Emanuela Leonardi

Cx26 deafness: mutation analysis and clinical variability.

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found. Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.

Familial temporal lobe epilepsy with psychic auras associated with a novel LGI1 mutation

Background: Autosomal dominant lateral temporal epilepsy (ADLTE) is characterized by focal seizures with auditory features or aphasia. Mutations in the LGI1 gene have been reported in up to 50% of ADLTE pedigrees. We report a family with temporal lobe epilepsy characterized by psychic symptoms associated with a novel LGI1 mutation. Methods: All participants were personally interviewed and underwent neurologic examination and video-EEG recordings. LGI1 exons were sequenced by standard methods. Mutant cDNA was transfected into human embryonic kidney 293 cells; both cell lysates and media were analyzed by Western blot. In silico modeling of the Lgi1 protein EPTP domain was carried out using the structure of WD repeat protein and manually refined. Results: Three affected family members were ascertained, 2 of whom had temporal epilepsy with psychic symptoms (déjà vu, fear) but no auditory or aphasic phenomena, while the third had complex partial seizures without any aura. In all patients, we found a novel LGI1 mutation, Arg407Cys, which did not hamper protein secretion in vitro. Mapping of the mutation on a 3-dimensional protein model showed that this mutation does not induce large structural rearrangements but could destabilize interactions of Lgi1 with target proteins. Conclusions: The Arg407Cys is the first mutation with no effect on Lgi1 protein secretion. The uncommon, isolated psychic symptoms associated with it suggests that ADLTE encompasses a wider range of auras of temporal origin than hitherto reported.

Fly cryptochrome and the visual system

Cryptochromes are flavoproteins, structurally and evolutionarily related to photolyases, that are involved in the development, magnetoreception, and temporal organization of a variety of organisms. Drosophila CRYPTOCHROME (dCRY) is involved in light synchronization of the master circadian clock, and its C terminus plays an important role in modulating light sensitivity and activity of the protein. The activation of dCRY by light requires a conformational change, but it has been suggested that activation could be mediated also by specific “regulators” that bind the C terminus of the protein. This C-terminal region harbors several protein–protein interaction motifs, likely relevant for signal transduction regulation. Here, we show that some functional linear motifs are evolutionarily conserved in the C terminus of cryptochromes and that class III PDZ-binding sites are selectively maintained in animals. A coimmunoprecipitation assay followed by mass spectrometry analysis revealed that dCRY interacts with Retinal Degeneration A (RDGA) and with Neither Inactivation Nor Afterpotential C (NINAC) proteins. Both proteins belong to a multiprotein complex (the Signalplex) that includes visual-signaling molecules. Using bioinformatic and molecular approaches, dCRY was found to interact with Neither Inactivation Nor Afterpotential C through Inactivation No Afterpotential D (INAD) in a light-dependent manner and that the CRY–Inactivation No Afterpotential D interaction is mediated by specific domains of the two proteins and involves the CRY C terminus. Moreover, an impairment of the visual behavior was observed in fly mutants for dCRY, indicative of a role, direct or indirect, for this photoreceptor in fly vision.

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions. A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times. The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

INGA: protein function prediction combining interaction networks, domain assignments and sequence similarity

Identifying protein functions can be useful for numerous applications in biology. The prediction of gene ontology (GO) functional terms from sequence remains however a challenging task, as shown by the recent CAFA experiments. Here we present INGA, a web server developed to predict protein function from a combination of three orthogonal approaches. Sequence similarity and domain architecture searches are combined with protein-protein interaction network data to derive consensus predictions for GO terms using functional enrichment. The INGA server can be queried both programmatically through RESTful services and through a web interface designed for usability. The latter provides output supporting the GO term predictions with the annotating sequences. INGA is validated on the CAFA-1 data set and was recently shown to perform consistently well in the CAFA-2 blind test. The INGA web server is available from URL: http://protein.bio.unipd.it/inga.

A Novel WT1 Gene Mutation in a Three-Generation Family with Progressive Isolated Focal Segmental Glomerulosclerosis

BACKGROUND AND OBJECTIVES Wilms tumor-suppressor gene-1 (WT1) plays a key role in kidney development and function. WT1 mutations usually occur in exons 8 and 9 and are associated with Denys-Drash, or in intron 9 and are associated with Frasier syndrome. However, overlapping clinical and molecular features have been reported. Few familial cases have been described, with intrafamilial variability. Sporadic cases of WT1 mutations in isolated diffuse mesangial sclerosis or focal segmental glomerulosclerosis have also been reported. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Molecular analysis of WT1 exons 8 and 9 was carried out in five members on three generations of a family with late-onset isolated proteinuria. The effect of the detected amino acid substitution on WT1 proteins structure was studied by bioinformatics tools. RESULTS Three family members reached end-stage renal disease in full adulthood. None had genital abnormalities or Wilms tumor. Histologic analysis in two subjects revealed focal segmental glomerulosclerosis. The novel sequence variant c.1208G>A in WT1 exon 9 was identified in all of the affected members of the family. CONCLUSIONS The lack of Wilms tumor or other related phenotypes suggests the expansion of WT1 gene analysis in patients with focal segmental glomerulosclerosis, regardless of age or presence of typical Denys-Drash or Frasier syndrome clinical features. Structural analysis of the mutated protein revealed that the mutation hampers zinc finger-DNA interactions, impairing target gene transcription. This finding opens up new issues about WT1 function in the maintenance of the complex gene network that regulates normal podocyte function.

Spectrum and Frequency of SLC26A4 Mutations Among Czech Patients with Early Hearing Loss with and without Enlarged Vestibular Aqueduct (EVA)

Mutations in SLC26A4 cause Pendred syndrome (PS) – hearing loss with goitre – or DFNB4 – non‐syndromic hearing loss (NSHL) with inner ear abnormalities such as Enlarged Vestibular Aqueduct (EVA) or Mondini Dysplasia (MD). We tested 303 unrelated Czech patients with early hearing loss (298 with NSHL and 5 with PS), all GJB2‐negative, for SLC26A4 mutations and evaluated their clinical and radiological phenotype. Among 115 available HRCT/MRI scans we detected three MD (2.6%), three Mondini‐like affections (2.6%), 16 EVA (13 bilateral – 19.2% and 15.6% respectively) and 61 EVA/MD‐negative scans (73.4%). We found mutation(s) in 26 patients (8.6%) and biallelic mutations in eight patients (2.7%) out of 303 tested. In 18 of 26 (69%) patients, no second mutation could be detected even using MLPA. The spectrum of SLC26A4 mutations in Czech patients is broad without any prevalent mutation. We detected 21 different mutations (four novel). The most frequent mutations were p.Val138Phe and p.Leu445Trp (18% and 8.9% of pathogenic alleles respectively). Among 13 patients with bilateral EVA, six patients (50%) carry biallelic mutations. In EVA ‐negative patients no biallelic mutations were found but 4.9% had monoallelic mutations. SLC26A4 mutations are present mostly in patients with EVA/MD and/or progressive HL and those with affected siblings.

Molecular Characterization of Large Deletions in the von Hippel-Lindau (VHL) Gene by Quantitative Real-Time PCR

AbstractIntroduction: Mutations of the von Hippel-Lindau (VHL) gene are responsible for VHL disease. This is a familial autosomal-dominant syndrome, predisposing to the development of benign and malignant tumors, including CNS and retinal hemangioblastomas, pheochromocytomas, and clear cell renal carcinomas.At least 30% of the disease-causing mutations in the VHL gene involve large alterations. Identification of these mutations is not possible using PCR-based mutational scanning methods. Quantitative Southern blot analysis has been traditionally employed for the detection of complete or partial deletions and more complex rearrangements of the gene. Methods: An alternative quantitative method was developed using a combination of quantitative Southern blot analysis and real-time PCR. With this approach, we studied 24 large VHL gene alterations to determine the exact nature of the mutations and to possibly characterize the boundaries of the deleted regions. Results: This combined molecular approach showed that all the VHL alterations studied were due to deletions, from which the position in the gene could be more precisely mapped. One of the samples that was completely characterized was found to carry an intragenic 2.2kb deletion with both 5′ and 3′ breakpoints located within Alu-repeat sequences. Conclusion: This is the first report on the molecular analysis of large VHL alterations. The results of our study and the complete characterization of a large deletion lead to the hypothesis that an Alu-mediated mechanism may be responsible for the common occurrence of large alterations in the VHL gene.

Identification and In Silico Analysis of Novel von Hippel‐Lindau (VHL) Gene Variants from a Large Population

Mutational inactivation of the VHL gene is the cause of von Hippel‐Lindau (VHL) disease, an autosomal dominant hereditary cancer syndrome predisposing to haemangioblastomas, pheochromocytomas and clear‐cell renal carcinomas. The gene product (pVHL) functions as an adapter in cellular processes including cell growth and apoptosis. VHL mutation analysis was carried out in 426 unrelated subjects with phenotypes ranging from VHL syndrome, to isolated VHL‐related tumours that could represent the first manifestation of the disease. A total of 111 individuals were found to carry alterations, with large deletions representing 40% of the variants. Eighteen of the 95 detected variants were novel, seemingly disease‐causing mutations; their pathogenic role has been evaluated in silico for effects on protein folding and interactions. Putative regions of interaction between pVHL and proteins involved in common pathways have been identified, assessing possible implications for the presence of mutations in these regions. All new variants predicted to truncate or cause complete pVHL loss of structure were associated with phenotypes consistent with VHL type 1. Seven of the new amino acid substitutions are disease‐causing mutations, one is a neutral variant, whereas the results for two remain ambiguous. Our combined molecular and in silico approach for the evaluation of putative disease‐causing mutations contributes to the interpretation of the potential pathogenicity of these novel variants.

A novel SACS mutation results in non-ataxic spastic paraplegia and peripheral neuropathy.

Mutations in the SACS gene are commonly associated with autosomal recessive spastic ataxia of Charlevoix‐Saguenay (ARSACS), a complex neurodegenerative disorder characterized by progressive degeneration of the cerebellum and spinal cord tracts. The aim of this study was to identify the genetic cause of the disease in an Italian family with spastic paraplegia and peripheral neuropathy.