The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of “off-the-shelf” CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells’ functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety

Alteration of nephrocystins and IFT-A proteins causes similar ciliary phenotypes leading to Nephronophthisis

International audienceNephronophtisis (NPH) is a kidney ciliopathy often associated with extra-renal defects and for which 12 genes (NPHP1-12) have been identified. NPHP1 and NPHP4 control the ciliary access at the transition zone and the velocity of some intraflagellar transport (IFT)/BBS proteins in C.elegans. Recently, in a collaborative effort, we have identified, in families with isolated NPH, mutations in TTC21B as well as in WDR19, which encode the retrograde IFT-A proteins IFT139 and IFT144, respectively. By ciliome sequencing of 1600 candidate genes from 14 NPH patients followed by Sanger sequencing of a cohort of 52 patients, we have found respectively 8 and 7 patients carrying pathogenic missense mutations in genes coding IFT-A proteins, including WDR35, TTC21B and IFT140, which could partially affect their function. Together, these results indicate that IFT-A are involved in nephronophtisis. Moreover, alteration of cilia length was observed in patient kidney, Nphp4-/- mice kidney tubules and NPHP1 or NPHP4 knockdown IMCD3 cell lines. In these cells, primary cilia present swellings at the distal region accompanied by an accumulation of IFT-B at the base and the tip, similar to what was observed in IFT-A mutants, suggesting a possible alteration of retrograde transport. Additionally, ARL13B, a small GTPase required for proper cilium shape and IFT stability, is absent along the axoneme of NPHP4-KD-IMCD cells. By controlling the entry of ciliary components at the transition zone, NPHP1 and NPHP4 may modulate IFT-A cargos thus participating in the same pathway (i.e. Wnt/PCP), alteration of which would lead to renal lesions observed in nephronophthisis

Genetic heterogeneity of polydactyly in Maine Coon cats

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Fibrose pulmonaire et pathogénicité de la mutation p.Val178Met de SFTPA1/SFTPA2

National audienceIntroductionLes mutations bi-alléliques de SFTPB sont généralement associées à des détresses respiratoires néonatale (DRNN) sévères chez des enfants nés à terme. L'imagerie thoracique montre des poumons d'emblée blancs, et l'analyse histologique pulmonaire retrouve typiquement des cloisons alvéolaires épaissies associées à un certain degré de protéinose alvéolaire. Nous rapportons l'observation d'un nouveau-né à terme ayant présenté une DRNN par hypertension pulmonaire supra-systémique sévère et résistante rapidement fatale. Une première enfant du couple, non consanguin, était décédée à J4 d'une DRNN en rapport avec une dysplasie alvéolo-capillaire (DAC) sans mésalignement des veines pulmonaires (MVP). Elle présentait par ailleurs une fente palatine. Le caryotype et la CGH array étaient normaux.InvestigationsLa radiographie thoracique initiale était normale. L'échographie cardiaque a par ailleurs mis en évidence un double arc aortique. La biopsie pulmonaire a montré des parois alvéolaires rigides, sans protéinose alvéolaire, avec des artères hypoplasiques et la persistance d'un lit capillaire compatibles avec une DAC sans MVP. Sur la base de ces éléments et des malformations associées chez les 2 enfants atteints, une mutation du gène FOXF1 était suspectée.RésultatsL'analyse moléculaire n'a pas retrouvé de mutation des séquences codantes et régions introniques flanquantes de FOXF1. En revanche, il a été mis en évidence que les deux s&urs atteintes étaient hétérozygotes composites pour les mutations non-sens c.111G > A p.(Trp37*) (exon 3, probablement pathogène), et décalant le cadre de lecture c.397delinsGAA p.(Pro133Glufs*95) (exon 5, pathogène) de SFTPB. Chacun des parents est hétérozygote pour une des mutations.Discussion et conclusionLes DRNN à terme avec anomalies de développement pulmonaire telles que les DAC sont rares et sont dans 60 à 80% des cas associées à des mutations du gène FOXF1 ou de ses régions régulatrices. Des malformations extra-respiratoires (cardiopathie congénitale, malrotations digestives, anomalies de la ligne médiane) sont fréquemment associées. Les mutations bi-alléliques de SFTPB sont associées à des pneumopathies interstitielles diffuses néonatales, mais n'ont été associées qu'une fois dans la littérature à une DAC. Ce cas clinique met en évidence la difficulté à cibler un seul gène dans la recherche étiologique des DRNN à terme, et étend le spectre phénotypique des mutations de SFTPB aux DAC

Functional characterization of tektin-1 in motile cilia and evidence for TEKT1 as a new candidate gene for motile ciliopathies

International audienceA child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia

COPA syndrome restricted to life-threatening alveolar hemorrhages: clinical, pathological, molecular and biological characterization

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Use of Ruxolitinib in COPA syndrome manifesting as life-threatening alveolar hemorrhage

International audienceCOPA (coatomer subunit α) syndrome is a newly recognised cause of interstitial lung disease in children and adults, frequently associated with arthritis and renal dysfunction. We report a 11-year-old girl with disease limited to major pulmonary haemosiderosis manifesting at the age of 2 years, due to a heterozygous p.(Arg233His) mutation in COPA Her interferon (IFN) signature was elevated (10.312 and 12.429, healthy <2.466), as was the level of serum IFNα (211 fg/mL, healthy <10 fg/mL). STAT1 phosphorylation in T lymphocytes and monocytes was increased as compared with healthy controls. Based on these results she was treated with the JAK1/2 inhibitor ruxolitinib, which resulted in reduction in IFN signalling and appeared to be associated with partial though incomplete decrease in the severity of her pulmonary disease. Patients with alveolar haemorrhage of unknown origin should be considered for COPA screening. Functional tests can help to personalise patient therapy