Chiara Angiari

Giant axon and neurofilament accumulation in Charcot–Marie–Tooth disease type 2E

The axonal type 2 Charcot–Marie–Tooth disease (CMT2) is phenotypically poorly characterized. Here the authors report a family with a Pro22Ser mutation in the neurofilament-light gene (NF-L; CMT2E) manifesting electrophysiologically as the demyelinating type 1 CMT (CMT1) and pathologically as an axonopathy with giant axons and accumulation of disorganized NF. NF-L should be investigated in CMT2 as well as in CMT1 not associated with the usual genes PMP22, Cx32, and P0.

Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

Background Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. Description Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC2(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80–1.00). Results and Significance CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.

Segmental conduction abnormalities and myelin thickenings in Val102/fs null mutation of MPZ gene

The authors report in patients with Val102/fs null mutation a possibly age dependent variability of clinical and electrophysiologic phenotype, segmental conduction abnormalities mainly in ulnar nerves at the elbow, and excessive myelin foldings and thickenings. The authors hypothesize that myelin thickenings at the paranodal region, in concurrence with compression at usual entrapment sites or minor repetitive trauma, may induce segmental conduction abnormalities.

Gene dosage sensitivity of a novel mutation in the intracellular domain of P0 associated with Charcot-Marie-Tooth disease type 1B.

Autosomal dominant Charcot-Marie-Tooth disease type 1B (CMT1B) is caused by heterozygous mutations in the extracellular domain of P0. Here, we investigated clinically, electrophysiologically and pathologically a pedigree with a novel mutation in the intracellular domain of P0 (P0ic). The mutational analysis included denaturing high performance liquid chromatography (DHPLC) and nucleotide sequencing. Two patients from subsequent generations were homozygous for an Asp195Tyr mutation in the intracellular domain of P0 (P0ic), whereas two healthy individuals with minimal electrophysiological changes were heterozygous for the same mutation. The authors conclude that mutations of P0ic may undergo a gene dosage effect manifesting semidominant inheritance.

Impaired CFTR function in mild cystic fibrosis associated with the S977F/T5TG12complex allele in trans with F508del mutation

BACKGROUND The S977F mutation (c.2930C>T) in the CFTR gene (CFTR/ABCC7) is extremely rare. We describe the case of an adult patient carrying the complex allele S977F/T5TG12 in trans with the F508del mutation. Mild respiratory manifestations arose in adulthood associated with azoospermia, acute pancreatitis, minor hemoptysis and Cl(-) levels ranging from 40 to 42 mEq/L. METHOD Diagnosis was confirmed by repeated NPD measurements, genetic DHPLC analysis and a recently described functional assay measuring cAMP-dependent cell depolarization in peripheral blood monocytes. RESULTS NPD measurements, DHPLC and monocyte functional assay (CF index=-18). Results were consistent with a CF phenotype. CONCLUSIONS The combined application of DHPLC and NPD analysis in the algorithm for CF diagnosis appears useful for the management of similar cases. In addition, the novel monocyte functional assay might contribute to improve our diagnostic capability, counseling and better treatment of these challenging clinical cases.

Functional Evaluation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in human monocytes

85 Expression of Unfolded Protein Response (UPR) protein markers are increased in primary and cystic fibrosis (CF) nasal polyposis (NP) L. Jeanson1,2, C. Guerrera1,4, M. Baudouin-Legros1,5, S. Amselem2, A. Coste3, E. Escudier2, A. Edelman1,5. 1U845, Inserm, Paris, France; 2U933, Inserm, Paris, France; 3U955, Inserm, Creteil, France; 4Plateau Proteome Necker, IFR 94, Paris, France; 5Faculte de Medecine, Universite Paris Descartes, Paris, France

Charcot‐marie‐tooth disease type 1: novel cases and novel mutations detected by DHPLC

Background: Charcot‐Marie‐Tooth disease type 1 (CMT1) and related Dejerine‐Sottas syndrome (DSS) and Hereditary Neuropathy with liability to Pressure Palsy (HNPP) have a high degree of genetic heterogeneity; mutation analysis performed by direct nucleotide sequencing is highly sensitive but time‐consuming and expensive. Objective: To evaluate the sensitivity of denaturing high performance liquid chromatography (DHPLC), a recently developed technology for fast mutational analysis. Methods: Optimal conditions for analysing Cx32, P0 and PMP22 genes by DHPLC were developed on the basis of 39 mutations (Cx32 = 21; P0 = 10; PMP22 = 8) detected in the period 1997–2002. Patients: During 2003, 44 patients fitting the clinical and electrophysiological criteria of CMT1, DSS or HNPP who were negative for the 17p11.2 duplication/deletion were analysed either by nucleotide sequencing or by DHPLC. Results: In the last year we identified a total of 3 mutations in Cx32, 3 mutations in MP0 (2 novels mutations, one recessive) and 2 mutations in PMP22 (2 novels nonsense mutations). Conclusions: DHPLC was capable of detecting all mutations identified by sequencing, thus appearing as a reliable approach for the mutational analysis of CMT1.

Are giant axons a pathological marker of charcot‐marie‐tooth neuropathy type 2E?

Background: According to electrophysiological and pathological criteria Charcot Marie Tooth (CMT) disease includes primary demyelinating forms (CMT1) and neuropathies with primary axonal loss (CMT2). In CMT1, genetic analysis provided some associations between characteristic lesions and different proteins. In CMT2, four genes were identified recently (CMT2A, B, D, E); the molecular diagnosis is complex and phenotypical hallmarks are lacking. Objectives: To describe the nerve biopsy in three pedigrees with CMT2E caused by mutations of the neurofilament‐light chain gene (NF‐L): two pedigrees from Campania sharing a Pro22Ser substitution in the head domain of protein and one pedigree from Apulia with a novel Leu268Prol substitution in the central rod domain. In all three pedigrees electrophysiology was consistent with a mixed, demyelinating and axonal neuropathy. Results: The three patients analysed revealed a primary axonopathy characterized by giant axonal swelling filled with densely packed neurofilaments and some atrophic axons. Conclusions: We propose that, in the diagnostic work up of CMT2, giant axons may orientate towards CMT2E. The pathological alterations detected correlate intuitively with an altered function of the neurofilaments which constitute the axonal cytoskeleton and are critical for radial growth and for axonal transport.