Restriction site Associated DNA Sequencing (RAD-Seq) is a technique characterized by the sequencing of specific loci along the genome that is widely employed in the field of evolutionary biology ...since it allows to exploit variants (mainly Single Nucleotide Polymorphism—SNPs) information from entire populations at a reduced cost. Common RAD dedicated tools, such as
STACKS
or
IPyRAD
, are based on all-vs-all read alignments, which require consequent time and computing resources. We present an original method, DiscoSnp-RAD, that avoids this pitfall since variants are detected by exploiting specific parts of the assembly graph built from the reads, hence preventing all-vs-all read alignments. We tested the implementation on simulated datasets of increasing size, up to 1,000 samples, and on real RAD-Seq data from 259 specimens of
Chiastocheta
flies, morphologically assigned to seven species. All individuals were successfully assigned to their species using both STRUCTURE and Maximum Likelihood phylogenetic reconstruction. Moreover, identified variants succeeded to reveal a within-species genetic structure linked to the geographic distribution. Furthermore, our results show that DiscoSnp-RAD is significantly faster than state-of-the-art tools. The overall results show that DiscoSnp-RAD is suitable to identify variants from RAD-Seq data, it does not require time-consuming parameterization steps and it stands out from other tools due to its completely different principle, making it substantially faster, in particular on large datasets.
Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been ...identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.
Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial ...chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing
, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the
genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in
subsp.
recipient cells to produce the designed
chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the
chassis confirmed the corresponding loss of H
O
production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent
subsp.
was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing
, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.
Holoprosencephaly (HPE) is the most common congenital cerebral malformation, characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been ...associated with HPE and are often inherited from an unaffected parent, underlying complex genetic bases. It is now emerging that HPE may result from a combination of multiple genetic events, rather than from a single heterozygous mutation. To explore this hypothesis, we undertook whole exome sequencing and targeted high‐throughput sequencing approaches to identify mutations in HPE subjects. Here, we report two HPE families in which two mutations are implicated in the disease. In the first family presenting two foetuses with alobar and semi‐lobar HPE, we found mutations in two genes involved in HPE, SHH and DISP1, inherited respectively from the father and the mother. The second reported case is a family with a 9‐year‐old girl presenting lobar HPE, harbouring two compound heterozygous mutations in DISP1. Together, these cases of digenic inheritance and autosomal recessive HPE suggest that in some families, several genetic events are necessary to cause HPE. This study highlights the complexity of HPE inheritance and has to be taken into account by clinicians to improve HPE genetic counselling.
ABSTRACT
Holoprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high ...heterogeneity, both in clinics and genetics. We have developed a novel targeted next‐generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling.
–FGF signaling plays a major role in the onset of HPE
–FGFR1 mutations are involved in isolated HPE
–Mutations in GLI2 are often associated with midline abnormalities
–Several cases of double‐mutations argue for digenic inheritance of HPE
Isolating genetic markers is often costly and time-consuming for non-model fungal species. However, these markers are of primary importance to identify the origin of invasive species and to infer ...their reproductive mode and dispersal ability. We slightly modified a recent molecular method to quickly isolate and validate single-nucleotide polymorphism (SNP) markers, from a first
Erysiphe alphitoides
draft genome, one of the main causal agent of oak powdery mildew in Europe. Although the draft assembly was strongly fragmented (555,289 contigs), we successfully isolated 1700 SNPs from 75 single-copy genes conserved in most fungal genomes. Ninety percent of them allowed to clearly distinguish the two main
Erysiphe
species reported on European oaks:
E. alphitoides
and
E. quercicola
. Thirty-six SNPs, located in distinct genes, were then validated using a strategy of MassArray genotyping on 95
E. alphitoides
isolates sampled in Europe. This genotyping showed that only monospore isolates had the expected haploid signature, whereas direct genotyping from field leaves showed signature of mixed infection. Considering haploid isolates, these markers led to the first results of population genetic diversity, and suggested that
E. quercicola
may have a more asexual reproduction than its sister species,
E. alphitoides
.
L’holoprosencéphalie (HPE) est la malformation congénitale cérébrale la plus fréquente chez l’Homme. Elle est caractérisée par une non-séparation plus ou moins importante des hémisphères cérébraux. ...Lorsqu’elle ne provient pas de défauts chromosomiques, une origine génétique est suspectée. Quatorze gènes sont impliqués, appartenant majoritairement aux voies de signalisation SHH, NODAL, FGF et NOTCH. Au total, les mutations retrouvées n’expliquent qu’environ un tiers des cas d’HPE non-chromosomique, et le mode de transmission n’est pas clairement établi. Afin d’améliorer le rendement diagnostique et le conseil génétique auprès des familles concernées, l’équipe GPLD (IGDR) cherche à identifier de nouveaux gènes impliqués. Dans ce but, le séquençage exomique de familles a été initié depuis 2013. Dans une famille consanguine, une mutation homozygote délétère dans le gène STIL a été identifiée. La protéine STIL est impliquée dans l’assemblage du cil primaire par lequel transite le signal SHH, dont la dysfonction est la première cause d’HPE. Dans une autre famille consanguine, une mutation homozygote candidate est présente dans FAT1, protocadhérine impliquée dans le développement cérébral et responsable d’HPE chez les modèles animaux. D’autres familles, non-consanguines, ont été analysées en trio. Les enfants de toutes ces familles portent une mutation dans un gène de l’HPE, héritée d’un de leur parent asymptomatique. Des mutations additionnelles ont été recherchées, en supposant une origine multigénique de l’HPE chez ces enfants. Un digénisme SHH/DISP1 est présent dans l’une d’elles, et des associations de mutations candidates ont été mises en évidence dans les autres familles, dont une impliquant également FAT1. En conclusion, ces travaux apportent de nouveaux éléments pour la compréhension des bases génétiques de l’HPE et notamment de nouveaux arguments en faveur d’une part importante de multigénisme. L’investigation de ces bases génétiques complexes nécessite le développement de nouvelles méthodes d’analyses, qui pourront être utiles à d’autres pathologies du développement pour lesquelles une origine multigénique est suspectée.
Holoprosencephaly (HPE) is the most common developmental disorder affecting the brain in humans. HPE is characterised by a lack of interhemispheric separation, on a varying scale of severity. When HPE is not due to chromosomal aberrations, a genetic origin is suspected. Alterations of fourteen genes have been implicated in HPE, mainly involved in SHH, NODAL, FGF and NOTCH signalling pathways, with an unclear mode of inheritance. In order to increase the molecular diagnosis yield and to improve genetic counselling, the goal of the GPLD team (IGDR) is to identify new genes. In one inbred family, a deleterious homozygous mutation in STIL gene has been identified. The STIL protein is involved in primary cilia assembly, through which SHH signalling transits. In another inbred family, a homozygous candidate mutation was located in FAT1, a protocadherin involved in brain development that causes HPE-like phenotypes in animal models. For other non-consanguineous families, exome sequencing data were analysed in trios. All children of these families have a previously identified mutation in a HPE gene that is transmitted from a healthy parent. The approach consisted in searching for additional genetic events, under the hypothesis of a multigenic inheritance. Thus, a digenic inheritance of mutations in SHH and DISP1 has been identified in one family. Further associations of candidate mutations have been identified in others, one also involving FAT1. In conclusion, this work provides new elements accounting for the understanding of HPE genetic bases and particularly new arguments in favour of a multigenic inheritance. The study of these complex genetics bases requires the development of new analytical methods that could be of use in relation to other developmental disorders in which a multigenic inheritance is suspected.
Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been ...identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.
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