The down-regulation of pank2 gene in zebrafish as a model of Pantothenate Kinase Associated Neurodegeneration.

open9siThe increased iron deposition is a hallmark of many neurodegenerative diseases, but its pathogenic role is still unclear. A strong link between iron and neurodegeneration is evident in a set of heterogeneous neurological disorders, known as Neurodegeneration with Brain Iron Accumulation (NBIA). The most common form of inherited NBIA is associated with mutations in hPank2 gene (PKAN). Pank2 is the rate limiting enzyme in CoA biosynthesis and its downregulation in mammalian cells leads to perturbation of cellular iron homeostasis. Here we explore Pank2 biological function in Danio rerio, and propose this system as an important new tool for the study of PKAN disease.openZizioli, Daniela; Tiso, Natascia; Busolin, Giorgia; Khatri, Deepak; Giuliani, Roberta; Borsani, Giuseppe; Monti, Eugenio; Argenton, Francesco; Finazzi, DarioZizioli, Daniela; Tiso, Natascia; Busolin, Giorgia; Khatri, Deepak; Giuliani, Roberta; Borsani, Giuseppe; Monti, Eugenio; Argenton, Francesco; Finazzi, Dari

Gallus gallus NEU3 sialidase as model to study protein evolution mechanism based on rapid evolving loops

<p>Abstract</p> <p>Background</p> <p>Large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. These loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. A good model to study this hypothesis is represented by sialidase enzymes. Indeed, the structure of sialidases is a β-propeller composed by anti-parallel β-sheets connected by loops that suit well with the rapid evolving loop hypothesis. These features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases.</p> <p>Results</p> <p><it>Gallus gallus (Gg) </it>genome contains one <it>NEU3 </it>gene encoding a protein with a unique 188 amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. The repeat region is located at the same position as the roughly 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved β-strands of the fifth blade of the sialidase β-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence.</p> <p>Conclusion</p> <p>In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved β-sheets and emerging on the opposite site of the catalytic crevice. These data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. Giving the peculiar organization of the loop region identified in Gg NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.</p

Mutation analysis by direct and whole exome sequencing in familial and sporadic tooth agenesis

Dental agenesis is one of the most common congenital craniofacial abnormalities. Dental agenesis can be classified, relative to the number of missing teeth (excluding third molars), as hypodontia (1 to 5 missing teeth), oligodontia (6 or more missing teeth), or anodontia (lack of all teeth). Tooth agenesis may occur either in association with genetic syndromes, based on the presence of other inherited abnormalities, or as a non-syndromic trait, with both familiar and sporadic cases reported. In this study, we enrolled 16 individuals affected by tooth agenesis, prevalently hypodontia, and we carried out direct Sanger sequencing of paired box 9 (PAX9) and Msh homeobox 1 (MSX1) genes in 9 subjects. Since no mutations were identified, we performed whole exome sequencing (WES) in the members of 5 families to identify causative gene mutations either novel or previously described. Three individuals carried a known homozygous disease mutation in the Wnt family member 10A (WNT10A) gene (rs121908120). Interestingly, two of these individuals were siblings and also carried a heterozygous functional variant in EDAR-associated death domain (EDARADD) (rs114632254), another disease causing gene, generating a combination of genetic variants never described until now. The analysis of exome sequencing data in the members of other 3 families highlighted new candidate genes potentially involved in tooth agenesis and considered suitable for future studies. Overall, our study confirmed the major role played by WNT10A in tooth agenesis and the genetic heterogeneity of this disease. Moreover, as more genes are shown to be involved in tooth agenesis, WES analysis may be an effective approach to search for genetic variants in familiar or sporadic tooth agenesis, at least in more severe clinical manifestations

Characterization of the AP-1 μ1A and μ1B adaptins in zebrafish (Danio rerio)

Protein transport between the trans-Golgi network and endosomes is mediated by transport vesicles formed by the adaptor-protein complex AP-1, consisting of the adaptins γ1, β1, μ1, σ1. Mammalia express μ1A ubiquitously and isoform μ1B in polarized epithelia. Mouse γ1 or μ1A 'knock out's revealed that AP-1 is indispensable for embryonic development. We isolated μ1A and μ1B from Danio rerio. Analysis of μ1A and μ1B expression revealed tissue-specific expression for either one during embryogenesis and in adult tissues in contrast to their expression in mammalia. μ1B transcript was detected in organs of endodermal derivation and "knock-down" experiments gave rise to embryos defective in formation of intestine, liver, and pronephric ducts. Development ceased at 7-8 dpf. μ1B is not expressed in murine liver, indicating loss of μ1B expression and establishment of alternative sorting mechanisms during mammalian development

Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development

Atypical Chemokine Receptor 3 Generates Guidance Cues for CXCL12-Mediated Endothelial Cell Migration

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12

Progressive Myoclonus Epilepsy Caused by a Homozygous Splicing Variant of SLC7A6OS

Exome sequencing was performed in 2 unrelated families with progressive myoclonus epilepsy. Affected individuals from both families shared a rare, homozygous c.191A > G variant affecting a splice site in SLC7A6OS. Analysis of cDNA from lymphoblastoid cells demonstrated partial splice site abolition and the creation of an abnormal isoform. Quantitative reverse transcriptase polymerase chain reaction and Western blot showed a marked reduction of protein expression. Haplotype analysis identified a similar to 0.85cM shared genomic region on chromosome 16q encompassing the c.191A > G variant, consistent with a distant ancestor common to both families. Our results suggest that biallelic loss-of-function variants in SLC7A6OS are a novel genetic cause of progressive myoclonus epilepsy. ANN NEUROL 2020Peer reviewe

Importance of Different Types of Prior Knowledge in Selecting Genome‐Wide Findings for Follow‐Up

Biological plausibility and other prior information could help select genome‐wide association ( GWA ) findings for further follow‐up, but there is no consensus on which types of knowledge should be considered or how to weight them. We used experts’ opinions and empirical evidence to estimate the relative importance of 15 types of information at the single‐nucleotide polymorphism ( SNP ) and gene levels. Opinions were elicited from 10 experts using a two‐round Delphi survey. Empirical evidence was obtained by comparing the frequency of each type of characteristic in SNP s established as being associated with seven disease traits through GWA meta‐analysis and independent replication, with the corresponding frequency in a randomly selected set of SNP s. SNP and gene characteristics were retrieved using a specially developed bioinformatics tool. Both the expert and the empirical evidence rated previous association in a meta‐analysis or more than one study as conferring the highest relative probability of true association, whereas previous association in a single study ranked much lower. High relative probabilities were also observed for location in a functional protein domain, although location in a region evolutionarily conserved in vertebrates was ranked high by the data but not by the experts. Our empirical evidence did not support the importance attributed by the experts to whether the gene encodes a protein in a pathway or shows interactions relevant to the trait. Our findings provide insight into the selection and weighting of different types of knowledge in SNP or gene prioritization, and point to areas requiring further research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96262/1/gepi21705.pd

SNP Prioritization Using a B ayesian Probability of Association

Prioritization is the process whereby a set of possible candidate genes or SNP s is ranked so that the most promising can be taken forward into further studies. In a genome‐wide association study, prioritization is usually based on the P ‐values alone, but researchers sometimes take account of external annotation information about the SNP s such as whether the SNP lies close to a good candidate gene. Using external information in this way is inherently subjective and is often not formalized, making the analysis difficult to reproduce. Building on previous work that has identified 14 important types of external information, we present an approximate B ayesian analysis that produces an estimate of the probability of association. The calculation combines four sources of information: the genome‐wide data, SNP information derived from bioinformatics databases, empirical SNP weights, and the researchers’ subjective prior opinions. The calculation is fast enough that it can be applied to millions of SNPS and although it does rely on subjective judgments, those judgments are made explicit so that the final SNP selection can be reproduced. We show that the resulting probability of association is intuitively more appealing than the P ‐value because it is easier to interpret and it makes allowance for the power of the study. We illustrate the use of the probability of association for SNP prioritization by applying it to a meta‐analysis of kidney function genome‐wide association studies and demonstrate that SNP selection performs better using the probability of association compared with P ‐values alone.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96317/1/gepi21704.pd