| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Craddock, Nick, et al.
(författare)
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Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls
- 2010
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 464:7289, s. 713-720
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Tidskriftsartikel (refereegranskat)abstract
- Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed,19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated similar to 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.
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| 3. |
- Rendtorff, Nanna D., et al.
(författare)
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Identification of p.A684V missense mutation in the WFS1 gene as a frequent cause of autosomal dominant optic atrophy and hearing impairment
- 2011
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Ingår i: American Journal of Medical Genetics. Part A. - : Wiley-Blackwell. - 1552-4825 .- 1552-4833. ; 155A:6, s. 1298-1313
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Tidskriftsartikel (refereegranskat)abstract
- Optic atrophy (OA) and sensorineural hearing loss (SNHL) are key abnormalities in several syndromes, including the recessively inherited Wolfram syndrome, caused by mutations in WFS1. In contrast, the association of autosomal dominant OA and SNHL without other phenotypic abnormalities is rare, and almost exclusively attributed to mutations in the Optic Atrophy-1 gene (OPA1), most commonly the p.R445H mutation. We present eight probands and their families from the US, Sweden, and UK with OA and SNHL, whom we analyzed for mutations in OPA1 and WFS1. Among these families, we found three heterozygous missense mutations in WFS1 segregating with OA and SNHL: p.A684V (six families), and two novel mutations, p.G780S and p.D797Y, all involving evolutionarily conserved amino acids and absent from 298 control chromosomes. Importantly, none of these families harbored the OPA1 p.R445H mutation. No mitochondrial DNA deletions were detected in muscle from one p.A684V patient analyzed. Finally, wolframin p.A684V mutant ectopically expressed in HEK cells showed reduced protein levels compared to wild-type wolframin, strongly indicating that the mutation is disease-causing. Our data support OA and SNHL as a phenotype caused by dominant mutations in WFS1 in these additional eight families. Importantly, our data provide the first evidence that a single, recurrent mutation in WFS1, p.A684V, may be a common cause of ADOA and SNHL, similar to the role played by the p.R445H mutation in OPA1. Our findings suggest that patients who are heterozygous for WFS1 missense mutations should be carefully clinically examined for OA and other manifestations of Wolfram syndrome.
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