Carmela Rinaldi

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.

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.

Expression of Sarcoglycans in the Human Cerebral Cortex: An Immunohistochemical and Molecular Study

The sarcoglycan (SG) complex (SGC) is a subcomplex within the dystrophin-glycoprotein complex (DGC) and is composed of several transmembrane proteins (α, β, δ, γ, ε and ζ). The DGC supplies a transmembranous connection between the subsarcolemmal cytoskeleton networks and the basal lamina in order to protect the lipid bilayer and to provide a scaffold for signaling molecules in all muscle cells. In addition to its role in muscle tissue, dystrophin and some DGC components are expressed in neurons and glia. Very little is known about the SG subunits in the central nervous system (CNS) and some data suggested the presence of ε and ζ subunits only. In fact, mutations in the ε-SG gene cause myoclonus-dystonia, indicating its importance for brain function. To determine the presence and localization of SGC in the human cerebral cortex, we performed an investigation using immunofluorescence, immunoblotting and reverse transcriptase polymerase chain reaction. The results showed that all SG subunits are expressed in the human cerebral cortex, particularly in large neurons but also in astrocytes. These data suggest that the SG subcomplex may be involved in the organization of CNS synapses.

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.

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

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.

Fish odor syndrome (trimethylaminuria) supporting the possible FMO3 down expression in childhood: a case report

IntroductionTrimethylaminuria is a rare inherited disorder due to decreased metabolism of dietary-derived trimethylamine by flavin-containing monooxygenase 3. Several single nucleotide polymorphisms of the flavin-containing monooxygenase 3 gene have been described and result in an enzyme with decreased or abolished functional activity for trimethylamine N-oxygenation thus leading to trimethylaminuria.Case presentationHere we investigated an Italian family in which the proband was a 7-year-old girl with suspected trimethylaminuria, by flavin-containing monooxygenase 3 gene direct sequencing and urinary determination of trimethylamine and trimethylamine N-oxide. Genetic analysis found that, as with her parents and one of her two brothers, the proband carried three polymorphisms: c.472 G>A p. E158K (rs 2266782) in exon 4, c.627+10 C>G (IVS5+10G>C) (rs 2066534) and c.485-21 G>A (IVS4-22G>A) (rs 1920149) in intronic regions.ConclusionsDespite the same genotypic condition only the girl had symptoms attributable to the trimethylaminuria. The suspicion is that she has transient childhood trimethylaminuria. Therefore, we bring attention to the importance of genetic testing and eventual determination of urinary trimethylamine and trimethylamine N-oxide as instruments to offer to clinicians in the management of these pediatric patients.

Sarcoglycan Complex in Human Normal and Pathological Prostatic Tissue: An Immunohistochemical and RT‐PCR Study

The sarcoglycan complex is a trans‐membrane system playing a key role in mechano‐signaling the connection from the cytoskeleton to the extracellular matrix. While β‐, δ‐, and ε‐sarcoglycans are widely distributed, γ‐ and α‐sarcoglycans are expressed exclusively in skeletal and cardiac muscle. Insufficient data are available on the distribution of sarcoglycans in nonmuscular tissue. In the present study, we used immunohistochemical and RT‐PCR techniques to study the sarcoglycans also in normal human glandular tissue, a type of tissue never studied in relation to the sarcoglycan complex, with the aim of verifying the real wider distribution of this complex. To understand the role of sarcoglycans, we tested specimens collected from patients affected by benign prostatic hyperplasia and adenocarcinoma. For the first time, our results showed that all sarcoglycans are detectable in normal samples both in epithelial and in myoepithelial cells; in pathological prostate, sarcoglycans appeared severely reduced in number or were absent. These data demonstrated that all sarcoglycans have a wider distribution suggesting a new unknown role for these proteins. The decreased number of sarcoglycans, containing cadherin domain homologs in samples of prostate affected by hyperplasia, and the absence of proteins in prostate biopsies, in cases affected by adenocarcinoma, could be responsible for the loss of adhesion between epithelial cells, which in turn facilitates the progression of benign tumors and the invasive potential of malignant tumors. Anat Rec, 297:327–336, 2014.