Clinical and genetic familial study of a large cohort of Italian children with idiopathic epilepsy.

Epilepsies are characterized by genetic heterogeneity and by the possible coexistence of different phenotypes in one family. Moreover, in different epilepsies, mutations in the same gene have been reported. We aimed to collect data in a large Italian cohort of 81 families with children affected by partial or generalized epilepsies and to evaluate the prevalence of several ion channel mutations. In particular, a clinical and genetic survey was performed and DNA regions known to be associated with several epilepsies were analysed by sequencing. We observed genetic complexity in all phenotype groups: any epileptic type may be transmitted as either autosomal dominant or recessive. No significant phenotype identity among generations and no differences among genders could be observed. Two missense mutations in SCN1A were identified in two GEFS+ probands confirming the importance of this channel for this epilepsy. Moreover, a previously unreported CLCN2 mutation was detected in a proband showing CAE. In conclusion, even in this highly heterogeneous cohort, the complexity of the epileptic condition was highlighted and mutations in the analysed candidate region of ion channel genes appear to explain only a minority of cases.

CHRNA2 mutations are rare in the NFLE population: evaluation of a large cohort of Italian patients.

OBJECTIVE: Forty-six nocturnal frontal lobe epilepsy (NFLE) patients (in which the involvement of the CHRNA4 and CHRNB2 genes coding for neuronal nicotinic acetylcholine receptor (nAChRs) subunits associated to the disease were previously excluded) were analyzed for the presence of mutations in the CHRNA2 gene coding for the alpha2 subunit of the same receptor, which has been recently associated with the disease. METHODS: Mutational screening was performed by sequencing two polymerase chain reaction-amplified CHRNA2 DNA fragments, spanning the whole exon 6 and exon 7, respectively, which code for approximately 75% of the mature protein and contain all four transmembrane domains contributing to the ion pore. RESULTS: No mutations were identified in the analyzed region of CHRNA2. CONCLUSIONS: These data demonstrate the rarity of the identified CHRNA2 mutations in NFLE patients, supporting the recently reported hypothesis of a restricted role for this gene in the disease.

Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.

BACKGROUND: Factor V deficiency is a rare autosomal recessive hemorrhagic disorder, associated with bleeding manifestations of variable severity. In the present study, we investigated the molecular basis of factor V deficiency in three patients, and performed a comprehensive analysis of the factor V gene (F5) splicing pattern. DESIGN AND METHODS: Mutational screening was performed by DNA sequencing. Wild-type and mutant F5 mRNA were expressed by transient transfection in COS-1 cells, followed by reverse-transcriptase polymerase chain reaction and sequencing. Real-time reverse-transcriptase polymerase chain reaction was used to evaluate degradation of mRNA carrying premature termination codons. RESULTS: Mutational screening identified three hitherto unknown splicing mutations (IVS8+6T>C, IVS21+1G>A, and IVS24+1_+4delGTAG). Production of mutant transcripts in COS-1 cells demonstrated that both IVS21+1G>A and IVS24+1_+4delGTAG cause the activation of cryptic donor splice sites, whereas IVS8+6T>C causes exon-8 skipping (F5-Delta 8-mRNA). Interestingly, F5-Delta 8-mRNA was also detected in wild-type transfected samples, human liver, platelets, and HepG2 cells, demonstrating that F5 exon-8 skipping takes place physiologically. Since F5-Delta 8-mRNA bears a premature termination codons, we investigated whether this transcript is subjected to nonsense-mediated mRNA decay degradation. The results confirmed the involvement of nonsense-mediated mRNA decay in the degradation of F5 PTC(+) mRNA. Moreover, a comprehensive analysis of the F5 splicing pattern led to the identification of two in-frame splicing variants resulting from skipping of exons 3 and 5-6. CONCLUSIONS: The functional consequences of three splicing mutations leading to FV deficiency were elucidated. Furthermore, we report the identification of three alternatively spliced F5 transcripts.

Congenital hypofibrinogenemia: characterization of two missense mutations affecting fibrinogen assembly and secretion.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (alphaCys45Phe), and one previously described (gammaAsn345Ser). The involvement of alphaCys45Phe and gammaAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (alphaCys45Phe) or by hampering hexamer secretion (gammaAsn345Ser).

Simultaneous genotyping of coagulation factor XI type II and type III mutations by multiplex real-time polymerase chain reaction to determine their prevalence in healthy and factor XI-deficient Italians.

BACKGROUND: Factor XI deficiency is a rare autosomal recessive coagulopathy, which is, however, common among Ashkenazi Jews, in whom the so-called type II (E117X) and type III (F283L) mutations account for 98% of alleles. In non-Jewish populations, a higher level of allelic heterogeneity has been reported. However, the type II mutation was found in individuals from England, Portugal, and Italy, and haplotype analysis confirmed its Jewish origin. The aims of this study were to develop a rapid and accurate assay for the simultaneous detection of type II/type III mutations and to determine the frequency of these mutations in a large Italian population of healthy individuals and in a cohort of factor XI-deficient Italian patients. DESIGN AND METHODS: Type II and III mutations were detected using a newly developed multiplex four-color real-time polymerase chain reaction assay. Haplotype analysis was performed by either DNA sequencing or fragment-length analysis. RESULTS: Both type II and type III mutations were found among 3879 healthy Italians with an allele frequency of 0.00064 and 0.00051, respectively. Among the 31 analyzed factor XI-deficient patients, the type II mutation was found in three individuals in the homozygous state and in eight individuals in the heterozygous state (one compound heterozygote type II/III). Haplotype analysis revealed the Jewish origin of both mutations. CONCLUSIONS: The newly developed assay is highly specific and reliable (0.02% false positives); and offers a useful means for the molecular diagnosis of factor XI deficiency. Type II and III mutations are present in the Italian population and should be searched for first in factor XI-deficient patients.

Non-random retention of protein-coding overlapping genes in Metazoa.

BACKGROUND: Although the overlap of transcriptional units occurs frequently in eukaryotic genomes, its evolutionary and biological significance remains largely unclear. Here we report a comparative analysis of overlaps between genes coding for well-annotated proteins in five metazoan genomes (human, mouse, zebrafish, fruit fly and worm). RESULTS: For all analyzed species the observed number of overlapping genes is always lower than expected assuming functional neutrality, suggesting that gene overlap is negatively selected. The comparison to the random distribution also shows that retained overlaps do not exhibit random features: antiparallel overlaps are significantly enriched, while overlaps lying on the same strand and those involving coding sequences are highly underrepresented. We confirm that overlap is mostly species-specific and provide evidence that it frequently originates through the acquisition of terminal, non-coding exons. Finally, we show that overlapping genes tend to be significantly co-expressed in a breast cancer cDNA library obtained by 454 deep sequencing, and that different overlap types display different patterns of reciprocal expression. CONCLUSION: Our data suggest that overlap between protein-coding genes is selected against in Metazoa. However, when retained it may be used as a species-specific mechanism for the reciprocal regulation of neighboring genes. The tendency of overlaps to involve non-coding regions of the genes leads to the speculation that the advantages achieved by an overlapping arrangement may be optimized by evolving regulatory non-coding transcripts.

Molecular characterization of two novel mutations causing factor XI deficiency: A splicing defect and a missense mutation responsible for a CRM+ defect.

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency.

Pseudo-exon activation caused by a deep-intronic mutation in the fibrinogen gamma-chain gene as a novel mechanism for congenital afibrinogenaemia.

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

Activation of NF-kappaB by IL-1beta blocks IL-6-induced sustained STAT3 activation and STAT3-dependent gene expression of the human gamma-fibrinogen gene.

Despite the essential role of the fibrinogen gamma-chain as a blood clotting factor, the fibrinogen gamma-chain contains a number of interaction sites to recruit other factors such as leukocytes important for prevention of pathogen entry and propagation of the repair process. Interleukin-6 (IL-6) is known as the major inducer of gamma-fibrinogen synthesis in hepatocytes, whereas IL-1beta has been shown to act as a potent inhibitor of gamma-fibrinogen expression. Studies on the rat fibrinogen gamma-chain promoter suggest that nuclear factor (NF)-kappaB replaces the signal transducer and activator of transcription (STAT) 3 from binding to overlapping NF-kappaB/STAT3 binding sites within the 5' regulatory region of the rat gamma-chain gene promoter. However, despite its physiological relevance, the underlying mechanism responsible for the inhibitory effect of IL-1beta in humans is still not understood and apparently more complex. In contrast to the mechanism described for the rat gene our results indicate that IL-1beta suppresses the IL-6-induced activation of the human gamma-fibrinogen gene particularly by blocking the late phase STAT3-tyrosine phosphorylation NF-kappaB-dependently but independent from de novo protein synthesis. Consequently, blocking NF-kappaB activation restores specifically late phase STAT3 activation as well as the induction of the human gamma-fibrinogen gene. In contrast, specifically early STAT3 activation could be restored by a block of the p38 mitogen-activated protein kinase (p38(MAPK)) pathway. In summary, our results indicate that expression of the gamma-fibrinogen gene is mainly controlled by the strength of late phase STAT3 activation, which in turn is negatively regulated by the extent of IL-1beta-mediated NF-kappaB activity.

Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient.

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.

Mutational screening of six afibrinogenemic patients: identification and characterization of four novel molecular defects.

Congenital afibrinogenemia (CAF) is a rare coagulation disorder characterized by very low or unmeasurable levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait (prevalence 1:1,000,000) and is invariantly associated with mutations affecting one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Fibrinogen is secreted by hepatocytes as a hexamer composed of two copies of each chain; the lack of one chain has been demonstrated to prevent its secretion. Most genetic defects causing afibrinogenemia are point mutations, whereas only three large deletions have been identified so far, all affecting the FGA gene. We here report the molecular characterization of six unrelated afibrinogenemic patients leading to the identification of eight different mutations, four hitherto unknown: a 4.1-Kb large deletion involving exon 1 of FGA (AC107385:g. 65682_69828del), two nonsense mutations (p.Trp229X in FGA and p.Trp266X in FGB), and an ins-del mutation (g.1787_1789del3ins12) in FGA. The molecular characterization of CAF-causing genetic defects increases our understanding on the genetic basis of this disease and might be helpful for prenatal screening purposes, as also demonstrated during this study.

Association of NOD2/CARD15 mutations on Crohn's disease phenotype in an Italian population.

AIMS: To confirm the prevalence of NOD2/CARD15 mutations in Italian inflammatory bowel disease patients and to define the role of the different mutations on Crohn's disease phenotype. PATIENTS AND METHODS: A total of 177 patients with Crohn's disease and 92 patients with ulcerative colitis and 164 control participants were investigated for the presence of Arg702Trp, Gly908Arg and Leu1007fsinsC NOD2/CARD15 mutations. Allele frequencies in Crohn's disease and ulcerative colitis patients were compared with those observed in the control population. Genotype-phenotype correlations with the major clinical features were also established and estimated risks (odds ratio with 95% confidence interval) for the mutations were calculated by logistic regression and multiple correspondent analysis. RESULTS: Gly908Arg and Leu1007fsinsC mutations were significantly more frequent in Crohn's disease patients compared with healthy controls (P<0.01 and <0.003 respectively). Indeed, using a logistic regression model adding terms for age (differently distributed between cases and controls) and sex, a significantly increased risk of having Crohn's disease compared with healthy controls was found for all NOD2 mutations: Leu1007fsinsC (odds ratio=7.35; 95% confidence interval: 1.73-31.3), Gly908Arg (odds ratio=5.70; 95% confidence interval: 1.37-23.7) and Arg702Trp (odds ratio=2.45; 95% confidence interval: 1.10-5.47). As far as the genotype-phenotype correlations are concerned, by multivariate conditional logistic regression methods, we found a significant association between Gly908Arg mutations and familial history of inflammatory bowel disease, between Leu1007fsinsC mutations and appendectomy and between Arg702Trp mutations and fibrostenotic phenotype of Crohn's disease. A nonsignificant association between Arg702Trp variants and ileal disease was also found (odds ratio=8, 95% confidence interval: 0.99-64.9). CONCLUSIONS: The results of the study confirm a significant association of CARD15 gene mutations in our Italian Crohn's disease population and the impact of different NOD2/CARD15 mutations on specific disease phenotypes.

Coagulation factor V gene analysis in five Indian patients: identification of three novel small deletions.

Congenital factor V (FV) deficiency is a rare coagulopathy associated with moderate to severe bleeding symptoms. A total of 34 mutations, all located in the FV gene (F5), have been described in patients with severe FV deficiency, only eight of them being of Asian descent. Sequencing of F5 in five unrelated Indian patients identified three novel small deletions in exon 13, all present in the homozygous state (g.50936-50937delAA or AG and g.51660delA, both occurring in two different patients, and g.52162delC). Besides widening the knowledge on the mutational spectrum of FV deficiency in Asian populations, these data will also be useful for purposes of prenatal diagnosis.

Fibrinogen Mumbai: intracellular retention due to a novel G434D mutation in the Bbeta-chain gene.

BACKGROUND AND OBJECTIVES: Afibrinogenemia and hypofibrinogenemia are rare inherited coagulation disorders characterized by hemorrhagic manifestations of variable entity and by plasma fibrinogen deficiency. So far, 57 mutations have been associated with these disorders, and 18 of these are missense mutations. The aim of this study was to characterize the molecular mechanism underlying severe hypofibrinogenemia in a proband from India. DESIGN AND METHODS: The mutational screening was accomplished by DNA sequencing of the three fibrinogen genes. The mutant protein was expressed in COS-1 cells, and intracellular and secreted mutant fibrinogen was analyzed by means of pulse-chase experiments. RESULTS: A novel homozygous G-->A transition in exon 8 (nucleotide position 8017) was found in the proband's fibrinogen Bbeta-chain gene. The resulting G434D missense mutation (fibrinogen Mumbai) involves a highly conserved amino acid residue, located in the C-terminal globular D domain. In vitro expression experiments demonstrated intracellular retention of the mutant fibrinogen and marked reduction of its secretion. INTERPRETATION AND CONCLUSIONS: The G434D substitution causes severe hypofibrinogenemia by impairing fibrinogen secretion. Expression data confirm the importance of Bbeta-chain D domain folding in the intracellular processing of fibrinogen.

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

A type II mutation (Glu117stop), induction of allele-specific mRNA degradation and factor XI deficiency.

The Glu117stop mutation in the factor XI (FXI) gene is the most common cause of FXI deficiency and might cause the disease either by poor secretion/stability of the truncated protein or by decreased mRNA levels. Platelet- and lymphocyte-derived mRNA from three Glu117stop heterozygotes were analyzed by reverse-transcriptase polymerase chain reaction and sequencing, demonstrating allele-specific reduction of FXI Glu117stop mRNA.

Two new putative susceptibility loci for ADNFLE.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) has been up to now considered a simple Mendelian trait caused by mutations in neuronal nicotinic acetylcholine receptor (nAChR) subunit genes. We previously demonstrated that in a three-generation Italian family the disease was unlinked to all known ADNFLE loci as well as to all known brain-expressed nAChR subunits. The genome-wide linkage analysis here presented performed on this family points to the existence of two new putative ADNFLE loci on chromosomes 3p22-p24 and 8q11.2-q21.1. These findings, together with several ADNFLE characteristics, suggest that this epilepsy could be, at least in the above family, a complex disorder. In particular, we propose and discuss the hypothesis of a digenic transmission of the disease.

In vivo RNA-RNA duplexes from human alpha3 and alpha5 nicotinic receptor subunit mRNAs.

Natural antisense transcripts, because of their potential to form double-stranded RNA (dsRNA) molecules, recently emerged as a mechanism acting on eukaryotic gene regulation at multiple levels. CHRNA3 and CHRNA5, coding for alpha3 and alpha5 subunits of the neuronal nicotinic acetylcholine receptor, have been reported to overlap at their 3'ends in human and bovine genomes. In the present paper, four CHRNA3 and three CHRNA5 human transcripts were characterised, leading to the identification of different antisense complementary regions. Since the two genes are coexpressed in some neuronal and non-neuronal tissues, we ventured on the in vivo identification of RNA-RNA duplexes in both humans and cattle. Using an RNase protection-based approach, CHRNA3/CHRNA5 duplexes were detected in human neuroblastoma SY5Y cells, but not in bovine cerebellum. A semi-quantitative analysis of overlapping transcript levels was performed by real-time RT-PCR. Possible consequences of sense-antisense interaction are discussed.