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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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| 5. |
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- 2019
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Journal article (peer-reviewed)
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| 6. |
- Lim, Che Kang
(author)
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Genetics of immunoglobulin a deficiency
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Caucasian populations. It is defined as a serum IgA level below 0.07 g/L with normal IgM and IgG levels in an individual older than four years of age. Approximately one-third of these patients present with recurrent respiratory and gastrointestinal tract infections, allergic disorders and autoimmune manifestations. High familial clustering and prevalence variation by ethnicity both suggest the existence of a strong genetic component of the disease. Traditionally, IgAD has been reported as permanent, and sub-normal IgA levels remain static and persist after 20 years of observation. However, a few cases of reversion have been observed. We thus investigated the frequency of reversal in children and more than one-fifth (>20%) of Swedish children who were diagnosed before 10 years of age, reversed their IgAD status. Our observation suggests that the diagnosis of IgAD should not be made before the early teens using a cutoff level of 0.07 g/L of IgA in serum. After suggesting improved diagnostic guidelines, we investigated the role of genetics in IgAD in a Swedish Twin cohort. Surprisingly, the prevalence of IgAD was found to be markedly increased in a twin cohort as compared with the normal Swedish adult population. Although the MHC is the main genetic factor associated with IgAD development, the MHC haplotypes were not the primary factor causing the differences observed. Nonetheless, risk-conveying MHC haplotypes including HLA-A*01, HLA-B*08 and HLA-DRB1*01 were found to be associated with significantly lower serum IgA concentration in the twin cohort. On the contrary, individuals who carried the protective HLA alleles B*07, DRB1*15 and DQB1*06 were found to have significantly higher mean IgA concentration. We then performed a comprehensive analysis within the MHC region in order to identify the potential susceptibility genes/loci within the MHC region. In our large-scale case-control study, we identified an independent MHC haplotype (HLA-DPB1*1301) in the class II region associated with IgAD. In addition, MHC recombination analysis suggested a region around 110 Kbp which may contain a portion of the ancestral block. However, verification using complete sequencing did not identify any differences. Nonetheless, identification of 4310 new variants from ancestral 8.1 haplotypes will provide valuable information for the investigation of other MHC associated diseases. We also identified novel genes/variants within the MHC class III region including AGER (rs1800625), RNF5 (rs3130349), BTNL2 (rs1980493) and HCG23 (rs3117097) that are associated with IgAD risk. Subsequently, we investigated the association of non-MHC genes using different MHC risk haplotypes as category factors. In total, 14 different genes/loci were identified as potentially associated with IgAD in individuals carrying different MHC risk alleles, including one from HLA-B*0801-DRB1*0301-DQB1*0201 (ancestral haplotype), three from the HLA-DRB1*0701-DQB1*0202 cohort, two from HLA-DRB1*01-DQB1*0501 and seven from patients who do not carry any susceptibility MHC allele. These findings suggest that the development of IgAD may be variable depending on the presence of potentially different genes within selected MHC susceptibility haplotypes that interact with the respective disease-causing non-MHC genes. Understanding the interaction between MHC and non-MHC genes and proteins may facilitate identification of the IgAD etiology. In summary, this thesis not only helped to identify the genetic basis of IgAD, but also improved the current diagnostic definition of the disease. Further work, including protein-protein interaction investigations, gene knock-in/out and expression analyses are required to validate the functional role of the novel associations described in this thesis. As IgAD has been shown to be markedly overrepresented among patients with autoimmune diseases, further potential studies will aim to identify the link between IgAD and autoimmunity which may ultimately result in improved patient care.
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| 7. |
- Lim, Che Kang, et al.
(author)
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Reversal of Immunoglobulin. A Deficiency in Children
- 2015
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In: Journal of Clinical Immunology. - : Springer Science and Business Media LLC. - 0271-9142 .- 1573-2592. ; 35:1, s. 87-91
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Journal article (peer-reviewed)abstract
- Immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in the general population. It is defined as a serum IgA level below or equal to 0.07 g/l with normal IgM and IgG levels in children over the age of 4. However, a few cases of reversal of IgAD at later ages have been observed previously, especially in pediatric patients. This study aimed at investigating the frequency of reversal in a large cohort of children and young adults in order to evaluate the present definition of IgAD. Clinical laboratory records from 654 pediatric IgA deficient patients, 4-13 years of age, were retrieved from five university hospitals in Sweden. Follow up in the children where IgA serum levels had been routinely measured was subsequently performed. In addition, follow up of the IgA-levels was also performed at 4, 8 and 16 years of age in children who were IgA deficient at the age of 4 years in a Swedish population-based birth cohort study in Stockholm (BAMSE). Nine out of 39 (23.1 %) children who were identified as IgAD at 4 years of age subsequently increased their serum IgA level above 0.07 g/L. The average age of reversal was 9.53 +/- 2.91 years. In addition, 30 out of the 131 (22.9 %) children with serum IgAD when sampled between 5 and 9.99 years of age reversed their serum IgA level with time. The BAMSE follow up study showed a reversal of IgAD noted at 4 years of age in 8 out of 14 IgAD children at 16 years of age (5 at 8 years of age) where 4 were normalized their serum IgA levels while 4 still showed low serum levels of IgA, yet above the level defining IgAD. The results indicate that using 4 years of age, as a cut off for a diagnosis of IgAD may not be appropriate. Our findings suggest that a diagnosis of IgAD should not be made before the early teens using 0.07 g/L of IgA in serum as a cut off.
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| 8. |
- Wang, Zhaoming, et al.
(author)
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Imputation and subset-based association analysis across different cancer types identifies multiple independent risk loci in the TERT-CLPTM1L region on chromosome 5p15.33
- 2014
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 23:24, s. 6616-6633
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Journal article (peer-reviewed)abstract
- Genome-wide association studies (GWAS) have mapped risk alleles for at least 10 distinct cancers to a small region of 63 000 bp on chromosome 5p15.33. This region harbors the TERT and CLPTM1L genes; the former encodes the catalytic subunit of telomerase reverse transcriptase and the latter may play a role in apoptosis. To investigate further the genetic architecture of common susceptibility alleles in this region, we conducted an agnostic subset-based meta-analysis (association analysis based on subsets) across six distinct cancers in 34 248 cases and 45 036 controls. Based on sequential conditional analysis, we identified as many as six independent risk loci marked by common single-nucleotide polymorphisms: five in the TERT gene (Region 1: rs7726159, P = 2.10 × 10(-39); Region 3: rs2853677, P = 3.30 × 10(-36) and PConditional = 2.36 × 10(-8); Region 4: rs2736098, P = 3.87 × 10(-12) and PConditional = 5.19 × 10(-6), Region 5: rs13172201, P = 0.041 and PConditional = 2.04 × 10(-6); and Region 6: rs10069690, P = 7.49 × 10(-15) and PConditional = 5.35 × 10(-7)) and one in the neighboring CLPTM1L gene (Region 2: rs451360; P = 1.90 × 10(-18) and PConditional = 7.06 × 10(-16)). Between three and five cancers mapped to each independent locus with both risk-enhancing and protective effects. Allele-specific effects on DNA methylation were seen for a subset of risk loci, indicating that methylation and subsequent effects on gene expression may contribute to the biology of risk variants on 5p15.33. Our results provide strong support for extensive pleiotropy across this region of 5p15.33, to an extent not previously observed in other cancer susceptibility loci.
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