Swift Large-scale Examination of Directed Genome Editing

Omar T. Hammouda, Frank Böttger, Joachim Wittbrodt, Thomas Thumberger

PLOS

 

Published: March 5, 2019

https://doi.org/10.1371/journal.pone.0213317

Abstract

In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.

 

Novel variants and clinical symptoms in four new ALG3‐CDG patients review of the literature, and identification of AAGRP‐ALG3 as a novel ALG3 variant with alanine and glycine‐rich N‐terminus

Human Mutation
Variation, Informatics and Disease
Published: May 8, 2019

Abstract

ALG3‐CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER‐mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3‐CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5GlcNAc2‐PP‐dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right‐descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3‐CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open‐reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP‐ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.

 

Cutis laxa, exocrine pancreatic insufficiency and altered cellular metabolomics as additional symptoms in a new patient with ATP6AP1-CDG.

Dimitrov B,  Himmelreich N, Hipgrave Ederveen AL, Lüchtenborg C, Okun JG, Breuer M, Hutter AM, Carl M, Guglielmi L, Hellwig A, Thiemann KC, Jost M, Peters V, Staufner C, Hoffmann GF, Hackenberg A, Paramasivam N, Wiemann S, Eils R, Schlesner M, Strahl S, Brügger B, Wuhrer M, Christoph Korenke G, Thiel C

Molecular Genetics and Metabolism

Abstract

 

A mutation in mannose‐phosphate‐dolichol utilization defect 1 reveals clinical symptoms of congenital disorders of glycosylation type I and dystroglycanopathy

Walinka van Tol, Angel Ashikov, Eckhard Korsch, Nurulamin Abu Bakar, Michèl A. Willemsen, Christian Thiel, Dirk J. Lefeber

Journal of Inherited Metabolic Disease

Published: September 30, 2019

https://doi.org/10.1002/jmd2.12060

Abstract

Congenital disorders of glycosylation type I (CDG‐I) are inborn errors of metabolism, generally characterized by multisystem clinical manifestations, including developmental delay, hepatopathy, hypotonia, and skin, skeletal, and neurological abnormalities. Among others, dolichol‐phosphate‐mannose (DPM) is the mannose donor for N‐glycosylation as well as O‐mannosylation. DOLK‐CDG, DPM1‐CDG, DPM2‐CDG, and DPM3‐CDG are defects in the DPM synthesis showing both CDG‐I abnormalities and reduced O‐mannosylation of alpha‐dystroglycan (αDG), which leads to muscular dystrophy‐dystroglycanopathy. Mannose‐phosphate‐dolichol utilization defect 1 (MPDU1) plays a role in the utilization of DPM. Here, we report two MPDU1‐CDG patients without skin involvement, but with massive dilatation of the biliary duct system and dystroglycanopathy characteristics including hypotonia, elevated creatine kinase, dilated cardiomyopathy, buphthalmos, and congenital glaucoma. Biochemical analyses revealed elevated disialotransferrin in serum, and analyses in fibroblasts showed shortened lipid linked oligosaccharides and DPM, and reduced O‐mannosylation of αDG. Thus, MPDU1‐CDG can be added to the list of disorders with overlapping biochemical and clinical abnormalities of CDG‐I and dystroglycanopathy.

 

The Fine Art of Destruction: A Guide to In‐Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions