Rosalia D'Angelo

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.

Sarcoglycan Subcomplex Expression in Normal Human Smooth Muscle

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other members of the dystrophin–glycoprotein complex (DGC) to provide a mechanosignaling connection from the cytoskeleton to the extracellular matrix. The SGC consists of four proteins (α, β, γ, and δ). A fifth sarcoglycan subunit, ∊-sarcoglycan, shows a wider tissue distribution. Recently, a novel sarcoglycan, the ζ-sarcoglycan, has been identified. All reports about the structure of SGC showed a common assumption of a tetrameric arrangement of sarcoglycans. Addressing this issue, our immunofluorescence and molecular results showed, for the first time, that all sarcoglycans are always detectable in all observed samples. Therefore, one intriguing possibility is the existence of a pentameric or hexameric complex considering ζ-sarcoglycan of SGC, which could present a higher or lower expression of a single sarcoglycan in conformity with muscle type—skeletal, cardiac, or smooth—or also in conformity with the origin of smooth muscle. (J Histochem Cytochem 55:831–843, 2007)

FMO3 allelic variants in Sicilian and Sardinian populations: trimethylaminuria and absence of fish-like body odor.

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria. The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype-phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals. Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K-308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population. Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath. Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.

CCM2 gene polymorphisms in Italian sporadic patients with cerebral cavernous malformation: A case-control study

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities that can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10). Among our group of CCM Italian patients, we selected a cohort of sporadic cases negative for mutations in CCM genes. In this cohort, five variants in CCM2 gene were detected, which proved to be the known polymorphisms in intronic regions (IVS2-36A>G and IVS8 +119 C>T) and in coding sequence (c.157 G>A in exon 2, c.358 G>A in exon 4 and c.915 G>A in exon 8). Therefore, we undertook a case-control study to investigate the possible association of these polymorphisms with sporadic CCMs. The five polymorphisms were identified in 91 CCM sporadic patients and in 100 healthy controls by direct sequencing methods using lymphocyte DNA. Polymorphisms IVS2-36A>G and c.915 G>A showed statistically significant differences in frequencies between patients and controls [(χ2, 6.583; P<0.037); (χ2, 14.205; P<0.001)]. The prevalence of the wild-type genotype was significantly lower in the CCM group than in the control sample. Patients with the A/G and G/G genotypes (IVS2-36A>G) had a significant increase for CCM risk (OR, 3.08; 95% CI, 1.5-5.9 and OR, 4.3; 95% CI, 1.4-22.6) and the same was observed for the polymorphism c.915 G> A (genotype G/A OR, 6.1; 95% CI, 3.0-12.6 and genotype A/A OR, 2.79). In addition, the polymorphisms c.358 G>A in exon 4 (χ2, 15.977; P<0.04) and c.915 G>A in exon 8 (χ2, 18.109; P<0.02) were significantly associated with different types of symptoms. Haplotype analysis, performed only on polymorphisms c.358 G>A (p.Val120Ile), c.915 G>A (p.Thr305 Thr) and IVS2-36A>G, shows that haplotype GAG (+--) significantly increased among CCM sporadic patients compared to the control group. Significant differences between patients and controls were observed only for IVS2-36A>G and c.915 G>A polymorphisms indicating their possible association with sporadic CCMs and an increased risk of CCM. On the other hand, polymorphisms c.358 G>A and c.915 G>A were associated with a more benign course of the disease. These data were confirmed by the haplotype GAG (+--) frequencies.

Sporadic cerebral cavernous malformations: report of further mutations of CCM genes in 40 Italian patients.

Cerebral cavernous malformations (CCMs) are vascular lesions characterized by abnormally enlarged capillary cavities, affecting the central nervous system. CCMs can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (K-Rev interaction trapped 1 (KRIT1)), CCM2 (MGC4607), and CCM3 (PDCD10). CCMs occur as a single or multiple malformations that can lead to seizures, focal neurological deficits, hemorrhagic stroke, and headache. However, patients are frequently asymptomatic. In our previous mutation screening, performed in a cohort of 95 Italian patients, both sporadic and familial, we have identified several mutations in CCM genes, three of which in three distinct sporadic patients. In this study, representing further molecular screening of the three CCM genes, in a south Italian cohort of CCM patients enrolled by us in the last three years, we report the identification of other four new mutations in 40 sporadic patients with either single or multiple CCM.

Identification of a novel CCM2 gene mutation in an Italian family with multiple cerebral cavernous malformations and epilepsy: a causative mutation?

Cerebral cavernous malformations (CCMs; OMIM 116860) are vascular anomalies mostly located in the central nervous system (CNS) and occasionally within the skin and retina. Main clinical manifestations are seizure, hemorrhage, recurrent headaches, focal neurological deficits and epileptic attacks. The CCMs can occur as sporadic or autosomal dominant conditions, although with incomplete penetrance and variable clinical expression. Familial CCMs were associated with causative mutations in the CCM1 [K-Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. This study reports the identification of a previously undescribed deletion mutation in CCM2 gene exon 5, in an Italian family with multiple cerebral cavernous malformations and epilepsy. Mutation c.502_503delAG results in a frame shift causing a TGA stop codon. This truncates the mutant CCM2 gene protein, the malcavernin, to 233 amino acids, respect to 444 amino acids of the wild-type malcavernin. By using real-time RT-PCR, we have found that the mRNA resulting from two nucleotides deletion showed a 70% reduction relative to the wild-type transcript, indicating that it may be subject to a degradation mechanism such as nonsense-mediated decay (NMD).

CCR5Δ32 Polymorphism Associated with a Slower Rate Disease Progression in a Cohort of RR-MS Sicilian Patients

Multiple sclerosis (MS) disease is carried through inflammatory and degenerative stages. Based on clinical feaures, it can be subdivided into three groups: relapsing-remitting MS, secondary progressive MS, and primary progressive MS. Multiple sclerosis has a multifactorial etiology with an interplay of genetic predisposition, environmental factors, and autoimmune inflammatory mechanism in which play a key role CC-chemokines and its receptors. In this paper, we studied the frequency of CCR5 gene Δ32 allele in a cohort of Sicilian RR-MS patients comparing with general Sicilian population. Also, we evaluate the association between this commonly polymorphism and disability development and age of disease onset in the same cohort. Our results show that presence of CCR5Δ32 is significantly associated with expanded disability status scale score (EDSS) but not with age of disease onset.

miRNAexpression profile of retinal pigment epithelial cells under oxidative stress conditions

Deep analysis of regulative mechanisms of transcription and translation in eukaryotes could improve knowledge of many genetic pathologies such as retinitis pigmentosa (RP). New layers of complexity have recently emerged with the discovery that ‘junk’ DNA is transcribed and, among these, miRNAs have assumed a preponderant role. We compared changes in the expression of miRNAs obtained from whole transcriptome analyses, between two groups of retinal pigment epithelium (RPE) cells, one untreated and the other exposed to the oxidant agent oxidized low‐density lipoprotein (oxLDL), examining four time points (1, 2, 4 and 6 h). We found that 23 miRNAs exhibited altered expression in the treated samples, targeting genes involved in several biochemical pathways, many of them associated to RP for the first time, such as those mediated by insulin receptor signaling and son of sevenless. Moreover, five RP causative genes (KLHL7, RDH11, CERKL, AIPL1 and USH1G) emerged as already validated targets of five altered miRNAs (hsa‐miR‐1307, hsa‐miR‐3064, hsa‐miR‐4709, hsa‐miR‐3615 and hsa‐miR‐637), suggesting a tight connection between induced oxidative stress and RP development and progression. This miRNA expression analysis of oxidative stress‐induced RPE cells has discovered new regulative functions of miRNAs in RP that should lead to the discovery of new ways to regulate the etiopathogenesis of RP.

PON I and GLO I Gene Polymorphisms and Their Association with Breast Cancer: A Case-Control Study in a Population from Southern Italy

Background: Many factors may play a role in the susceptibility to the breast cancer. Oxidative stress may be one of these. Polymorphisms of genes such as paraoxonase I (PON I) and glyoxalase I (GLO I) may influence individual susceptibility to breast cancer. In the present study, we have conducted a case-control study in order to examine the possible relation between GLO I A111E and PON I Q192R/L55M polymorphisms with the risk of breast cancer. Methods: The three polymorphisms were characterized in 144 breast cancer postmenopausal patients and in 152 healthy women by PCR/RFLP methods using DNA from lymphocytes. Results: Among the three polymorphisms, only PON I L55M polymorphism was associated with the patient’s age and, more precisely, the heterozygous genotype that is more represented in women aged between 51-69 years. In addition, we found that individuals with the PON192 Q/R - R/R genotypes and PON55 L/M - M/M genotypes had a significantly higher risk of breast cancer compared with the other genotypes. The genotypes PON55 L/M and PON192 Q/R showed significant association with lymph nodes positivity (p < 0.001) and with a high nuclear grading (p < 0.001), respectively. Conversely the genotypes GLO I AE/EE were associated with a low nuclear grading. Conclusions: We believe that the combination of the three polymorphisms may be a more predictive factor for the risk of this neoplasia in each single examined case.

Further data on a 9.1-kb insertion-deletion polymorphism: survey of Mediterranean populations.

ABSTRACT We report the distribution of a previously described 9.1-kb insertion-deletion polymorphism located on chromosome 22. We analyzed 1,844 individuals sampled from 26 Mediterranean populations in mainland Italy, Sicily, Sardinia, Tunisia, Libya, Morocco, Egypt, Greece, and Albania. The 9.1 kb − allele is the prevalent allele in the North African (range, 0.53–0.56), Greek (0.51), and Albanian (0.66) populations, whereas the 9.1 kb + allele is most frequent in a mainland Italian town (0.55) and in all Sicilian and Sardinian towns and villages thus far tested, with marked fluctuation ranges of 0.53–0.78 and 0.56–0.80, respectively. In tests for Hardy-Weinberg equilibrium the genotype frequencies observed in Athens and in four of the nine towns in Sicily (but in none of the towns in Sardinia) departed highly significantly from the expected values. Identical results were found in the same towns for a second insertion-deletion polymorphism located on chromosome 22q13 at a distance compatible with a low incidence of recombination. The data, which are in good agreement with the different histories of the two islands (Sardinia and Sicily), are consistent with a west-east differentiation in Sicily and support the evidence for ancient gene flow from the Iberian peninsula to Sardinia.