I112M SOD1 mutation causes ALS with rapid progression and reduced penetrance in four Mediterranean families.

We evaluated a possible genotype-phenotype correlation and looked for a founder effect in four Mediterranean families carrying the I112M SOD1 mutation. The structural characteristics of the mutated protein were also analysed. Clinical data of FALS subjects from four families were evaluated. Mutational analysis of the SOD1 gene was carried out by direct sequencing. A haplotype study was carried out using 11 polymorphic markers flanking the SOD1 gene. Structural analysis was performed by means of homology modelling and molecular graphics methods. The clinical pattern of 17 FALS patients was characterized by prevalent spinal onset, mean age at onset of 47.1 years and mean duration of 20.7 months. Several obligate carriers were observed. These findings indicate that the I112M mutation is consistently associated with a uniform, fast-progressing phenotype with reduced penetrance of the disease. The haplotype analysis did not show a common haplotype among the Spanish families and the Italian family; however, a possible common founder could be hypothesized for Spanish families. From a structural viewpoint, mutation at codon 112 seems to confer a severe phenotype, probably related to altered protein functionality.

Mutation analysis of oxisterol-binding-protein gene in patients with age-related macular degeneration.

Allelic variants of several genes are increasingly recognized as susceptibility factors in age-related macular degeneration (AMD). Because of its metabolic characteristics the macula is sensitive to oxidative damage, and supplementation with antioxidants has been shown to be effective in slowing the progression of disease in AMD patients. The oxisterol-binding-protein (OSBP2) gene is expressed mainly in the retinal pigmented epithelium underlying the macular region. Its product specifically binds and transports oxisterols, the cytotoxic effects of which may be involved in macular damage. The aim of this study was to search for allelic variants of OSBP2 gene, as well as to evaluate several risk factors in 24 patients with AMD; 17 with nonexudative (NE) and 7 with neovascular (NV) form. Total cholesterol was elevated in 66% of the patients, high-density lipoprotein (HDL) cholesterol was reduced in 12%; vitamin A or vitamin E deficiency was not observed. OSBP2 gene analysis was performed in AMD patients and in 110 control subjects by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Six allelic variants were detected: 2 nonpolymorphic unique exonic variants in 2 AMD subjects and 4 polymorphic variants (2 exonic and 2 intronic). These data indicate a possible role of OSBP2 gene in the pathogenesis of oxidative damage to the macula induced by oxysterols in AMD patients.

Intrafamilial clinical heterogeneity associated with a novel mutation of the retinal degeneration slow/peripherin gene.

AIMS: To identify the phenotypic variations in 6 related individuals affected by a novel mutation in the retinal degeneration slow/peripherin gene. METHODS: Ten family members underwent ophthalmologic assessment with slit-lamp biomicroscopy, dilated fundus examination, fundus photography, autofluorescence imaging and electrophysiological tests. Genomic DNA was extracted from blood samples of all family members (n = 15) using the standard salting-out procedure. RESULTS: The novel C165R mutation was identified in 8 individuals. Of these 8 patients, only 6 gave consent to the clinical study. They had a retinal disease characterized by an adulthood onset of symptoms, and their best corrected visual acuity was between 20/50 and 20/20. Fundus examination showed that 3 patients had typical fundus flavimaculatus: 1 had butterfly-shaped pattern dystrophy and 2 had incipient retinal changes. CONCLUSION: We identified a novel mutation of the retinal degeneration slow/peripherin gene in a family affected by different patterns of retinal dystrophy. This is the first report of an association of fundus flavimaculatus with butterfly-shaped pattern dystrophy.

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

Amyotrophic 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.

Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients [corrected].

In the present study the entire NF1 coding region was analyzed for mutations in 132 unrelated Italian NF1 patients. Using PTT, SSCP, and DNA sequencing, we found 8 novel mutations. Clinical diagnosis of NF1 was established according to the NIH consensus criteria. We detected 59 truncated fragments, and 46 of them were characterized by SSCP and direct sequencing. Eight mutations represent novel changes that contribute to the germline mutational spectrum of the NF1 gene. In two patients, premature termination was due to substitutions at nucleotide c.3982C>T (Q1298X) and c.7411C>T (Q2471X), respectively. Two other mutations were caused by the deletions (1756delA, 4699delA), and two by the insertions (c.5266_5267insT, c.7464_7465insTCCA) of a small number of nucleotides. Lastly, we found 2 splice-site mutations (c.2252-2A>C, c.2251+1G>A).

Ten novel mutations in the human neurofibromatosis type 1 (NF1) gene in Italian patients.

The entire NF1 coding region was analyzed for mutations in a panel of 108 unrelated Italian NF1 patients. Using PTT, SSCP, and DNA sequencing, we found 10 mutations which have never been reported before. Clinical diagnosis of NF1 was established according to the NIH consensus criteria in 100 individuals, while 8 were young children with only multiple cafè-au-lait spots. We detected 46 truncated fragments, and 24 of them were fully characterized by SSCP and direct sequencing. Of the 24, 14 were known mutations (R304X, R681X, Q682X, R1306X, R1362X, R1513X, R1748X, Q1794X, R1947X, Y2264X, R2237X, 2674delA, 6789delTTAC, 2027insC). The other 10 mutations represent novel changes that contribute to the germline mutational spectrum of the NF1 gene (K810X, Q2595X, 6772delT, 7190delCT, 7331delA, 1021insTT, 3921insT, 4106insTA, 7149insC, 2033insCG / 2034delA). PTT in a large number of Italian NF1 patients supports the usefulness of this method for characterization of mutations in disorders where the responsible gene is very large and the disease-causing mutations often create a stop codon. In agreement with previous reports, no mutational hotspots within the NF1 gene were detected.

Inverse correlation between p16INK4A expression and NF-kappaB activation in melanoma progression.

Expression of p16INK4A, the product of the melanoma susceptibility gene CDKN2A, has been shown to decrease in correlation with tumor progression. P16INK4A is a key regulator of cell-cycle function, and likely interacts with a variety of targets alongside cyclin-dependent kinases (CDKs). One such target is nuclear factor KB (NF-kappaB), a pleiotropic transcription factor that plays a crucial role in apoptosis, oncogenesis and cell cycle control. NF-kappaB p65 has been shown to be activated in melanoma cell lines but few studies decribe its expression in the tissue. In the present study we focused on synchronous expression of p16INK4A and NF-kappaB p65 and their functional activation in melanoma cell lines and biopsy tissue. Activation of NF-kappaB p65, as observed by electrophoretic mobility shift assay in cell lines, was correlated with expression and cellular localization of the active and inactive forms of its inhibitor, IkappaB-alpha. In melanocytic lesions, p16INK4A and NF-kappaB p65 expression were inversely correlated with levels of the nuclear component of NF-kappaB p65 increasing from nevi to primary melanomas and metastases.

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.

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.1682-1683 delTA in CCM1; c.48A > G; c.82-83dupAG in CCM2; and c.395 + 1G > A in CCM3 genes [corrected].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.

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.