A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

The ethylene pathway contributes to root hair elongation induced by the beneficial bacteria Phyllobacterium brassicacearum STM196.

International audienceIn Arabidopsis roots, some epidermal cells differentiate into root hair cells. Auxin regulates root hair positioning, while ethylene controls cell elongation. Phyllobacterium brassicacearum STM196, a beneficial strain of plant growth promoting rhizobacteria (PGPR) isolated from the roots of field-grown oilseed rape, stimulates root hair elongation in Arabidopsis thaliana seedlings. We investigated the role of ethylene in the response of root hair cells to STM196 inoculation. While we could not detect a significant increase in ethylene biosynthesis, we could detect a slight activation of the ethylene signalling pathway. Consistent with this, an exhaustive survey of the root hair elongation response of mutants and transgenic lines affected in the ethylene pathway showed contrasting root hair sensitivities to STM196. We propose that local ethylene emission contributes to STM196-induceed root hair elongation

GSH threshold requirement for NO-mediated expression of the Arabidopsis AtFer1 ferritin gene in response to iron.

International audienceIron treatment of Arabidopsis cultured cells promotes a rapid NO burst within chloroplasts, necessary for up-regulation of the AtFer1 ferritin gene expression. The same occurs in Arabidopsis leaf chloroplasts, and is dependent upon the GSH content of plants. A leaf GSH concentration threshold between 10 and 50 nmol GSHg(-1) FW is required for full induction of AtFer1 gene expression in response to iron

Looking for the function(s) of the plastidial BolA1 and BolA4 proteins in Arabidopsis thaliana

International audienc

The AtNFS2 gene from Arabidopsis thaliana encodes a NifS-like plastidial cysteine desulphurase.

NifS-like proteins are cysteine desulphurases required for the mobilization of sulphur from cysteine. They are present in all organisms, where they are involved in iron-sulphur (Fe-S) cluster biosynthesis. In eukaryotes, these enzymes are present in mitochondria, which are the major site for Fe-S cluster assembly. The genome of the model plant Arabidopsis thaliana contains two putative NifS-like proteins. A cDNA corresponding to one of them was cloned by reverse-transcription PCR, and named AtNFS2. The corresponding transcript is expressed in many plant tissues. It encodes a protein highly related (75% similarity) to the slr0077-gene product from Synechocystis PCC 6803, and is predicted to be targeted to plastids. Indeed, a chimaeric AtNFS2-GFP fusion protein, containing one-third of AtNFS2 from its N-terminal end, was addressed to chloroplasts. Overproduction in Escherichia coli and purification of recombinant AtNFS2 protein enabled one to demonstrate that it bears a pyridoxal 5'-phosphate-dependent cysteine desulphurase activity in vitro, thus being the first NifS homologue characterized to date in plants. The putative physiological functions of this gene are discussed, including the attractive hypothesis of a possible role in Fe-S cluster assembly in plastids

Iron-sulphur cluster assembly in plants: distinct NFU proteins in mitochondria and plastids from Arabidopsis thaliana.

Recent results are in favour of a role for NFU-like proteins in Fe-S cluster biogenesis. These polypeptides share a conserved CXXC motif in their NFU domain. In the present study, we have characterized Arabidopsis thaliana NFU1-5 genes. AtNFU proteins are separated into two classes. NFU4 and NFU5 are part of the mitochondrial type, presenting a structural organization similar to Saccharomyces cerevisiae Nfu1p. These proteins complement a Delta isu1 Delta nfu1 yeast mutant and NFU4 mitochondrial localization was confirmed by green fluorescent protein fusion analysis. AtNFU1-3 represent a new class of NFU proteins, unique to plants. These polypeptides are made of two NFU domains, the second having lost its CXXC motif. AtNFU1-3 proteins are more related to Synechocystis PCC6803 NFU-like proteins and are localized to plastids when fused with the green fluorescent protein. NFU2 and/or NFU3 were detected in leaf chloroplasts by immunoblotting. NFU1 and NFU2 are functional NFU capable of restoring the growth of a Delta isu1 Delta nfu1 yeast mutant, when addressed to yeast mitochondria. Furthermore, NFU2 recombinant protein is capable of binding a labile 2Fe-2S cluster in vitro. These results demonstrate the presence of distinct NFU proteins in Arabidopsis mitochondria and plastids. Such results suggest the existence of two different Fe-S assembly machineries in plant cells

Subcellular localization of ferritin mRNA in Arabidopsis thaliana mutants impaired in mRNA decay

Subcellular localization of ferritin mRNA in Arabidopsis thaliana mutants impaired in mRNA decay. 3e journées scientifiques et techniques du réseau des microscopistes Inra : " De l'imagerie multiple à l'imagerie multimodale

How are Fe-S cofactors and proteins assembled in plant cells? focus on NFUs, involved in the late steps of the maturation process in organelles

International audienc

How are Fe-S cofactors and proteins assembled in plant cells? focus on NFUs, involved in the late steps of the maturation process in organelles

International audienc

Decrypting the chloroplastic [fe-s] cluster assembly machinery using a label free quantification interactomic strategy

Decrypting the chloroplastic [fe-s] cluster assembly machinery using a label free quantification interactomic strategy. Congrès de la Société Française d'Electrophorèse et d'Analyse Protéomique (SFEAP