DiscoSnp-RAD: de novo detection of small variants for RAD-Seq population genomics

International audienceRestriction 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 : identification of new genes and redefinition of the inheritance pattern usinghigh-throughput sequencing approaches

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

Human mitochondrial DNA sequencing by Oxford Nanopore MinION

International audienc

DiscoSnp-RAD: de novo detection of small variants for population genomics

We present an original method to de novo call variants for Restriction site associated DNA Sequencing (RAD-Seq). 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 SNPs) information from entire populations at a reduced cost. Common RAD dedicated tools, as STACKS or IPyRAD, are based on all-versus-all read comparisons , which require consequent time and computing resources. Based on the variant caller DiscoSnp, initially designed for shotgun sequencing, DiscoSnp-RAD avoids this pitfall as variants are detected by exploring the De Bruijn Graph built from all the read datasets. We tested the implementation on RAD data from 259 specimens of Chiastocheta flies, morphologically assigned to 7 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 structuration and the existence of two populations linked to their geographic distributions. Furthermore, our results show that DiscoSnp-RAD is at least one order of magnitude faster than state-of-the-art tools. The overall results show that DiscoSnp-RAD is suitable to identify variants from RAD data, and stands out from other tools due to his completely different principle, making it significantly faster, in particular on large datasets

Genome Engineering of the Fast-Growing Mycoplasma feriruminatoris toward a Live Vaccine Chassis.

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 Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris 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 M. feriruminatoris chassis confirmed the corresponding loss of H2O2 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 Mycoplasma capricolum subsp. capripneumoniae 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 M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases

Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings.

International audienceHoloprosencephaly (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

Integrated clinical and omics approach to rare diseases novel genes and oligogenic inheritance in holoprosencephaly

International audienceHoloprosencephaly is a pathology of forebrain development characterized by high phenotypic heterogeneity. The disease presents with various clinical manifestations at the cerebral or facial levels. Several genes have been implicated in holoprosencephaly but its genetic basis remains unclear different transmission patterns have been described including autosomal dominant, recessive and digenic inheritance. Conventional molecular testing approaches result in a very low diagnostic yield and most cases remain unsolved. In our study, we address the possibility that genetically unsolved cases of holoprosencephaly present an oligogenic origin and result from combined inherited mutations in several genes. Twenty-six unrelated families, for whom no genetic cause of holoprosencephaly could be identified in clinical settings [whole exome sequencing and comparative genomic hybridization (CGH)-array analyses], were reanalysed under the hypothesis of oligogenic inheritance. Standard variant analysis was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis. Statistical validation was performed on 248 ancestrally similar control trios provided by the Genome of the Netherlands project and on 574 ancestrally matched controls provided by the French Exome Project. Variants of clinical interest were identified in 180 genes significantly associated with key pathways of forebrain development including sonic hedgehog (SHH) and primary cilia. Oligogenic events were observed in 10 families and involved both known and novel holoprosencephaly genes including recurrently mutated FAT1, NDST1, COL2A1 and SCUBE2. The incidence of oligogenic combinations was significantly higher in holoprosencephaly patients compared to two control populations (P < 10-9). We also show that depending on the affected genes, patients present with particular clinical features. This study reports novel disease genes and supports oligogenicity as clinically relevant model in holoprosencephaly. It also highlights key roles of SHH signalling and primary cilia in forebrain development. We hypothesize that distinction between different clinical manifestations of holoprosencephaly lies in the degree of overall functional impact on SHH signalling. Finally, we underline that integrating clinical phenotyping in genetic studies is a powerful tool to specify the clinical relevance of certain mutations

p.Gly717Glu cannot fully restore STIL depletion in synchronized U2OS cells.

<p>(A) Protocol used to assay the centriole duplication potential of WT and mutant STIL proteins. U2OS were treated with STIL RNAi, and transfected 24h later with GFP-STIL WT or GFP-STIL p.Gly717Glu constructs in aphidicolin containing medium (4 μg/ml). Cells were fixed and counted 36h later to allow centriole duplication. (B) Percentages of S phase cells containing <4 or ≥4 centrioles following control RNAi (scrambled) and STIL RNAi, followed or not by transfection with GFP-STIL WT or GFP-STIL p.Gly717Glu (p<0,001***). (C) Examples of S phase-arrested cells following different treatments. A control cell with 4 centrioles (top left panel), a STIL RNAi treated cell with 2 centrioles (top right panel), a STIL-depleted cell expressing GFP-STIL WT with 4 centrioles (bottom left), and a STIL-depleted cell expressing GFP-STIL p.Gly717Glu with 2 centrioles are displayed (bottom right). Centrin is shown in red (and in monochrome in the insets), DNA is blue (top panels) and GFP is green (bottom panels). The white arrowheads indicate the centriole region. The bar represents 10 μm.</p

Pedigree of the consanguineous family, brain MRI of the affected siblings, Sanger validation of the c.2150G>A (p.Gly717Glu) <i>STIL</i> mutation, and schematic report of all <i>STIL</i> mutations reported so far.

<p>(A) Pedigree of the inbred family. Closed symbols indicate individuals affected with holoprosencephaly. Family members marked with an asterisk were analyzed by whole exome sequencing. (B) Coronal (on the left) and axial (on the right) brain MRI in individuals II3 and II5 at 12 and 5 years old respectively. II3: lobar HPE, the arrow in the coronal section shows the corpus callosum, and the arrows in the axial section show the absence of visualization of frontal horns, and a partial agenesis of the corpus callosum; II5: semi-lobar HPE, the arrow on axial MRI shows the absence of occipital lobe and a large unilateral temporal and occipital fluid cavity communicating. (C) Sanger validation was performed for the 3 available individuals I2, II3 and II5. The c.2150G>A mutation in <i>STIL</i> revealed a segregation with HPE in the two affected children. (D) Distribution of mutations previously reported in the literature on STIL protein. All mutations were present in a homozygous state [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117418#pone.0117418.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117418#pone.0117418.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117418#pone.0117418.ref029" target="_blank">29</a>] except those represented under the protein, which were two compound heterozygous mutations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117418#pone.0117418.ref028" target="_blank">28</a>]. The p.Leu1218* mutation was found twice in two different families. The mutation reported in this study is p.Gly717Glu (in red) and is located in the central domain of the protein. Three important domains were represented here, the CPAP binding domain from amino acid 429 to 448, the coiled-coil domain (CC) from amino acid 720 to 750 and the KEN box, located between amino acids 1243–1245.</p

Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway

International audienceHoloprosencephaly (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