Generierung und Charakterisierung zweier Mauslinien für die Untersuchung der Rolle von Strumpellin in der Hereditären Spastischen Paraplegie Typ SPG8

Das motorische Nervensystem ist für die Kontrolle von Körperhaltung und Willkürbewegungen zuständig. Seine zellulären Hauptbestandteile sind obere und untere Motoneurone. Diese Nervenzellen haben extrem lange axonale Fortsätze, deren lebenslanger Erhalt damit eine besondere Herausforderung für die Zellen ist. Die erblichen Axonopathien des oberen Motoneurons werden als Hereditäre Spastische Paraplegien (HSP) bezeichnet. Hauptsymptome der HSP sind zunehmende Schwäche und Spastik der Beine. Derzeit sind über 50 Gene und mehr als 70 Genloci mit einem HSP Phänotyp assoziiert. Die HSP Typ SPG8 ist eine autosomal dominant vererbte Form, die bisher mit neun publizierten Mutationen im Gen KIAA0196 bzw. dem entsprechenden Genprodukt Strumpellin Protein assoziiert ist. Die zu Beginn der Arbeit vorliegenden Daten zum Pathomechanismus bei SPG8 waren inkonsistent. Die bestehende Hypothese zum Mutationsmechanismus war Haploinsuffizienz. In der vorliegenden Arbeit wurden zwei KIAA0196 Mauslinien generiert und charakterisiert. Es handelte sich dabei um einen Knockin einer als humanpathogen beschriebenen Variante (p.N471D) und einen konventionellen Knockout. Ziel dieser Ansätze war die Aufklärung des Mutations- und Pathomechanismus bei SPG8. ..

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Doublet-Mediated DNA Rearrangement-A Novel and Potentially Underestimated Mechanism for the Formation of Recurrent Pathogenic Deletions

Deletions and duplications of genomic DNA contribute to evolution, phenotypic diversity, and human disease. The underlying mechanisms are incompletely understood. We identified deletions of exon 10 of the SPAST gene in two unrelated families with hereditary spastic paraplegia. We excluded a founder event, but observed that the breakpoints map to identical repeat regions. These regions likely represent an intragenic doublet, that is, an enigmatic class of local duplications. The fusion sequences for both deletions are compatible with recombination-based as well as with replication-based mechanisms. Searching the literature, we identified a partial SLC24A4 deletion that involved two copies of another doublet, and was likely formed in an analogous way. Comparing the SPAST and the SLC24A4 doublets with doublets identified previously suggested that many additional doublets have a high potential for triggering rearrangements. Considering that doublets are still being formed in the human genome, and that they likely create high local instability, we suggest that a two-step mechanism consisting of doublet generation and subsequent doublet-mediated deletion/duplication may underlie certain copy-number changes for which other mechanisms are currently assumed. Further studies are necessary to delineate the significance of the thus-far understudied doublets for the formation of copy-number variation. (c) 2016 Wiley Periodicals, Inc

Maternal mosaicism for IDUA deletion clarifies recurrence risk in MPS I.

Mucopolysaccharidosis I (MPS I) is a rare autosomal recessive multisystem lysosomal storage disorder. It is caused by biallelic loss-of-function variants in IDUA, encoding alpha-l iduronidase. Here, we describe an individual affected by MPS I due to a paternally inherited deletion of IDUA exons 1 and 2, c.(?_-88)_(299+1_300-1)del and a whole-gene deletion of IDUA (?_-88?)_(*136?)del secondary to maternal somatic mosaicism. We define a previously unreported mutational mechanism for this disorder

“Missing mutations” in MPS I: Identification of two novel copy number variations by an IDUA‐specific in house MLPA assay

Background: Mucopolysaccharidosis type I (MPS I) is a rare, recessively inherited lysosomal storage disorder, characterized by progressive multi-systemic disease. It is caused by a reduced or absent alpha-l iduronidase (IDUA) enzyme activity secondary to biallelic loss-of-function variants in the IDUA. Over 200 causative variants in IDUA have been identified. Nevertheless, there is a fraction of MPS I patients with only a single mutated IDUA allele detectable. Methods: As genetic testing of MPS I is usually based on sequencing methods, copy number variations (CNVs) in IDUA can be missed and therefore presumably remain underdiagnosed. The aim of this study was the detection of CNVs using an IDUA-specific in house multiplex ligation-dependent probe amplification (MLPA) assay. Results: A total of five unrelated MPS I patient samples were re-analyzed after only a single heterozygous IDUA mutation c.979G>C (p.A327P), c.1469T>C (p.L490P), c.1598C>G (p.P533R), c.1205G>A (p.W402X), c.973-7C>G (p.?) could be identified. We detected a novel splice site variant c.973-7C>G (p.?), as well as two novel CNVs, a large deletion of IDUA exon 14 and 3'UTR c.(1828 + 1_1829-1)_(*1963_?)del, and a large duplication extending from IDUA exon 2 to intron 12 c.(157 + 1_158-1)_(1727 + 1_1728-1)dup. Conclusion: Together with the CNVs we previously identified, a total of four pathogenic IDUA CNVs have now been reported

The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

International audienceBackground : The hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency.Methods : We generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients. Results : Homozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established. Conclusions : Our data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models

Additional file 2: Figure S2. of The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

Visualization of lysosomes (LAMP-1), early endosomes (EEA1), and autophagosomes/lysosomes (LC3-RFP) by immunofluorescence (LAMP-1, EEA1) and transfection of an autofluorescent marker (LC3-RFP) in primary adult mouse fibroblast cultures. Scale bars: 20 μm. (TIFF 1055 kb

Additional file 1: Figure S1. of The spectrum of KIAA0196 variants, and characterization of a murine knockout: implications for the mutational mechanism in hereditary spastic paraplegia type SPG8

KIAA0196-specific MLPA probes. Shown are exons against which probes were designed, plus neighbouring intronic sequence. The MLPA probe binding sequence is underlined. White underlined space indicates the ligation site. Marked in yellow is the stop codon in the terminal exon 29, while the nucleotide marked in red represents SNP rs188863489 which served as a positive control. Stretches of Ns indicate intronic sequence masked out by repeat masker [ http://www.repeatmasker.org/ ]. (DOC 28 kb