Paola Origone

SOD1 mutations in amyotrophic lateral sclerosis. Results from a multicenter Italian study.

AbstractAmyotrophic Lateral Sclerosis (ALS), the most common form among motoneuron diseases, is characterized by a progressive neurodegenerative process involving motor neurons in the motor cortex, brain stem and spinal cord. Sporadic (SALS) accounts for the majority of patients but in about 10% of ALS cases the disease is inherited (FALS), usually as an autosomal dominant trait.In the present study we show the results of a referred based multicenter study on the distribution of SOD1 gene mutations in the largest cohort of Italian ALS patients described so far. Two hundred and sixty–four patients (39 FALS and 225 SALS) of Italian origin were studied. In 7 out of 39 FALS patients we found the following SOD1 gene mutations: i) a new G12R missense mutation in exon 1, found in a patient with a slowly progressive disease course; ii) the G41S mutation, in four unrelated patients with rapidly progressive course complicated with cognitive decline in two of them; iii) the L114F mutation, in a patient with a slowly progressive phenotype; iv) the D90A mutation, in a heterozygous patient with atypical phenotype. In addition, in one SALS patient a previously reported synonymous variant S59S was identified. In 17 (3 FALS and 14 SALS) out of 264 patients (6.4 %) the polymorphism A→C at position 34 of intron 3 (IVS3: + 34 A→C) was found, and in one FALS patient a novel variant IVS3 + 62 T→C was identified.The frequency of SOD1 gene mutations (17.9 %) in FALS cases was comparable with that found in other surveys with a similar sample size of ALS cases. No SOD1 gene mutations have been identified in SALS cases. Within FALS cases, The most frequent mutation was the G41S identified in four FALS.

Mutation Analysis of CCM1, CCM2 and CCM3 Genes in a Cohort of Italian Patients with Cerebral Cavernous Malformation

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K‐Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three‐gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at‐risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified, nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255‐4delGTA; c.1681‐1682delTA in CCM1; c.48A > G; c.82‐83insAG in CCM2; and c.396G > A in CCM3 genes. The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at‐risk relatives.

Enlarging clinical spectrum of FALS with TARDBP gene mutations: S393L variant in an Italian family showing phenotypic variability and relevance for genetic counselling

Objective: The present study was aimed to enlarge the Italian ALS sample analysed for TARDBP gene mutations. Methods: Genomic DNA from 47 patients, 70 FTD patients and 158 controls was extracted from peripheral blood samples according to a standard protocol. The five coding exons (2–6) of the TARDBP gene and the flanking exon-intron boundaries were analysed by direct sequencing. Using ClustalW2 human TDP-43, protein sequence was aligned with TDP-43 protein sequence of different species. Results: A heterozygous c.1178 C→T transition that leads to a change p.S393L was observed in a 75-years-old male patient and in his two affected siblings. A patients brother had died at 69 years of age after a two-year history of ALS. In FTD patients no mutations were found. Conclusions: We describe a further Italian family with FALS, in which a variant (p.S393L) of the TARDBP gene was identified. Clinical course and phenotypic variability in three affected siblings is presented and relevance for genetic counselling of patients and their families is underlined. At the present state of knowledge, we suggest that the same guidelines established for SOD1 molecular testing could be proposed also for TARDBP analysis in FALS.

Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia

Leukemic cells with double minute (DM) chromosomes from an ANLL(M1) patient were found to carry 10-15 fold amplified c-myc sequences. The linked pvt-1-like locus was amplified at the same level, suggesting that the c-myc amplicon is at least 300 kb in size.

Homozygous inactivation of NF1 gene in a patient with familial NF1 and disseminated neuroblastoma

Neurofibromatosis type 1 (NF1) patients are susceptible to tumor development. In the present study we describe a child with NF1 and disseminated neuroblastoma whose death resulted from disease progression. The mother had café‐au‐lait spots suggesting a familial NF1. Neuroblastoma cells showed MYCN amplification and chromosome 1p36 deletion, common features associated with tumor progression in this malignancy. The NF1 gene displayed a germline T → C transition of intron 14 in both the proband and mother DNA. This mutation, not yet previously described, occurs in a splicing donor site and produces a new mRNA variant observed together with normal NF1 mRNA. Furthermore, the SSCP analysis of the NF1 gene in tumor cells showed a somatic deletion encompassing the intron 26 and 27b of the paternal NF1 allele. Hence, neuroblastoma cells displayed both somatic and germline mutation of the NF1 gene. Our data suggest that, although rare, neuroblastoma in patients with NF1 may display homozygous gene inactivation.

Lack of association of PON polymorphisms with sporadic ALS in an Italian population.

Paraoxonase (PON) gene polymorphisms have been associated with susceptibility to sporadic amyotrophic lateral sclerosis (ALS). We have investigated the role of the previously associated single nucleotide polymorphisms rs854560, rs662, and rs6954345 in 350 ALS patients and 376 matched controls from Italy. No significant association was observed at genotype and haplotype level. Our data suggest that PON polymorphisms are not involved in ALS pathogenesis in an Italian population.

Search for loss of heterozygosity and mutation analysis of KRIT1 gene in CCM patients

Cerebral cavernous malformation (CCM) is a vascular disorder of the brain characterized by dilated capillary-like sinusoid chambers lined by a single layer of endothelium and thin fibrous adventitia but lacking other vessel wall components and intervening neural structures [Clatterbuck et al., 2001]. Spinal, cutaneous, retinal, and vertebral cavernous angiomas have also been observed. Both sporadic and familial forms have been identified. The familial form exhibits autosomal dominant inheritance with variable clinical expression and incomplete penetrance with three known loci on chromosomes 7q21.2 (CCM1), 7p15-p13 (CCM2), and 3q25.2-q27 (CCM3). The first gene identified on CCM1 locus is KRIT1 encoding KREV-1/RAP1 interaction trapped 1 protein (KRIT1), which is responsible for 40% or more of familial cases. Recently a novel gene MGC4607 has been isolated on the CCM2 locus [Liquori et al., 2003]. The KRIT1 gene has been reported to include 16 coding exons [Sahoo et al., 2001]. KRIT1 is a protein of 736 aminoacids and a molecular mass of 81 kDa containing N-terminal ankyrin repeats, one FERM domain, one trans-membrane domain, and a region at the C-terminal interacting with RAP1A [Sahoo et al., 2001; Marini et al., 2003]. The combina-

Clinical epidemiology of ALS in Liguria, Italy.

Abstract Our objective was to assess the incidence and trends of amyotrophic lateral sclerosis (ALS) in Liguria, a north-west region of Italy, utilizing a prospective design. Liguria (1,615,064 residents in 2010) is the site of a multicentre-multisource prospective population based registry called LIGALS (Liguria Amyotrophic Lateral Sclerosis Registry). All incident ALS cases during the period 2009–2010 were enrolled and followed up. Cases were identified using several concurrent sources. ALS diagnosis was based on the revised El Escorial criteria. One hundred and four cases were enrolled, generating an annual crude incidence of 3.22/100,000 (95% CI 2.66−3.90), with a male/female ratio of 1.34. The annual standardized incidence, age and gender adjusted to the 2001 Italian population, was 2.51. At last observation on 1 March 2012, 45% of patients registered in the LIGALS had died, with a median survival of 45 months from symptoms onset. According to capture-recapture estimation, three patients were unobserved. For both genders, demographic and clinical features were collected. In conclusion, comparing these data to those of epidemiological studies with a similar prospective design, the occurrence of ALS is similar. The observed crude incidence was higher compared to other Italian studies, due in part to a very careful case ascertainment and in part to a high percentage of the elderly in Liguria.

Identification of a novel KRIT1 mutation in an Italian family with cerebral cavernous malformation by the protein truncation test

Familial cerebral cavernous malformation (CCM) exhibits autosomal dominant inheritance and is characterized by vascular disorders of the brain, which can lead to seizures, focal neurological deficits, hemorrhagic stroke, and migraine. Three CCM loci have been mapped, but the gene for only one locus--KRIT1 coding for Krev-1/rap1 interaction trapped 1 (KRIT1) protein, which is responsible for more than 40% of familial cases--has been identified. To date, a total of 72 mutations have been described, with one founder effect in the Mexican/Hispanic community. We report the case of an Italian family with CCM that has a novel KRIT1 gene mutation leading to a truncated KRIT1 protein. The protein truncation test (PTT) has been used as a rapid method of identifying germline mutations in the KRIT1 gene.

An Italian dominant FALS Leu144Phe SOD1 mutation: Genotype-phenotype correlation

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease. Mutations of the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of autosomal dominant familial ALS (FALS). RESULTS: We examined the clinical features of the first Italian FALS with the Leu144Phe SOD1 mutation. Seven affected members were identified in a six‐generation pedigree. A slowly progressive course (20.4±14.6 years) was observed in five patients. One patient died of cardiac failure two years after the onset of the disease. The propositus is still alive. Neurological manifestations began in the legs in all patients, while bulbar signs were absent or appeared late in the course of the disease. DISCUSSION: There is evidence of a correlation between this mutation and a slowly progressive phenotype of ALS. Moreover this rare mutation might derive from a common ancestor.