelopathies, with particular reference to Episodic Ataxia type 2. Overall SCA6 appears to share features with both types of disorders, and the question as to whether it belongs to polyglutamine disorders or to channelopathies remains unanswered at present.

In this work we focus on a microsatellite-defined Y-chromosomal lineage (network 1.2) identified by us and reported in previous studies, whose geographic distribution and antiquity appear to be compatible with the Neolithic spread of farmers. Here, we set network 1.2 in the Y-chromosomal phylogenetic tree, date it with respect to other lineages associated with the same movements by other authors, examine its diversity by means of tri- and tetranucleotide loci and discuss the implications in reconstructing the spread of this group of chromosomes in the Mediterranean area. Our results define a tripartite phylogeny within HG 9 (Rosser et al. 2000), with the deepest branching defined by alleles T (Haplogroup Eu10) or G (Haplogroup Eu9) at M172 (Semino et al. 2000), and a subsequent branching within Eu9 defined by network 1.2. Population distributions of HG 9 and network 1.2 show that their occurrence in the surveyed area is not due to the spread of people from a single parental population but, rather, to a process punctuated by at least two phases. Our data identify the wide area of the Balkans, Aegean and Anatolia as the possible homeland harbouring the largest variation within network 1.2. The use of recently proposed tests based on the stepwise mutation model suggests that its spread was associated to a population expansion, with a high rate of male gene flow in the Turkish-Greek area.

Tn this work we focus on a microsatellite-defined Y-chromosomal lineage (network 1.2) identified by us and reported in previous studies, whose geographic distribution and antiquity appear to be compatible with the Neolithic spread of farmers. Here, we set network 1.2 in the Y-chromosomal phylogenetic tree, date it with respect to other lineages associated with the same movements by other authors, examine its diversity by means of tri- and tetranucleotide loci and discuss the implications hi reconstructing the spread of this group of chromosomes in the Mediterranean area. Our results define a tripartite phylogeny wit-bin HG 9 (Rosser et al. 2000) with the deepest branching defined by alleles T (Haplogroup Eu 10) or G (Haplogroup Eu9) at M172 (Semino et al. 2000), and a subsequent branching within Eu9 defined by network 1.2. Population distributions of HG 9 and network 1.2 show that their occurrence in the surveyed area is not due to the spread of people from a single parental population but, rather, to a process punctuated by at least two phases. Our data identify the wide area of the Balkans, Aegean and Anatolia as the possible homeland harbouring the largest variation within network 1.2. The use of recently proposed tests based on the stepwise mutation model suggests that its spread was associated to a population expansion, xvith a high rate of male gene flow in the Turkish Greek area.