Exonic deletions of FXN and early-onset Friedreich ataxia.

BACKGROUND: Friedreich ataxia (FA) is the most frequent type of autosomal recessive cerebellar ataxia, occurring at a mean age of 16 years. Nearly 98% of patients with FA present with homozygous GAA expansions in the FXN gene. The remaining patients are compound heterozygous for an expansion and a point mutation. Patients who are compound heterozygous for an exonic deletion and an expansion are exquisitely rare. OBJECTIVES: To describe 6 patients affected with FA due to an exonic deletion mutation (FAexdel) and to compare these 6 patients with FAexdel with 46 patients consecutively diagnosed with typical FA due to homozygous GAA expansion and whose small expansions were within the same range as that of the expansions of the patients with FAexdel. DESIGN: Description of a series. SETTING: Academic research. PATIENTS: Six patients with FAexdel and 46 patients with typical FA. INTERVENTION: FXN gene analysis, including assessments of GAA expansion and exon sequencing and determination of exonic copy numbers using multiplex ligation-dependent probe amplification. RESULTS: We identified 6 patients with FA who presented with the combination of 1 GAA expansion and 1 FXN exonic deletion. The mean (SD) age at onset of the disease was earlier for patients with FAexdel (7 [4] years [range, 3-12 years]) than for patients with typical FA (15 [5] years [range, 6-30 years]) (P = .001), and the median time to confinement to wheelchair was shorter for patients with FAexdel (20 years) than for patients with typical FA (28 years) (P = .002). There was no difference between the mean (SD) size of the expansion for the patients with FAexdel (780 [256] GAA triplet repeat sequences [range, 340-1070 GAA triplet repeat sequences]) and the mean (SD) size of the short expansion for the patients with typical FA (634 [163] GAA triplet repeat sequences [range, 367-1000 GAA triplet repeat sequences]) (P = .10). The mean disease duration before becoming wheelchair bound was shorter for patients with FAexdel (9 years) than for patients with typical FA (13 years), and the incidence of cardiomyopathy was higher for patients with FAexdel (84%) than for patients with typical FA (68%). However, these differences were not significant, probably owing to the small size of the FAexdel group. The other extraneurological signs, such as scoliosis or diabetes mellitus, were particularly frequently observed in the FAexdel group. One patient presented at 9 years of age with severe angina and marked cardiomyopathy that confined her to a wheelchair. Three patients had disabling autonomic disturbances. It appears that exonic deletion significantly contributes to the clinical picture of patients with FA. CONCLUSIONS: Friedreich ataxia due to an exonic deletion mutation corresponds to an early onset and severe variant of FA. FXN should be investigated for exonic deletion in patients with early-onset FA in which only 1 GAA expansion without a point mutation is found. Patients with FAexdel have to be carefully observed using cardiological, orthopaedic, endocrinological, gastroenterological, and ophthalmological data. Friedreich ataxia due to an exonic deletion mutation should be suspected in young patients presenting with severe scoliosis.

Use of SNP array analysis to identify a novel TRIM32 mutation in limb-girdle muscular dystrophy type 2H.

Molecular diagnosis of monogenic diseases with high genetic heterogeneity is usually challenging. In the case of limb-girdle muscular dystrophy, multiplex Western blot analysis is a very useful initial step, but that often fails to identify the primarily affected protein. We report how homozygosity analysis using a genome-wide SNP array allowed us to solve the diagnostic enigma in a patient with a moderate form of LGMD, born from consanguineous parents. The genome-wide scan performed on the patient's DNA revealed several regions of homozygosity, that were compared to the location of known LGMD genes. One such region indeed contained the TRIM32 gene. This gene was previously found mutated in families with limb-girdle muscular dystrophy type 2H (LGMD2H), a mild autosomal recessive myopathy described in Hutterite populations and in 4 patients with a diagnosis of sarcotubular myopathy. A single missense mutation was found in all these patients, located in a conserved domain of the C-terminal part of the protein. Another missense mutation affecting the N-terminal part of TRIM32, observed in a single consanguineous Bedouin family, was reported to cause the phenotypically unrelated and genetically heterogeneous Bardet-Biedl syndrome, defining the BBS11 locus. Sequencing of TRIM32 in our patient revealed a distal frameshift mutation, c.1753_1766dup14 (p.Ile590Leu fsX38). Together with two recently reported mutations, this novel mutation confirms that integrity of the C-terminal domain of TRIM32 is necessary for muscle maintenance.

Identification of transfected cell types following non-viral gene transfer to the murine lung.

BACKGROUND: Identification of the cell types transfected following gene transfer is an important factor in the selection of appropriate gene transfer agents (GTAs). Due to the relatively low gene expression mediated by non-viral GTAs, current methodologies for the detection and identification of transfected cells in the lung have proven insensitive and unreliable. We have investigated the use of the green fluorescent protein (GFP) to identify transfected cells in a mouse lung model. METHODS: Direct visualisation of GFP fluorescence in frozen histological sections was used in conjunction with a panel of cell type specific antibodies to investigate the distribution and level of gene expression in mouse lungs following instillation of non-viral GTAs. RESULTS: Despite considerable tissue autofluorescence, dose-dependent expression of GFP was detected following instillation of as little as 25 microg naked plasmid DNA (pDNA). Naked pDNA and pDNA complexed with polyethylenimine appeared to transfect mainly ciliated cells and Clara cells of the conducting airway, whereas expression mediated by pDNA complexed with the cationic lipid GL67 was found predominantly in type I pneumocytes. CONCLUSIONS: Direct visualisation of GFP expression was used to detect transfected cell types in the mouse lung. In contrast with observations made using beta-galactosidase as a reporter, gene expression from several non-viral GTAs was readily demonstrated and no false GFP-positive cells were ever detected in untreated lung tissues. Lung delivery of different GTAs resulted in GFP expression in different cell types, confirming the importance of identification of transfected cells when screening and selecting GTAs for disease targets.