60 research outputs found
Horny layer molecular structure: A new theory for hydrophilic molecules free diffusion through the skin
No full textAuthor shows a novel molecular theory capable to describe the passage of hydrophilic molecules and solutes through the horny layer (HL). According to the theory of the "gear wheels" the hydrophilic groups of the intercorneal lipids and especially ceramydes and free fatty acids, components of the lipidic domains, are oriented toward the hydrophilic head of the horny cells, represented by the aminoacidic appendages and humectants substances derived from the epidermal cells proteins catabolism (NMF). This mutual orientation would create aqueous zone placed between the surface of lipids domains surface and the surface of corneocytes so creating aqueous channels in which hydrophilic molecules can be solved in andflow toward the deepest skin layer moved by a mechanism similar to a "gear wheel" device. The molecules and solutes, so, could move for free diffusion or attracted by the aqueous gradient taking place through the adjacent different water content zones. This molecular theory could explain the penetration of high molecular weight hydrophilic molecules such as hyaluronic acid (HA) through the skin, thanks to the capability to hydrate in the water fulfilled channels placed in the space between lipids bi-lamellar domains and horny cells surface. It has been demonstrated that HA can penetrate deeply and quickly through the skin reaching dermal layer. Previous scientfic investigations on the HL structure clarified that skin surface exposure to a prolonged contact with moisture or water solutions produces a dramatic alteration of the HL inducing formation of "aqueous cisternae" and a geometric-structural alteration of the bi-layers inter-corneal lipids. This evidence supports the hypothesis according to which water can penetrate through the shin by mean of hydrophilic paths and then "enlarge" horny cells. Furthermore, this theory would open the path of investigation of new promising and performing new class of skin absorption enhancers even for hydrophilic drugs and cosmetics
Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing
No full textDisease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations
Depletion of mitochondrial DNA in fibroglast cultures from patients with POLG1 mutations is a consequence of catalytic mutations
No full textWe investigated clinical and cellular phenotypes of 24 children with mutations in the catalytic (alpha) subunit of the mitochondrial DNA (mtDNA) gamma polymerase (POLG1). Twenty-one had Alpers syndrome, the commonest severe POLG1 autosomal recessive phenotype, comprising hepatoencephalopathy and often mtDNA depletion. The cellular mtDNA content reflected the genotype more closely than did clinical features. Patients with tissue depletion of mtDNA all had at least one allele with either a missense mutation in a catalytic domain or a nonsense mutation. Four out of 12 patients exhibited a progressive, mosaic pattern of mtDNA depletion in cultured fibroblasts. All these patients had mutations in a catalytic domain in both POLG1 alleles, in either the polymerase or exonuclease domain or both. The tissue mtDNA content of patients who had two linker mutations was normal, and their phenotypes the mildest. Epilepsy and/or movement disorder were major features in all 21. Previous studies have implicated replication stalling as a mechanism for mtDNA depletion. The mosaic cellular depletion that we have demonstrated in cell cultures may be a manifestation of severe replication stalling. One patient with a severe cellular and clinical phenotype was a compound heterozydote with both polymerase and exonuclease domain in trans. This suggests that POLG1 requires both polymerase and 3'-5' exonuclease activity in the same molecule. This is consistent with current functional models for eukaryotic DNA polymerases, which alternate between polymerizing and editing modes, as determined by competition between these two active sites for the 3' end of the DNA
Novel mutation in the RNASEH1 gene in a chronic progressive external ophthalmoplegia patient
No full textDiagnosis of ‘possible’ mitochondrial disease: an existential crisis
No full textPrimary genetic mitochondrial diseases are often difficult to diagnose, and the term 'possible' mitochondrial disease is used frequently by clinicians when such a diagnosis is suspected. There are now many known phenocopies of mitochondrial disease. Advances in genomic testing have shown that some patients with a clinical phenotype and biochemical abnormalities suggesting mitochondrial disease may have other genetic disorders. In instances when a genetic diagnosis cannot be confirmed, a diagnosis of 'possible' mitochondrial disease may result in harm to patients and their families, creating anxiety, delaying appropriate diagnosis and leading to inappropriate management or care. A categorisation of 'diagnosis uncertain', together with a specific description of the metabolic or genetic abnormalities identified, is preferred when a mitochondrial disease cannot be genetically confirmed
() Quantification of PicoGreen staining of proportion of mosaic MDS cell cultures A–C that appeared depleted over time
No full textThe number of cells that appeared depleted of mtDNA increased over 45 days in the patients but not the controls (200 cells were counted at each time point). Numbers were significantly higher in patients than controls at all time points, other than 28 days (0.05< < 0.0016). () Quantification of the numbers of nucleoids visible by PicoGreen staining of mosaic MDS cell cultures A–C and controls [same experiment as () and ]. Nucleoid numbers drop with time in MDS cultures ( < 0.001 and <p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p
Author Correction: Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans
No full textEMQN best practice guidelines for genetic testing in dystrophinopathies.
No full textDystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed
() MDS cells are mosaic for expression of mtDNA and mitochondrial transcription factor A (TFAM) and have reduced mtDNA synthesis compared with controls
No full text(a) Control fibroblasts co-labelled with anti-DNA antibody (green) and Mitotracker red, showing orange-yellow labelling were there two signals co-localize. (b) Patient A fibroblasts co-labelled with anti-DNA/Mitotracker. (DAPI was used to visualize nuclei blue and an asterisk denotes a cell with no anti-DNA signal). (c) Patient B fibroblasts co-labelled with anti-DNA/Mitotracker. (note: a reduced anti-DNA signal is shown by the predominantly red co-localization with Mitotracker). (d) Patient C fibroblasts co-labelled with anti-DNA/Mitotracker (arrow shows area of residual anti-DNA/mitotracker co-labelling within a depleted cell with reduced Mitotracker labelling). (e) Control fibroblasts co-labelled with anti-TFAM antibody (green) and Mitotracker red. (f) Patient A fibroblasts co-labelled with anti-TFAM/Mitotracker (asterisks show TFAM depleted cells with reduced Mitotracker labelling). (g) Patient B fibroblasts co-labelled with anti-TFAM/Mitotracker. (h) Patient C fibroblasts co-labelled with anti-TFAM/Mitotracker. (i–l) Fibroblasts pulsed with Bromodeoxyuridine (BrdU) for 220 min, and Br-DNA immuno-detected. (i) BrdU labelling of normal fibroblasts, (j) BrdU labelling of patient A cells (inset shows magnified area of cytoplasm showing weak BrdU labelling). (k) BrdU labelling of patient B cells. (l) BrdU labelling of patient C cells (note: asterisks mark ‘ρ° type’ cells with very little or no mtDNA labelling). Bar 20 µM. () Expression of mtDNA encoded cytochrome oxidase subunit I (COXI) in MDS patients is reduced, as is COX activity, but not SDH activity. Cytochrome oxidase subunit I (COXI) expression was monitored using immuno-cytochemistry. (a) COXI labelling of normal control fibroblasts. (b) COXI labelling of patient A fibroblasts (COXI depleted cells are asterisked). (c) COXI labelling of patient B fibroblasts. (d) COXI labelling of patient C fibroblasts. (e–h) Histochemical demonstration of COX activity in fibroblasts. (e) COX activity of control. (f) COX activity of patient A cells (asterisks denote cells with markedly reduced activity). (g) COX activity of patient B cells. (h) COX activity of patient C cells. (i–l) Histochemical demonstration of SDH activity in fibroblasts. (i) SDH activity of control cells. (j) SDH activity of patient A cells. (k) SDH activity of patient B cells. (l) SDH activity of patient C cells. Bars 20 µM. () Tetramethyl-rhodamine-methyl ester (TMRM)/PicoGreen co-labelling of mosaic MDS fibroblast mitochondrial membrane potential. (a) PicoGreen labelling of normal control fibroblasts. (b) Co-staining of the same cells with TMRM. (c) Co-localization of the two signals in (a) and (b). (d and e) PicoGreen/TMRM co-labelling of later passage Patient A's fibroblasts. (f) Co-localization of the two signals in (d) and (e). (g and h) PicoGreen/TMRM co-labelling of later passage Patient B's fibroblasts. (i) Co-localization of the two signals. (j and k) PicoGreen/TMRM co-staining of late passage Patient C's cells. (l) Co-localization of the two signals. (m and n) PicoGreen/TMRM co-staining of late passage Patient D's cells. (o) Co-localization of the two signals. Bars 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p
Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease
No full textOBJECTIVE: Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and establish clinically meaningful genotype-phenotype correlations. METHODS: RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evidence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously reported patients harboring RNASEH1 mutations was subsequently undertaken. RESULTS: Pathogenic c.424G>A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 genetic analysis was undertaken in 50 additional Indian probands with variable clinical presentations associated with multiple mtDNA deletions, but no further RNASEH1 mutations were confirmed. RNASEH1-related mitochondrial disease was characterized by PEO (100%), cerebellar ataxia (57%), and dysphagia (50%). The ataxia neuropathy spectrum phenotype was observed in 1 patient. Although the c.424G>A p.Val142Ile mutation underpins all reported RNASEH1-related mitochondrial disease, haplotype analysis suggested an independent origin, rather than a founder event, for the variant in our families. CONCLUSIONS: In our cohort, RNASEH1 mutations represent the fourth most common cause of adult mendelian PEO associated with multiple mtDNA deletions, following mutations in POLG, RRM2B, and TWNK. RNASEH1 genetic analysis should also be considered in all patients with POLG-negative ataxia neuropathy spectrum. The pathophysiologic mechanisms by which the c.424G>A p.Val142Ile mutation impairs human RNase H1 warrant further investigation
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