Maria Luisa Tenchini

Autosomal dominant nocturnal frontal lobe epilepsy - A critical overview

Abstract.Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an idiopathic epilepsy, with a spectrum of clinical manifestations, ranging from brief, stereotyped, sudden arousals to more complex dystonic–dyskinetic seizures. Video–polysomnography allows a correct differential diagnosis. There is no difference between sporadic nocturnal frontal lobe epilepsy (NFLE) and ADNFLE in the clinical and neurophysiological findings. ADNFLE is the first idiopathic epilepsy for which a genetic basis has been identified. Mutations have been found in two genes (CHRNA4 and CHRNB2) coding for neuronal nicotinic receptor subunits (α4 and β2, respectively). Contrasting data have been reported on the effect of these mutations on the functionality of the receptor.Moreover, the incomplete data on the neuronal network/s in which this receptor is involved, make difficult the understanding of the genotype–phenotype correlation. This is an overview on the clinical and genetic aspects of ADNFLE including a discussion of some open questions on the role of the neuronal nicotinic receptor subunit mutations in the pathogenesis of this form of epilepsy.

The molecular basis of quantitative fibrinogen disorders

Summary.  Hereditary fibrinogen disorders include type I deficiencies (afibrinogenemia and hypofibrinogenemia, i.e. quantitative defects), with low or unmeasurable levels of immunoreactive protein; and type II deficiencies (dysfibrinogenemia and hypodysfibrinogenemia, i.e. qualitative defects), showing normal or altered antigen levels associated with reduced coagulant activity. While dysfibrinogenemias are in most cases autosomal dominant disorders, type I deficiencies are generally inherited as autosomal recessive traits. Patients affected by congenital afibrinogenemia or severe hypofibrinogenemia may experience bleeding manifestations varying from mild to severe. This review focuses on the genetic bases of type I fibrinogen deficiencies, which are invariantly represented by mutations within the three fibrinogen genes (FGA, FGB, and FGG) coding for the three polypeptide chains Aα, Bβ, and γ. From the inspection of the mutational spectrum of these disorders, some conclusions can be drawn: (i) genetic defects are scattered throughout the three fibrinogen genes, with only few sites appearing to represent relative mutational hot spots; (ii) several different types of genetic lesions and pathogenic mechanisms have been described in affected individuals (including gross deletions, point mutations causing premature termination codons, missense mutations affecting fibrinogen assembly/secretion, and uniparental isodisomy associated with a large deletion); (iii) the possibility to express recombinant fibrinogen mutants in eukaryotic cells is rapidly shedding light into the molecular mechanisms responsible for physiologic and pathologic properties of the molecule; (iv) though mutation analysis of the fibrinogen cluster does not yield precise information for predicting genotype/phenotype correlations, it still provides a valuable tool for diagnosis confirmation, identification of potential carriers, and prenatal diagnosis.

Inherited defects of coagulation factor V: the hemorrhagic side

Summary.  Coagulation factor V (FV) is the protein cofactor required in vivo for the rapid generation of thrombin catalyzed by the prothrombinase complex. It also represents a central regulator in the early phases of blood clot formation, as it contributes to the anticoagulant pathway by participating in the downregulation of factor VIII activity. Conversion of precursor FV to either a procoagulant or anticoagulant cofactor depends on the local concentration of procoagulant and anticoagulant enzymes, so that FV may be regarded as a daring tight‐rope walker gently balancing opposite forces. Given this dual role, genetic defects in the FV gene may result in opposite phenotypes (hemorrhagic or thrombotic). Besides a concise description on the structural, procoagulant and anticoagulant properties of FV, this review will focus on bleeding disorders associated with altered levels of this molecule. Particular attention will be paid to the mutational spectrum of type I FV deficiency, which is characterized by a remarkable genetic heterogeneity and by an uneven distribution of mutations throughout the FV gene.

Frontal lobe epilepsy and mutations of the corticotropin-releasing hormone gene

Nocturnal frontal lobe epilepsy up to now has been considered a channelopathy caused by mutations in the α4 and β2 subunits of the neuronal nicotinic acetylcholine receptor. However, these mutations account for only a minority of patients, and the existence of at least a new locus for the disease has been demonstrated. In one Italian nocturnal frontal lobe epilepsy family, we identified two new putative loci on chromosomes 3 and 8, where several candidate genes are mapped. In particular, on chromosome 8, corticotropin‐releasing hormone gene (CRH) appears to be a good candidate. We therefore searched for CRH mutations in the proband. The study allowed the identification of a nucleotide variation in the promoter that was subsequently detected in all affected and obligate carrier members of the same family, in two sporadic cases, in all affected members of an additional compliant family, and in the proband of a noncompliant family. Moreover, a different mutation in the promoter was detected in a familial case. In vitro experiments showed altered levels of gene expression. CRH alterations could explain several autosomal dominant nocturnal frontal lobe epilepsy clinical features. Ann Neurol 2006

Multidrug resistance 1 gene polymorphism and susceptibility to inflammatory bowel disease

Background Several studies have evaluated the role of the multidrug resistance 1 gene (MDR1) polymorphism, which encodes the membrane‐bound efflux transporter P‐glycoprotein 170, in determining susceptibility to and disease behavior in inflammatory bowel disease (IBD), but with conflicting results. Methods A total of 211 patients with Crohns disease (CD), 97 patients with ulcerative colitis (UC), and 212 control subjects were investigated for the presence of MDR1 G2677T/A and C3435T polymorphisms. Genotype frequencies of CD and UC patients were compared to those observed in a control population. Genotype–phenotype correlations with major clinical features were also established and estimated risks (odds ratio [OR] with 95% confidence interval [CI]) for the mutations were calculated by a logistic regression analysis and multiple correspondent analysis. Results No significant difference was observed for genotype frequencies for both MDR1 G2677T/A and C3435T polymorphisms on overall disease susceptibility for either CD or UC patients compared with control subjects. A significant association was found between the MDR1 C3435T polymorphism and patients with ileo‐colonic CD (OR = 3.34; 95% CI: 1.34–8.27). Interestingly, a negative association was found between MDR1 C3435T polymorphism in patients with a positive family history for IBD (OR = 0.44; 95% CI: 0.20–0.95) and articular manifestations (OR = 0.29; 95% CI: 0.13–0.68). Both susceptible and protective effects were identified. No significant association between G2677T/A polymorphism and any specific subphenotypes was found, nor was there any association with subphenotypic categories of UC and both single nucleotide polymorphisms. Conclusions The results of our study suggest that MDR1 gene polymorphism could have a role in determining susceptibility to IBD. The variability of this possible effect in the several studies reported so far may be the indirect expression of the complex role played by the MDR1 gene and its product, P‐glycoprotein 170, in the regulation of host–bacteria interactions and in the pathogenesis of IBD. (Inflamm Bowel Dis 2007)

Culture techniques for human keratinocytes

The two main approaches developed for in vitro culture of human keratinocytes are reviewed and discussed. The older technique is based on the use of a serum-containing medium and of a feeder-layer of lethally irradiated mouse fibroblasts; the second relies upon serum-free media, in the absence of a feeder-layer. While the former technique is most widely used for the preparation of epithelial sheets for grafting, the latter is best suited for studies on the biological properties of keratinocytes.

Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites.

Congenital afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in afibrinogenemia, 17 in the Aalpha, 6 in the gamma, and only 2 in the Bbeta fibrinogen-chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bbeta-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bbeta-chain gene splicing mutations described in afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bbeta-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase-polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bbeta chain for fibrinogen assembly and secretion.

Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bβ-chain gene

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.

A novel two base pair deletion in the factor V gene associated with severe factor V deficiency

We studied a family in which the proband, a 13‐year‐old boy, had unmeasurable plasma levels of coagulation factor V antigen and activity. Clinical symptoms were severe, with several episodes of haemorrhages in the mucosal tracts (gastrointestinal, nose and urinary) and recurrent haemarthroses that caused permanent arthropathy. Sequence analysis of the factor V gene demonstrated the presence of a novel 2 base pair (bp) homozygous deletion in exon 13 at positions 2833–2834. This mutation, present in the heterozygous state in the asymptomatic mother and absent in the healthy brother, introduced a frameshift and a premature stop at codon 900. This would predict the synthesis of a truncated factor V molecule, lacking part of the B domain and the complete light chain. Because of the existence of a surveillance mechanism that selectively recognizes and degrades mRNA molecules carrying premature termination codons, we analysed the relative abundance of mutant vs. wild‐type mRNA molecules in the platelets of the heterozygous probands mother. The mutant mRNA was significantly reduced in amount (mutant/wild‐type ratio 0·35). This is the first reported mutation in the factor V gene causing severe factor V deficiency, the effect of which was quantitatively analysed at mRNA level.

Congenital afibrinogenaemia caused by uniparental isodisomy of chromosome 4 containing a novel 15-kb deletion involving fibrinogen Aα-chain gene

Among rare inherited deficiencies of coagulation factors, congenital afibrinogenaemia is characterised by the lack of fibrinogen in plasma. In the last few years, several genetic defects underlying afibrinogenaemia (mostly point mutations) have been described in the fibrinogen gene cluster. In this study, the molecular basis responsible for afibrinogenaemia in a Thai proband was defined. Point mutation screening was accomplished by directly sequencing the three fibrinogen genes. The impossibility to amplify fibrinogen Aα-chain gene (FGA) exons 5 and 6 suggested the presence of a homozygous deletion. A specific long-range PCR assay enabled the identification of a novel 15-kb deletion, representing the largest afibrinogenaemia-causing deletion described so far. Direct sequencing of the deletion junction allowed mapping of the breakpoints in FGA intron 4 and in the intergenic region between Aα- and Bβ-chain genes. Since the mutation was inherited only from the mother and nonpaternity was ruled out, a maternal uniparental disomy (UPD) was hypothesised. UPD test, carried out with markers covering the whole chromosome 4, revealed that maternal isodisomy was responsible for homozygosity of the 15-kb deletion in the proband. The apparently normal phenotype of the proband, except for afibrinogenaemia, suggests that UPD for chromosome 4 is clinically silent. This represents the first case of a documented complete isodisomy of chromosome 4 causing the phenotypic expression of a recessive disorder. In silico analyses of the regions surrounding the breakpoints suggested that the 15-kb deletion might have originated from an inappropriate repair of a double-strand break by the nonhomologous end joining mechanism.