Familly with two different cases of post- and pre-natal L1 syndrome; When hydrocephaly become "multidisciplinary headache"

open11openBukvic, Nenad; Boaretto, Francesca; Loverro, Giuseppe; Susca, Francesco C.; Lovaglio, Rosaura; Patruno, Margherita; Bukvic, Dragoslav; Starcevic, Srdjan; Vazza, Giovanni; Mostaciuollo, Maria Luisa; Resta, NicolettaBukvic, Nenad; Boaretto, Francesca; Loverro, Giuseppe; Susca, Francesco C.; Lovaglio, Rosaura; Patruno, Margherita; Bukvic, Dragoslav; Starcevic, Srdjan; Vazza, Giovanni; Mostaciuollo, Maria Luisa; Resta, Nicolett

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Individuazione e caratterizzazione di geni implicati nelle paraparesi spastiche ereditarie

Hereditary spastic paraplegias (HSP) represents a group of single-gene disorders characterised by degeneration of the corticospinal tract axons, leading to slowly progressive lower extremity spasticity and weakness. HSP is termed as ‘complicated’ if additional symptoms such as dementia, extrapyramidal disturbance or peripheral neuropathy occur. So far 39 different chromosomal loci have been identified for HSP, which is inherited both as autosomal dominant, recessive and X-linked trait. Mutations in 18 genes involved in intracellular trafficking, axonal transport and impaired mitochondrial function have been identified in HSP patients. Such a scenario make then very difficult to decide what HSP form and consequently which gene have to be investigated in an affect subject. During the last three years many isolated patients and some families with a complicated HSP phenotype, have been studied in our lab in order to identify the genomic locus and/or the gene involved in the disease. We first investigated a subgroup of ten subjects with a specific severe phenotype characterized by the following major features: hydrocephalus, mental retardation, spasticity of the legs, and adducted thumbs (CRASH syndrome). Such patients were investigated by direct sequencing for mutations in L1CAM (Neural cell adhesion molecule L1) coding regions, a gene involved in CRASH syndrome. Five novel and one already known mutations have been detected in six unrelated patients. The large majority of the identified mutations were localized in the extracellular domain, which plays a primary role in the homo- and heterophilic protein-protein interactions. In the patients without causative mutations in the L1CAM coding region a duplication analysis using Multiplex Ligation-dependent Probe Amplification and Real Time-PCR has been performed. None of the analyzed patients showed such a riarrangement. In the second part of the present work, three families affected by a complicated form of recessive HSP were investigated. In the first one characterized by HSP with mental impairment, a linkage analysis for the known HSP loci have been performed. The candidate genes within the most reliable positive region have been direct sequenced without any significant result. Further studies are mandatory for identifying the molecular event responsible for HSP in such family. Haplotype analysis of the second family, in which two sibling were affected by HSP with suspected thin corpus callosum, was performed within the region 15q21.1 between the markers D15S994 and D15S978 where the SPG11 gene is located. As the two affected members shared the same genotype, the SPG11 gene was investigated by direct sequencing. Apparently both subjects were found to carry an homozygous 3 bp insertion at the splice site of exon 39, (c.7000-3_-4insAGG). However, further investigations demonstrated that they were compound heterozygous with the previously described insertion in one allele and an 2,6kb intragenic deletion between the 36 and the 39 introns (c.6754_7152del1397) in the other one. The third family with eleven brothers born from consanguineus parents, was already studied by genome-wide search and fine mapping in a previous project. Such analysis allowed the identification of a region cosegregating with the disease, located on chromosome 21q11.2-q21.1. Only the three subjects affected by HSP complicated by mild mental retardation and distal motor neuropathy, share the same genotype at this locus. By the candidate genes study, a single nucleotide substitution in the 3’UTR of STCH gene (Heat shock protein 70 family member 13) that cosegregated with HSP have been detected. Such variation was never detected in 300 healthy subjects from the same population. In order to evaluate if such variant might affect transcription as well as RNA stability the mouse embryonic spinal cord-neuroblastoma cell line (NSC34) was transfected using a vector expressing the luciferase gene fused with the wild type or mutant STCH 3’UTR. Preliminary results suggest that the STCH 3’UTR single nucleotide substitution significantly affects the luciferase activity. In order to understand if such substitution could lead to an activation of a cryptic miRNA target site using in silico and in vitro approach, we performed a selection of putative miRNAs able to interact with the STCH mutated 3’UTR. Such analysis suggest hsa-miR-134, hsa-miR-194, hsa-miR-637, hsa-miR-758 e hsa-miR-924 might be involved in the pathogenic role of such variant. However further functional studies are mandatory for drawing final conclusions.Le paraparesi spastiche ereditarie (HSP) sono un gruppo di disordini neurodegenerativi caratterizzate da progressiva spasticità e debolezza degli arti inferiori. Nelle forme complicate si possono osservare altre manifestazioni neurologiche o non neurologiche associate alla spasticità. I dati presenti in letteratura sulle HSP ad oggi riportano 39 loci mappati su diversi cromosomi e sono descritte sia forme a trasmissione autosomica dominante, che recessiva, che X-linked. Nei pazienti affetti da HSP sono state trovate mutazioni in 18 diversi geni coinvolti nel trafficking intracellulare, nel trasporto assonale e in anomalie nel funzionamento dei mitocondri. In un tale scenario in cui la quantità di informazioni raccolte sono molte, non è facile scegliere quale strada intraprendere per determinare da quale forma di HSP un paziente risulti affetto, né tanto meno accrescere, con dati di rilievo, le conoscenze generali. In questi tre anni sono stati studiati molti casi isolati ed alcuni casi familiari che presentavano un fenotipo di HSP complicata, con l’intenzione di individuare il locus e/o il gene coinvolto nei diversi pazienti. In primo luogo è stato analizzato un campione di 10 soggetti con uno grave fenotipo caratterizzato da spasticità agli arti inferiori, idrocefalo, ritardo mentale e pollici addotti (sindrome di CRASH). In questi individui, mediante sequenziamento diretto sono state studiate le regioni codificanti del gene L1CAM, associato alla sindrome di CRASH. Cinque nuove mutazioni sono state trovate in altrettanti pazienti non correlati, più una descritta in precedenza. La maggior parte delle mutazioni identificate in questo studio sono localizzate nella porzione extracellulare della proteina matura che svolge un ruolo primario nelle iterazioni omo- ed etero-filiche proteina-proteina. Nei pazienti privi di mutazioni puntiformi nelle regioni codificanti è stata condotta un’analisi di duplicazione del gene L1CAM mediante differenti metodiche (Multiplex Ligation-dependent Probe Amplification e Real Time-PCR). Nessuno degli individui analizzati si è dimostrato essere portatore di tali riarrangiamenti. Nella seconda parte di questo lavoro si è proceduto con un’indagine molecolare su 3 famiglie affette da paraparesi spastica complicata a trasmissione autosomica recessiva. Nella prima famiglia (Fam. 1) con HSP associata a deficit cognitivo è stata eseguita un’indagine preliminare di esclusione dei loci coinvolti nella HSP. Sono stati valutati geni candidati nella regione di linkage più promettente, ma non sono state trovate mutazioni causative. Ulteriori analisi saranno necessarie per comprendere quale forma di paraparesi spastica sia responsabile della patologia negli affetti di questa famiglia. Nella seconda famiglia (Fam. 2) a cui è stata diagnosticata, in due fratelli, una forma di HSP complicata da sospetto assottigliamento del corpo calloso si è proceduto con la caratterizzazione della regione 15q21.1 dove mappa il geneSPG11 tra i marcatori D15S994 e D15S978. Data la condivisione del genotipo negli individui affetti si è proceduto con il sequenziamento del gene SPG11. Apparentemente i due fratelli affetti risultavano omozigoti per un’inserzione di tre paia di basi a livello del sito di splicing dell’esone 39 (c.7000-3_-4insAGG; NM_025137). Ulteriori analisi hanno dimostrato che sono invece portatori di due mutazioni diverse, la mutazione descritta inizialmente ed una delezione di 2,76 kb tra gli introni 36 e 39 (c.6754_7152del1397). Per il terzo nucleo familiare, 11 fratelli nati da genitori consanguinei (Fam. 3), in passato erano già state eseguite delle analisi (genome-wide search e fine mapping) per cui era stata individuata una regione di omozigosità sul cromosoma 21. Solo gli individui affetti, tre fratelli con HSP complicata da un lieve ritardo mentale e neuropatia periferica, in questa regione condividevano l’assetto genotipico. L’analisi dei geni candidati ha portato all’individuazione di una sostituzione di un singolo nucleotide (T?C) sul 3’UTR del gene STCH (Heat shock protein 70 family member 13) localizzato in posizione 21q11.2 che cosegrega con la patologia e non risulta presente in 300 individui sani analizzati. Uno studio funzionale preliminare ha permesso di valutare gli effetti di questa variazione nella linea cellulare murina motoneurone-simile NSC34. Usando un vettore modificato esprimente il gene della luciferasi fuso con il 3’UTR del gene STCH si è potuto osservare una differente attività trascrizionale evidenziata come minor attività della luciferasi in presenza dell’allele mutato. Tenuto conto che tale sostituzione nucleotidica potrebbe attivare un sito target di appaiamento criptico per un microRNA è stata eseguita un’analisi in silico ed in vitro per selezionare i microRNA che potrebbero interagire con il trascritto di tale gene. Al momento appaiono candidati i miRNA: hsa-miR-134, hsa-miR-194, hsa-miR-637, hsa-miR-758 e hsa-miR-924

Novel mutations in the L1CAM gene support the complexity of L1 syndrome

X-linked hydrocephalus, MASA syndrome, X-linked complicated Spastic Paraplegia Type I and X-linked partial agenesis of the corpus callosum are the four rare diseases usually referred to L1 syndrome, caused by mutations in the L1CAM gene. By direct sequencing of L1CAM in 16 patients, we were able to identify seven mutations, five of which were never described before. Patients' phenotype evaluation revealed a correlation between the number of clinical features typical of L1 syndrome and the chance to find causative mutation. Our findings support that L1CAM mutations are associated with widely heterogeneous phenotypes, however the occurrence of several clinical features remains the best criterion for planning molecular testing both in familial and apparently sporadic cases

A novel missense mutation in the L1CAM gene in a boy with L1 disease

A novel missense mutation of the L1CAM gene (Xq28) is described in an adult patient affected with severe mental retardation, spastic paraparesis, adducted thumbs, agenesis of corpus callosum and microcephaly (L1 disease). We detected a transition c2308G-->A in exon 18 that caused an amino acid change in codon 770. The patient's mother and two sisters were heterozygous for the same mutation. This newly described mutation predicts the substitution of an aspartate by asparagine (D770N) in the second fibronectin (Fn2) domain of the extracellular portion of the mature L1 protein. Even if amino acid substitution does not significantly change the physico-chemical properties of the Fn2 domain, it seems clear that the integrity of this domain is required to maintain the biological functions of the protein. The feature peculiar to this patient is the decelerated head growth post-natally, leading to microcephaly. Mutations of L1CAM associated with prolonged survival may hamper post-natal brain and head growth