Aldo Amato

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.

Costameric proteins in human skeletal muscle during muscular inactivity.

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin–glycoprotein complex and vinculin–talin–integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive‐motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive‐motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of α7B‐integrin, β1D‐integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of α7A‐integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of α7B‐ and β1D‐integrin mRNA and a normal, or slightly higher than normal, level of α7A‐integrin mRNA in the skeletal muscle fibres of the patients with sensitive‐motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with α7B‐integrin being replaced by α7A‐integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down‐regulation of integrins and agrin in muscular dystrophies.

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)

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.

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.

Immunohistochemical localization of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta)

The present immunohistochemical study provides the first evidence of the presence of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta) a lower invertebrate. The entire epidermis was labelled for calmodulin which is in agreement with its ubiquitous occurrence. Immunopositivity for calbindin D28K was limited to mucous cells, while that for S-100 protein was present only in neuroendocrine-like small granular cells. Finally, labelling for parvalbumin was specifically present in the subcutaneous nerve plexus. S-100 protein is considered to be a marker of neuroendocrine cells, at least in lower invertebrates such as Annelida. Although calbindin D28K is considered to be a marker of these cells in vertebrates, the same function cannot be attributed in Lumbricus terrestris. However, we can conclude that S-100 protein, as a regulatory protein, is phylogenetically older than calbindin D28K. We assume that the latter has an autoregulatory function in secretory processes. In agreement with previous data, we suggest that small granular cells exert a paracrine action in osmoregulatory and secretory processes.

Age dependent switching role of cyclin D1 in breast cancer.

Background: Cyclin D1 gene (CCND1) plays pivotal roles in the development of several human cancers, including breast cancer, functioning as an oncogene. The aim of this study was to better understand the molecular dynamics of ductal carcinomas with regard to proliferation and the ageing process. Methods: 130 cases of ductal breast cancer in postmenopausal women, aged 52–96 in 3 age classes were selected. Tumoral tissues preserved in formaldehyde solution and subsequently embedded in paraffin were subjected to analysis Fluorescence in situ Hybridization (FISH), Reverse Transcription-Polymerase Chain Reaction (RT- PCR) and immuno-histochemical tests. The molecular variables studied were estimated in relation to the patients’ age. Results: The results obtained suggest that the increment of the levels of cyclin D1 in intra-ductal breast tumors in older woman that we have examined is significantly associated with a lower proliferation rate. Conclusion: Cyclin D1, which characterizes tumor in young women as molecular director involved in strengthening tumoral proliferation mechanisms, may be seen as a potential blocking molecular switch in corresponding tumours in old women.

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.