Utilisation du séquençage à haut débit dans l’identification des gènes prédisposant à l’épilepsie et aux syndromes neurocutanés

Epilepsy is a common neurological disorder affecting 1-3 % of the population. It is characterized by transitory neurological symptoms caused by excessive or synchronized abnormal neuronal activity—a seizure. Many factors can contribute to the etiology of epilepsy. It can be caused by genetic factors, morphological abnormalities, altered levels of biochemical molecules, excessive immune response in the brain, or by infections. Importantly, these factors are not mutually exclusive and can be caused by genetic aberrations. Thus, epilepsy associated with genetic mutations can be part of a complex symptomatology including several monogenic disorders such as progressive myoclonus epilepsies and neurocutaneous syndromes. The first objective of this thesis is to use modern sequencing methods in order to identify genetic causes of rare forms of epilepsies and neurocutaneous syndrome. Firstly, we studied Kufs disease, an adult form of neuronal ceroid lipofuscinosis, part of the progressive myoclonus epilepsies. The study of two American families and one sporadic case led us to identify two causative mutations (p.L116del, p.L115R) in the gene DNAJC5. Other families with this rare but fatal syndrome were reported with mutations in that gene, corroborating our findings. The gene DNAJC5 encodes for the cysteine string protein alpha (CSPα), a presynaptic protein involved in neurodegeneration. Secondly, using the same methods, we discovered the causative mutation for the Giroux-Barbeau syndrome, a neurocutaneous syndrome characterized by spinocerebellar ataxia and erythrokeratodermia variabilis. Thus, following the study of a large French Canadian family, we found the p.L168F mutation in the gene ELOVL4. This gene encodes an enzyme involved in the metabolism of very long chain fatty acid. These fatty acids participate in a wide variety of physiological functions, including skin barrier formation and peroxisome β-oxidation. The second objective of this work is to use whole exome sequencing in the context of molecular diagnostics. To achieve that, we studied a large family presenting skin lesions at the extremities following exposure to cold. Initially diagnosed as a primary form of cryofibrinogenemia, our work showed that these patients were affected with an atypical form of chilblain lupus. In turn, the phenotypical resemblance as well as the presence of the p.D18N mutation in the gene TREX1 led to that new diagnosis. This study shows the role of whole exome sequencing in the diagnosis of rare disease and widen the phenotypic spectrum associated with TREX1 mutations. The last objective of this research is to apply whole exome sequencing in the discovery of genetic mechanisms predisposing to genetic generalized epilepsy (GGE). For this study, we recruited 7 large families with GGE. We showed that variants found in the genes GLI2, MBD5, CELSR2, CELSR3, TNIK and CACNA1G could participate in the genetic etiology in our families. However, because of the numbers of variants identified, other factors could contribute to the epileptic phenotype in our families. Other large scale studies are necessary in order to validate the implication of those genes in epilepsy

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice

Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylation in vitro and in vivo. Immunofluorescence microscopy and ATAC-See revealed the association of this modification with active chromatin. Brpf1 deletion obliterates the acylation in mouse embryos and fibroblasts. Moreover, we identify BRPF1 variants in 12 previously unidentified cases of syndromic intellectual disability and demonstrate that these cases and known BRPF1 variants impair H3K23 propionylation. Cardiac anomalies are present in a subset of the cases. H3K23 acylation is also impaired by cancer-derived somatic BRPF1 mutations. Valproate, vorinostat, propionate and butyrate promote H3K23 acylation. These results reveal the dual functionality of BRPF1-KAT6 complexes, shed light on mechanisms underlying related developmental disorders and various cancers, and suggest mutation-based therapy for medical conditions with deficient histone acylation

Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylati

Endo-MitoEGFP mice: a novel transgenic mouse with fluorescently marked mitochondria in microvascular endothelial cells.

Blood vessel-specific fluorescent transgenic mice are excellent tools to study the development of the vasculature and angiogenic processes. There is growing interest in the biological processes relevant to endothelial cells but limited tools exist to selectively evaluate subcellular functions of this cell type in vivo. Here, we report a novel transgenic animal model that expresses mitochondrially targeted enhanced green fluorescent protein (EGFP) via the Hb9 promoter, a homeobox transcription factor with limited known involvement in the vasculature. Random integration of the transgene, containing the entire mouse Hb9 promoter, was found to be expressed in a variety of vascularised tissues. Further inspection revealed that Mito-EGFP localizes to the endothelial cells (ECs) of a subset of microvascular blood vessels, especially in the central nervous system (CNS), heart, spleen, thymus, lymph nodes and skin. We demonstrate the utility of this novel transgenic mouse, named Endo-MitoEGFP, in the detection, imaging, and isolation of microvascular ECs and evaluation of EC mitochondrial function isolated from adult animals. These transgenic mice will be useful to studies of ECs in development, physiology, and pathology

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicles cycling and axon outgrowth

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2 \u200aknockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2 \u200aas a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD

Additional file 3: of Novel heterozygous pathogenic variants in CHUK in a patient with AEC-like phenotype, immune deficiencies and 1q21.1 microdeletion syndrome: a case report

Figure S3. Growth chart. A) Weight for age (kg). B) Length for age (cm). (TIFF 602 kb

Additional file 1: of Novel heterozygous pathogenic variants in CHUK in a patient with AEC-like phenotype, immune deficiencies and 1q21.1 microdeletion syndrome: a case report

Figure S1. DNA alignment of NGS data using Integrative Genomics Viewer (IGV). IGV snapshot of exon 13 of CHUK gene (NM_001278.3), located on chromosome 10, showing that the two variants (c.1365del, p.Arg457Aspfs*6; c.1388C > A, p.Thr463Lys) are present on different reads, indicating that they occurred on different chromosome (in trans). (TIFF 307 kb

Additional file 2: of Novel heterozygous pathogenic variants in CHUK in a patient with AEC-like phenotype, immune deficiencies and 1q21.1 microdeletion syndrome: a case report

Figure S2. The protein is composed of a protein kinase domain (orange), a leucine zipper region (green) and the NEMO binding-region (blue). Missense (gray) and loss of function (red) pathogenic variants found in the CHUK gene. Reported in this study (Bold). (TIFF 128 kb