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Phakomatosis pigmentovascularis (PPV) is a diagnosis which denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement including central nervous system disease, asymmetrical growth and a predisposition to malignancy. Using a tightly phenotyped group and high depth next generation sequencing of affected tissues we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of PPV type III or spilorosea. Within an individual the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to demonstrate that the same variants can cause either the specific pigmentary or vascular phenotypes alone, as well as driving melanoma development within the pigmentary lesion. Protein conformational modelling highlights that while variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modelling of the missense variants confirms downstream MAPK pathway overactivation, and widespread disruption of human endothelial cell angiogenesis. Importantly, PTPN11-mosaic patients theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of naevus spilus and capillary malformation syndromes, paving the way for better clinical management.

To describe clinical and molecular findings in a French multicenter cohort of fetuses with prenatal diagnosis of congenital abnormality and suspicion of a localized overgrowth disorder (LOD) suggestive of genetic variants in the PI3K-AKT-mTOR signaling pathway.We analyzed retrospectively data obtained between 1 January 2013 and 1 May 2020 from fetuses with brain and/or limb overgrowth referred for molecular diagnosis of PI3K-AKT-mTOR pathway genes by next-generation sequencing (NGS) using pathological tissue obtained by fetal autopsy. We also assessed the diagnostic yield of amniotic fluid.During the study period, 21 subjects with LOD suspected of being secondary to a genetic variant of the PI3K-AKT-mTOR pathway were referred for analysis. Of these, 17 fetuses had brain overgrowth, including six with isolated megalencephaly (MEG) and 11 with hemimegalencephaly (HMEG). Of the six with MEG, germline variants were identified in four cases, in either PIK3R2, AKT3 or MTOR, and a postzygotic PIK3R2 variant was found in the other two cases. Of the 11 with HMEG, a postzygotic PIK3CA variant was found in three fetuses with extracerebral features of PIK3CA-related overgrowth spectrum, and in seven fetuses with isolated HMEG. No pathogenic variant was identified in the 11Isolated MEG or HMEG may lead to identification of genetic variants in the PI3K-AKT-mTOR signaling pathway. Cases of limb overgrowth and LM or isolated HMEG are likely associated with PIK3CA variants. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

PRDM16 (positive regulatory domain 16) is localized in the critical region for cardiomyopathy in patients with deletions of chromosome 1p36, as defined by Gajecka et al., American Journal of Medical Genetics, 2010, 152A, 3074-3083, and encodes a zinc finger transcription factor. We present the first fetal case of left ventricular non-compaction (LVNC) with a PRDM16 variant. The third-trimester obstetric ultrasound revealed a hydropic fetus with hydramnios and expanded hypokinetic heart. After termination of pregnancy, foetopathology showed a eutrophic fetus with isolated cardiomegaly. Endocardial fibroelastosis was associated with non-compaction of the myocardium of the left ventricle. Exome sequencing (ES) identified a de novo unreported p.(Gln353*) heterozygous nonsense variant in PRDM16. ES also identified two rare variants of unknown significance, according to the American College of Medical Genetics and Genomics guidelines, in the titin gene (TTN): a de novo missense p.(Lys14773Asn) variant and a c.33043+5A>G variant inherited from the mother. Along with the PRDM16 de novo probably pathogenic variant, TTN VOUS variants could possibly contribute to the severity and early onset of the cardiac phenotype. Because of the genetic heterogeneity of cardiomyopathies, large panels or even ES could be considered as the main approaches for the molecular diagnosis, particularly in fetal presentations, where multiple hits seem to be common.

Importance Sirolimus is increasingly being used to treat various vascular anomalies, although evidence of its efficacy is lacking. Objective To assess the efficacy and safety of sirolimus for children with slow-flow vascular malformations to better delineate the indications for treatment. Design, setting and participants This multicenter, open-label, observational-phase randomized clinical trial included 59 children aged 6 to 18 years with a slow-flow vascular malformation who were recruited between September 28, 2015, and March 22, 2018, in 11 French tertiary hospital centers. Statistical analysis was performed on an intent-to-treat basis from December 4, 2019, to November 10, 2020. Interventions Patients underwent an observational period, then switched to an interventional period when they received oral sirolimus (target serum levels, 4-12 ng/mL). The switch time was randomized from month 4 to month 8, and the whole study period lasted 12 months for each patient. Main outcomes and measures The primary outcome was change in the volume of vascular malformations detected on magnetic resonance imaging scan (with centralized interpretation) per unit of time (ie, between the interventional period and the observational period). Secondary outcomes included subjective end points: pain, bleeding, oozing, quality of life, and safety. Results Among the participants (35 girls [59.3%]; mean [SD] age, 11.6 [3.8] years), 22 (37.3%) had a pure venous malformation, 18 (30.5%) had a cystic lymphatic malformation, and 19 (32.2%) had a combined malformation, including syndromic forms. Variations in the volume of vascular malformations detected on magnetic resonance imaging scans associated with the duration period were not overall significantly different between the interventional period and the observational period (all vascular malformations: mean [SD] difference, -0.001 [0.007]; venous malformations: mean [SD] difference, 0.001 [0.004]; combined malformations: mean [SD] difference, 0.001 [0.009]). However, a significant decrease in volume was observed for children with pure lymphatic malformations (mean [SD] difference, -0.005 [0.005]). Overall, sirolimus had positive effects on pain, especially for combined malformations, and on bleeding, oozing, self-assessed efficacy, and quality of life. During sirolimus treatment, 56 patients experienced 231 adverse events (5 serious adverse events, none life-threatening). The most frequent adverse event was an oral ulcer (29 patients [49.2%]). Conclusions and relevance This observational-phase randomized clinical trial allows for clarifying the goals of patients and families when starting sirolimus therapy for children older than 6 years. Pure lymphatic malformations seem to be the best indication for sirolimus therapy because evidence of decreasing lymphatic malformation volume per unit of time, oozing, and bleeding and increasing quality of life was found. In combined malformations, sirolimus significantly reduced pain, oozing, and bleeding. Benefits seemed lower for pure venous malformations than for the 2 other subgroups, also based on symptoms. Trial registration ClinicalTrials.gov Identifier: NCT02509468; clinicaltrialsregister.eu Identifier: 2015-001096-43.

BRAF postzygotic activating mutations have been found in 50% of cases of syringocystadenoma papilliferum (SCAP)1 and in phacomatosis pigmentokeratotica (PPK)2,3 , also possibly caused by HRAS4 mutations. BRAF is a RAS-activating serine/threonine kinase of the MAP kinase pathway, resulting in cell growth and proliferation. BRAF mutations, particularly p.(Val600Glu), are frequently identified in melanoma and other human cancers5 . We report clinical presentations of three patients with postzygotic BRAF mutations in affected skin, identified by next generation sequencing (NGS).

Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS. KEY MESSAGES: Cohen syndrome patients' neutrophils display normal morphology and functions. Cohen syndrome patients' neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors' neutrophils. No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients' neutrophils. SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.

Background Postzygotic mutations in FGFR2 have been identified in mosaic forms of acne, keratinocytic epidermal nevi, nevoid acanthosis nigricans / rounded and velvety epidermal nevus and in two fetuses with papillomatous pedunculated sebaceous nevus (PPSN). Objectives To determine the clinical and genetic characteristics of children with cerebriform, papillomatous, and pedunculated variants of sebaceous nevi. Methods Infants diagnosed with sebaceous nevi characterized by a cerebriform, papillomatous, and/or pedunculated morphology over a 10-year period (2010 - 2019) at three pediatric dermatology centers in Switzerland and France were included in this case series. Clinical and histological characteristics were assessed. Next generation sequencing was used to assess for FGFR2 mutations. Results All nevi were located on the head, with a rounded or linear shape and a typical cerebriform, sometimes papillomatous and pedunculated, surface. No associated extracutaneous anomalies were found. Nevi harbored postzygotic mutations in the transmembrane domain of FGFR2 in 6/8 children (75%), either the known specific p.(Cys382Arg) mutation in 5 cases, or a novel mutation, p.(Val395Asp), in one. Conclusions We found an exquisite genotype-phenotype correlation in these rare nevi, with specific postzygotic mutations in the transmembrane domain of FGFR2. As not all lesions were truly papillomatous and pedunculated, the term cerebriform sebaceous nevus (CSN) appears more suitable than PPSN to describe this entity. The cerebriform pattern of CSN is reminiscent of cutis gyrata, as seen in Beare-Stevenson syndrome, which is caused by closely related germline FGFR2 mutations. While clinically impressive, CSN seem to carry a good prognosis and a low risk for extracutaneous associations.

Background The molecular basis of McCune Albright syndrome (MAS) is a recurrent GNAS Postzygotic gain of function sporadic mutation, resulting in a mosaic disease. Most of girls present precocious puberty, caused by the development of recurrent ovarian cysts with autonomous Hyperestrogenic stimulation. After menarche, the majority of patients with ovarian GNAS mutation have menstrual disturbances and infertility. Objectives We wanted to focus on the fertility of MAS females and propose an appropriate management, by a detailed case report and an exhaustive review of the literature on fertility and pregnancy in MAS females. Results We present the case of a 29-year-old MAS female, who had previously undergone a unilateral ovariectomy and was managed by in vitro fertilization (IVF). Eight oocytes with many morphological abnormalities were retrieved. The GNAS mutation was found at a low frequency in follicular cells. The ovarian histopathological examination showed developing follicles of all stages, strongly expressing AMH by immunohistochemistry. In addition, AMH was high (45.5 pmol/L) and the AMH / AFC ratio (5.69 pmol/L per follicle) was much higher than in PCOS and control groups (2.16, and 1.34 respectively). Conclusions Ovarian and endometrial involvement can be responsible for infertility in MAS women. IVF and oophorectomy may be useful in management. The genetic characterization of the different tissues may have a prognostic utility. Moreover, we suggest that the AMH could be a marker of the ovarian activity in MAS. Further studies are needed to clarify the potential oocyte abnormalities and the risk of miscarriages in order to guide genetic counseling.

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional corepressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

X-linked intellectual disability (XLID) is a genetically heterogeneous condition involving more than 100 genes. To date, 35 pathogenic variants have been reported in the lysine specific demethylase 5C (KDM5C) gene. KDM5C variants are one of the major causes of moderate to severe XLID. Affected males present with short stature, distinctive facial features, behavioral disorders, epilepsy, and spasticity. For most of these variants, related female carriers have been reported, but phenotypic descriptions were poor. Here, we present clinical and molecular features of 19 females carrying 10 novel heterozygous variants affecting KDM5C function, including five probands with de novo variants. Four heterozygous females were asymptomatic. All affected individuals presented with learning disabilities or ID (mostly moderate), and four also had a language impairment mainly affecting expression. Behavioral disturbances were frequent, and endocrine disorders were more frequent in females. In conclusion, our findings provide evidence of the role of KDM5C in ID in females highlighting the increasing implication of XLID genes in females, even in sporadic affected individuals. Disease expression of XLID in females should be taken into consideration for genetic counseling.

IntroductionPigmentary mosaicism (PM) manifests by pigmentation anomalies along Blaschko’s lines and represents a clue toward the molecular diagnosis of syndromic intellectual disability (ID). Together with new insights on the role for lysosomal signalling in embryonic stem cell differentiation, mutations in the X-linked transcription factor 3 (TFE3) have recently been reported in five patients. Functional analysis suggested these mutations to result in ectopic nuclear gain of functions.Materials and methodsSubsequent data sharing allowed the clustering of de novo TFE3 variants identified by exome sequencing on DNA extracted from leucocytes in patients referred for syndromic ID with or without PM.ResultsWe describe the detailed clinical and molecular data of 17 individuals harbouring a de novo TFE3 variant, including the patients that initially allowed reporting TFE3 as a new disease-causing gene. The 12 females and 5 males presented with pigmentation anomalies on Blaschko’s lines, severe ID, epilepsy, storage disorder-like features, growth retardation and recognisable facial dysmorphism. The variant was at a mosaic state in at least two male patients. All variants were missense except one splice variant. Eleven of the 13 variants were localised in exon 4, 2 in exon 3, and 3 were recurrent variants.ConclusionThis series further delineates the specific storage disorder-like phenotype with PM ascribed to de novo TFE3 mutation in exons 3 and 4. It confirms the identification of a novel X-linked human condition associated with mosaicism and dysregulation within the mechanistic target of rapamycin (mTOR) pathway, as well as a link between lysosomal signalling and human development.

The MTOR gene encodes the mechanistic target of rapamycin (mTOR), which is a core component of the PI3K-AKT-mTOR signaling pathway. Postzygotic MTOR variants result in various mosaic phenotypes, referred to in OMIM as Smith-Kinsgmore syndrome or focal cortical dysplasia. We report here the case of a patient, with an MTOR mosaic gain-of-function variant (p.Glu2419Lys) in the DNA of 41% skin cells, who received compassionate off-label treatment with everolimus for refractory epilepsy. This 12-year-old-girl presented with psychomotor regression, intractable seizures, hypopigmentation along Blaschko's lines (hypomelanosis of Ito), asymmetric regional body overgrowth, and ocular anomalies, as well as left cerebral hemispheric hypertrophy with some focal underlying migration disorders. In response to the patient's increasingly frequent epileptic seizures, everolimus was initiated (after approval from the hospital ethics committee) at 5 mg/day and progressively increased to 12.5 mg/day. After 5 months of close monitoring (including neuropsychological and electroencephalographic assessment), no decrease in seizure frequency was observed. Though the physiopathological rationale was good, no significant clinical response was noticed under everolimus treatment. A clinical trial would be needed to draw conclusions, but, because the phenotype is extremely rare, it would certainly need to be conducted on an international scale.

Introduction Des mutations postzygotiques de FGFR2 ont ete identifiees dans des formes d’acne en mosaique, dans le naevus epidermique keratinocytaire, l’acanthosis nigricans naevoide ou RAVEN (rounded and velvety epidermal nevus) et le naevus sebace papillomateux et pedoncule (NSPP) [1] . Objectifs Determiner les caracteristiques cliniques et genetiques du NSPP et d’une forme de naevus epidermique cerebriforme (NEC), proches sur le plan clinique. Patients et methodes Nourrissons diagnostiques avec des NSPP et NEC sur une periode de 10 ans (2010-2019) dans deux centres de dermatologie pediatrique en Suisse et en France. Les caracteristiques cliniques et histologiques ont ete evaluees. Le sequencage de nouvelle generation a ete utilise pour determiner la frequence et les caracteristiques des variations post-zygotiques de FGFR2. Resultats Tous les naevus etaient situes sur le cuir chevelu ou le visage, avec une forme lobulee, arrondie ou lineaire, et une surface souvent cerebriforme, avec absence d’anomalies extra cutanees. Des variations post-zygotiques dans le domaine transmembranaire de FGFR2 ont ete identifiees chez 6/8 enfants (75 %), la variation recurrente p.(Cys382Arg) (5/6) ainsi que la variation p.(Val395Asp), non encore rapportee (1/6). Conclusion Nous proposons la denomination « naevus epidermique cerebriforme » comme terme plus inclusif pour ces naevus rares, secondaires aux memes anomalies moleculaires, a savoir des variations post-zygotiques specifiques, codant le domaine transmembranaire de FGFR2. Le profil cerebriforme du NEC rappelle celui du cutis verticis gyrata (CVG), present dans le syndrome de Beare-Stevenson (BSS) [2] . Etant donne que le BSS est cause par des variations constitutionnelles proches de FGFR2, le NEC pourrait etre considere comme une forme circonscrite de CVG, a la fois cliniquement et genetiquement.

Introduction L’atteinte cutanee de la maladie de Kawasaki (MK) se presente generalement sous la forme d’un exantheme maculo-papuleux ou morbilliforme. Toutefois, de rares cas de MK pustuleuses ont ete rapportes. A premiere vue evocatrice d’un psoriasis pustuleux generalise (PPG) ou d’un syndrome DITRA (deficience en antagoniste du recepteur de l’IL36), la MK pustuleuse peut entrainer des difficultes diagnostiques. Nous rapportons la survenue d’une pustulose aseptique annulaire au cours d’une MK, et discutons un mecanisme physiopathologique commun entre ces deux entites aux cadres nosologiques distincts. Observations Un garcon de 5 ans etait adresse aux urgences pour un exantheme pustuleux annulaire febrile associe a un erytheme palmo-plantaire persistant depuis cinq jours. L’atteinte cutanee etait evocatrice d’un psoriasis pustuleux generalise mais la presence d’une cheilite, d’une conjonctivite bilaterale nous orienta vers le diagnostic de MK pustuleuse. La biopsie cutanee montrait une pustulose cornee et sous-cornee sans signe de vascularite. L’echographie cardiaque transthoracique mettait en evidence deux anevrismes coronaires. Le traitement consistait en une perfusion unique d’immunoglobulines polyvalentes intraveineuses et l’administration orale d’acide salicylique. Le patient etait apyretique en moins de 36 heures et l’ensemble des symptomes regressaient en quelques jours. L’ETT de controle a 6 semaines s’etait normalisee. Le sequencage d’exome n’avait pas mis en evidence de mutation pathogene majeure. Discussion L’association d’une PA et d’une MK a ete rapportee dans seulement 11 observations. Malgre leur appartenance a des cadres nosologiques differents (les vascularites pour la MK et les MAI pour le DITRA), l’hypothese d’un mecanisme physiopathologique commun est discutee. Le DITRA et la MK presentent de nombreuses similitudes cliniques et biologiques mais egalement genetiques. L’utilisation du sequencage d’exome pour le diagnostic des maladies auto-inflammatoires (MAI) infantiles a permis d’integrer certains PPG parmi les MAI mais egalement d’ameliorer les connaissances physiopathologiques d’autres affections. La decouverte de mutations pathogenes ITPKC ou ORAI-1 dans la MK et les fortes concentrations seriques d’IL-1β observees en phase aigue de la MK suggerent un mecanisme principalement medie par l’immunite innee chez des enfants genetiquement predisposes, ce qui est egalement observe dans les MAI. De meme, dans le DITRA, le deficit en recepteur antagoniste IL36-Ra entraine, via l’activation de la voie NFkB, une surproduction de pro-IL1β et, apres clivage, d’IL-1β. Ces donnees ouvrent egalement la voie de nouvelles pistes therapeutiques dans la MK. Comme l’utilisation des anti-IL1 dans les MAI infantiles se revele extremement efficace, on pourrait proposer ces traitements dans la MK, notamment dans les formes resistantes ou atypiques.

IF 5.340; International audience; The main identified function of BCL2 protein is to prevent cell death by apoptosis. Mice knock-out for Bcl2 demonstrate growth retardation, severe polycystic kidney disease (PKD), gray hair and lymphopenia, and die prematurely after birth. Here, we report a 40-year-old male referred to for abdominal and thoracic aortic dissection with associated aortic root aneurysm, PKD, lymphocytopenia with a history of T cell lymphoblastic lymphoma, white hair since the age of 20, and learning difficulties. PKD, which was also detected in the father and sister, was related to an inherited PKD1 mutation. The combination of PKD with gray hair and lymphocytopenia was also reminiscent of Bcl2-/- mouse phenotype. BCL2 gene transcript and protein level were observed to be dramatically decreased in patient peripheral blood T-cells and in his aorta vascular wall cells, which was not detected in parents and sister T-cells, suggesting an autosomal recessive inheritance. Accordingly, spontaneous apoptosis of patient T-cells was increased and could be rescued through stimulation with an anti-CD3 antibody. Direct sequencing of BCL2 gene exons, promoter and 3'UTR region as well as BCL2 mRNA sequencing failed in identifying any pathogenic variant. Array-CGH was also normal and whole exome sequencing of the patient, parents and sister DNA did not detect any significant variant in genes encoding Bcl2-interacting proteins. miRNA array identified an up-regulation of miR-181a, which is a known regulator of BCL2 expression. Altogether, miR-181a-mediated decrease in BCL2 gene expression could be a modifying factor that aggravates the phenotype of a PKD1 constitutive variant.

International audience; Macrozoospermia is characterized by a high proportion of abnormal spermatozoa with enlarged heads. So far, it has been associated with mutations only in the Aurora Kinase C gene (AURKC) in some cases. Although many publications have reported failure to conceive in couples with macrozoospermia, a few others have described successful pregnancies, thus raising questions as to whether ICSI and AURKC genetic screening should be recommended in all patients with macrozoospermia. First, we report on two monozygotic twins presenting macrozoospermia for whom the genetic status was explored (Aurora Kinase C sequencing) and whole semen and gradient-selected spermatozoa were analyzed, using Fluorescent In Situ Hybridization (FISH), Electron Microscopy and flow cytometry. Additionally, FISH analysis was performed on individually selected uniflagellate spermatozoa with normal sized heads. Second, we also provide an updated review of patients with macrozoospermia gathering the percentage of enlarged head spermatozoa, the genetic status and pregnancy outcomes. Both twins carried a homozygous mutation of AURKC. Spermocytograms showed means of 86% and 83.5% of enlarged head forms. FISH analyses showed that normal head size, uniflagellate spermatozoa had an aneuploid or polyploid nucleus despite a high level of selection. SEM analysis also showed special intranuclear inclusions in enlarged head spermatozoa. Our data together with cases reported in the literature allowed us to recommend that the AURKC gene should be sequenced when the sperm contains 30% or more of enlarged head spermatozoa, and when a mutation is found, ART should not be performed. Our analyses provide information that could greatly help practitioners in their decision-making with regard to optimal care of patients with macrozoospermia.

Introduction Le syndrome de Maffucci (SM) est une maladie genetique extremement rare (prevalence Observation Il s’agissait d’une patiente de 51 ans, aux antecedents d’encephalopathie vasculaire sans etiologie trouvee. Elle presentait depuis l’enfance des anomalies osseuses uniquement localisees a gauche, a type d’enchondromes, certains ayant fait l’objet d’une exerese (chondrosarcome). Les anomalies cutanees etaient apparues vers l’âge de 20 ans, de maniere strictement unilaterale gauche, allant du visage au pied. On notait des lesions hypertrophiques papillomateuses, hyperkeratosiques et vesiculeuses, en particulier sur la jambe et le pubis. On notait egalement quelques macules d’allure capillaire du tronc. L’echographie doppler et l’imagerie en resonance magnetique (IRM) concluaient a des malformations lymphatiques microkystiques etendues superficielles et profondes. Il existait des anomalies post-thrombotiques sans malformations veineuses. Les lesions etaient douloureuses, suintantes et le siege de surinfections recidivantes justifiant antibiotherapie au long court, contention, sclerotherapie et drainages. L’analyse moleculaire montrait une variation heterozygote faux-sens du gene IDH1 (Arg132Cys), presente a l’etat post-zygotique dans 8 % des alleles de l’ADN de la biopsie cutanee, confirmant le diagnostic de SM car consideree comme causale de la maladie. Une IRM corps entier est programmee afin d’eliminer des lesions malignes associees et un traitement systemique par sirolimus est envisage. Discussion Notre patiente presente un phenotype particulier. En effet, la disposition des lesions est classiquement asymetrique mais les atteintes hemicorporelles etendues semblent exceptionnelles. La presence de malformations lymphatiques quasi exclusive n’a ete decrite a notre connaissance que chez 2 patient,s sans confirmation moleculaire. Il est important de faire le diagnostic de SM pour rechercher des neoplasies associees. Une mutation des genes IDH 1 et IDH2 est retrouvee dans 80 % des SM et explique le risque de neoplasie squelettique (chondrosarcome) ou non squelettique (gliome), pouvant atteindre 100 % des patients, expliquant l’interet de la surveillance. Le traitement par sirolimus (inhibiteur m-TOR) a ete essaye chez 3 patients sans malformations lymphatiques avec des resultats discordants (rationnel : activation de la voie AKT-mTOR induite par la mutation). Conclusion Ce cas clinique apporte de donnees nouvelles concernant le phenotype du SM, affection tres rare et a haut risque de neoplasie associee.

PurposeMarfanoid habitus (MH) combined with intellectual disability (ID) (MHID) is a clinically and genetically heterogeneous presentation. The combination of array CGH and targeted sequencing of genes responsible for Marfan or Lujan–Fryns syndrome explain no more than 20% of subjects.MethodsTo further decipher the genetic basis of MHID, we performed exome sequencing on a combination of trio-based (33 subjects) or single probands (31 subjects), of which 61 were sporadic.ResultsWe identified eight genes with de novo variants (DNVs) in at least two unrelated individuals (ARID1B, ATP1A1, DLG4, EHMT1, NFIX, NSD1, NUP205 and ZEB2). Using simulation models, we showed that five genes (DLG4, NFIX, EHMT1, ZEB2 and ATP1A1) met conservative Bonferroni genomewide significance for an excess of the observed de novo point variants. Overall, at least one pathogenic or likely pathogenic variant was identified in 54.7% of subjects (35/64). These variants fell within 27 genes previously associated with Mendelian disorders, including NSD1 and NFIX, which are known to be mutated in overgrowth syndromes.ConclusionWe demonstrated that DNVs were enriched in chromatin remodelling (p=2×10−4) and genes regulated by the fragile X mental retardation protein (p=3×10−8), highlighting overlapping genetic mechanisms between MHID and related neurodevelopmental disorders.

International audience; Purpose Lanosterol synthase (LSS) gene was initially described in families with extensive congenital cataracts. Recently, a study has highlighted LSS associated with hypotrichosis simplex. We expanded the phenotypic spectrum of LSS to a recessive neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome. It is a rare autosomal recessive condition characterized by hypotrichosis and intellectual disability (ID) or developmental delay (DD), frequently associated with early-onset epilepsy and other dermatological features. Methods Through a multicenter international collaborative study, we identified LSS pathogenic variants in APMR individuals either by exome sequencing or LSS Sanger sequencing. Splicing defects were assessed by transcript analysis and minigene assay. Results We reported ten APMR individuals from six unrelated families with biallelic variants in LSS. We additionally identified one affected individual with a single rare variant in LSS and an allelic imbalance suggesting a second event. Among the identified variants, two were truncating, seven were missense, and two were splicing variants. Quantification of cholesterol and its precursors did not reveal noticeable imbalance. Conclusion In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclization of (S)-2,3-oxidosqualene into lanosterol. Our data suggest LSS as a major gene causing a rare recessive neuroectodermal syndrome.

Introduction Le syndrome des hamartomes et tumeurs lie a PTEN (PHTS) comporte parfois des malformations arterio-veineuses (MAV) pour lesquelles les options therapeutiques telles que l’embolisation ou la resection chirurgicale sont limitees. Le developpement en cancerologie d’inhibiteurs de la voie PI3K-AKT-MTOR permet d’escompter leur repositionnement comme medicaments cibles sur l’anomalie genetique. Nous rapportons la premiere observation de reponse a un traitement par alpelisib (BYL719), inhibiteur specifique de la sous-unite alpha de la PI3-kinase. Observations Un patient de 27 ans etait suivi depuis l’âge de 16 ans pour des MAV des parties molles du membre inferieur gauche, a l’origine d’une hemarthrose du genou en 2009, paravertebrales gauches T3-T4, et pelvienne, a l’origine d’une varicocele, d’une hydrocele et de thromboses fessieres. Elles ont ete regulierement embolisees jusqu’a l’âge de 23 ans. Un carcinome renal a cellules claires, decouvert sur l’imagerie, avait ete traite par nephrectomie droite a 17 ans. Une macrocrânie et une pigmentation genitale avaient fait rechercher et identifier une mutation germinale faux-sens de PTEN. Ces MAV multiples au stade III de Schobinger etaient principalement compliquees d’un hyperdebit cardiaque sans decompensation mais avec un index cardiaque (IC) a 5,7 L/min/m2 a l’âge de 21 ans (risque de passage au stade IV), motivant l’introduction d’un traitement par sirolimus 1 mg/j, interrompu apres 7 mois puis repris a 2 mg/j, et a nouveau interrompu en l’absence de reduction de l’IC. Apres obtention d’une autorisation temporaire d’utilisation nominative par l’ANSM, l’introduction de l’aleelisib a 150 mg/j a ete suivie d’une stabilite des MAV sans reduction de leur debit, d’une regression des douleurs et surtout d’une reduction de 40 % de l’IC, passant de 6,6 L/min/m2 a 4,0 L/min/m2 en 3 mois, se maintenant apres 6 mois de traitement. Discussion Bien que l’alpelisib ait ete exclusivement reserve jusqu’ici aux patients atteints de cancer du sein ou de syndromes hypertrophiques du spectre PROS et porteurs de mutations activatrices de PIK3CA, notre observation suggere qu’il puisse aussi exister une reponse des MAV chez des patients porteurs de mutations inactivatrices de PTEN, suppresseur de tumeur regulant la voie PI3K-AKT-MTOR. L’amelioration observee de l’IC nous semble attribuable a l’alpelisib, en l’absence de reponse anterieure aux autres traitements. Il s’agit donc d’une nouvelle option therapeutique medicamenteuse adaptee aux resultats de l’analyse genetique, de la meme maniere que des MAV liees a des mutations en mosaique de la voie RAS-MAPK ont repondu a des inhibiteurs de MEK. Cette observation illustre l’interet d’une demarche dite de « medecine personnalisee », ciblee sur l’identification d’une affection predisposante et l’analyse genetique, pour le traitement des malformations arterio-veineuses, qui restent toutefois un defi therapeutique.

The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.

Introduction La determination de l’etiologie genetique des anomalies de la pigmentation cutanee en mosaique est un enjeu a double titre: d’une part, elle necessite des methodes adaptees a la detection de variations genetiques a faible taux et confinees au tissu atteint, et d’autre part elle est un element d’orientation determinant pour la prise en charge des patients (mise en evidence d’une cible therapeutique, etablissement de la strategie de surveillance, conseil genetique). Le sequencage d’exome (ES) est en 2019 la methode de reference pour la detection des mutations ponctuelles post-zygotiques (mSNV), mais la detection d’anomalies chromosomiques en mosaique (mCNV) repose habituellement sur la CGH-array ou le caryotype, difficilement realisables en routine a partir d’une biopsie de peau sans culture. Nous avons developpe une strategie utilisant les donnees d’ES permettant la detection des mSNV et des mCNV, afin d’eviter la multiplication des prelevements invasifs et des examens couteux, et d’ameliorer le rendement diagnostic et la prise en charge de ces patients. Materiel et methodes Au sein de notre cohorte de 128 patients avec anomalie pigmentaire, nous avons analyse par ES 56 patients, pour lesquels les explorations chromosomiques (caryotypes sur sang ou sur fibroblastes cultives, ou CGH-array) etaient demeurees negatives. A l’aide d’un algorithme mis au point au sein de l’equipe, nous avons recherche systematiquement, sur les donnees d’ES en trio (peau + sang des parents), les mutations ponctuelles en mosaique, les variations de nombre de copie chromosomique, les disomies et les triploidies. Resultats Nous avons detecte des mCNV chez 17 patients (30 % de la cohorte: 5 trisomies, 6 tetrasomies, 2 monosomies et 4 triploidies), et des mSNV chez 12 (21 %, dans 6 genes differents). Parmi ces patients, l’identification de la cause genetique permet d’envisager une adaptation therapeutique pour 28 %, de mettre en evidence un risque familial de recurrence pour 25 %, ou d’hypofertilite pour 75 % (mutation presumee letale a l’etat constitutionnel). Des variations entrainant egalement un risque tumoral eleve, necessitant une surveillance adaptee, ont ete identifiees chez 2 patients. Conclusion Nous avons confirme par une methode originale et sensible la frequence des remaniements chromosomiques en mosaique associes aux anomalies de la pigmentation cutanee. Nous apportons la preuve de concept que l’ES a visee diagnostique permet la detection des mSNV et mCNV en un seul examen, a partir d’un seul prelevement, ameliorant significativement le rendement diagnostique. Face a l’errance diagnostique qui affecte souvent ces patients porteurs de maladies rares et complexes, la generalisation de cette approche devrait permettre l’amelioration de leur prise en charge.

International audience; PurposeCongenital anomalies and intellectual disability (CA/ID) are a major diagnostic challenge in medical genetics—50% of patients still have no molecular diagnosis after a long and stressful diagnostic “odyssey.” Solo clinical whole-exome sequencing (WES) was applied in our genetics center to improve diagnosis in patients with CA/ID.MethodsThis retrospective study examined 416 consecutive tests performed over 3 years to demonstrate the effectiveness of periodically reanalyzing WES data. The raw data from each nonpositive test was reanalyzed at 12 months with the most recent pipeline and in the light of new data in the literature. The results of the reanalysis for patients enrolled in the third year are not yet available.ResultsOf the 416 patients included, data for 156 without a diagnosis were reanalyzed. We obtained 24 (15.4%) additional diagnoses: 12 through the usual diagnostic process (7 new publications, 4 initially misclassified, and 1 copy-number variant), and 12 through translational research by international data sharing. The final yield of positive results was 27.9% through a strict diagnostic approach, and 2.9% through an additional research strategy.ConclusionThis article highlights the effectiveness of periodically combining diagnostic reinterpretation of clinical WES data with translational research involving data sharing for candidate genes.

International audience

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.

Over one hundred VPS13B mutations are reported in Cohen syndrome (CS). Most cases exhibit a homogeneous phenotype that includes intellectual deficiency (ID), microcephaly, facial dysmorphism, slender extremities, truncal obesity, progressive chorioretinal dystrophy, and neutropenia. We report on a patient carrying two VPS13B splicing mutations with an atypical phenotype that included microcephaly, retinopathy, and congenital neutropenia, but neither obesity nor ID. RNA analysis of the IVS34+2T_+3AinsT mutation did not reveal any abnormal splice fragments but mRNA quantification showed a significant decrease in VPS13B expression. RNA sequencing analysis up- and downstream from the IVS57+2T>C mutation showed abnormal splice isoforms. In contrast to patients with typical CS, who express only abnormal VPS13B mRNA and truncated protein, a dose effect of residual normal VPS13B protein possibly explains the incomplete phenotype in the patient. This observation emphasizes that VPS13B analysis should be performed in cases of congenital neutropenia associated with retinopathy, even in the absence of ID, therefore extending the VPS13B phenotype spectrum. © 2013 Wiley Periodicals, Inc.

Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS. We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested. We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10−5). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10−25), regardless of the phenotype. Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions. Genet Med advance online publication 02 February 2017

Mutations in the gene encoding laminin α2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin α2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin α7β1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin α2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin αIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin α7β1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin α7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin α7Bβ1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin α7Bβ1D signaling in skeletal muscle.

International audience; Today, there is growing interest in the potential epigenetic risk related to assisted reproductive technologies (ART). Much evidence in the literature supports the hypothesis that adverse pregnancy outcomes linked to ART are associated with abnormal trophoblastic invasion. The aim of this review is to investigate the relationship between epigenetic dysregulation caused by ART and subsequent placental response. The dialogue between the endometrium and the embryo is a crucial step to achieve successful trophoblastic invasion, thus ensuring a non-complicated pregnancy and healthy offspring. However, as described in this review, ART could impair both actors involved in this dialogue. First, ART may induce epigenetic defects in the conceptus by modifying the embryo environment. Second, as a result of hormone treatments, ART may impair endometrial receptivity. In some cases, it results in embryonic growth arrest but, when the development of the embryo continues, the placenta could bring adaptive responses throughout pregnancy. Amongst the different mechanisms, epigenetics, especially thanks to a finely tuned network of imprinted genes stimulated by foetal signals, may modify nutrient transfer, placental growth and vascularization. If these coping mechanisms are overwhelmed, improper maternal-foetal exchanges occur, potentially leading to adverse pregnancy outcomes such as abortion, preeclampsia or intra-uterine growth restriction. But in most cases, successful placental adaptation enables normal progress of the pregnancy. Nevertheless, the risks induced by these modifications during pregnancy are not fully understood. Metabolic diseases later in life could be exacerbated through the memory of epigenetic adaptation mechanisms established during pregnancy. Thus, more research is still needed to better understand abnormal interactions between the embryo and the milieu in artificial conditions. As trophectoderm cells are in direct contact with the environment, they deserve to be studied in more detail. The ultimate goal of these studies will be to render ART protocols safer. Optimization of the environment will be the key to improving the dialogue between the endometrium and embryo, so as to ensure that placentation after ART is similar to that following natural conception.

Like genetic mutations, DNA methylation anomalies or epimutations can disrupt gene expression and lead to human diseases. However, unlike genetic mutations, epimutations can in theory be reverted through developmental epigenetic reprograming, which should limit their transmission across generations. Following the request for a parental project of a patient diagnosed with Silver-Russell syndrome (SRS), and the availability of both somatic and spermatozoa DNA from the proband and his father, we had the exceptional opportunity to evaluate the question of inheritance of an epimutation. We provide here for the first time evidence for efficient reversion of a constitutive epimutation in the spermatozoa of an SRS patient, which has important implication for genetic counseling.

Cohen Syndrome (CS) is a rare autosomal recessive disorder, with defective glycosylation secondary to mutations in the VPS13B gene, which encodes a protein of the Golgi apparatus. Besides congenital neutropenia, retinopathy and intellectual deficiency, CS patients are faced with truncal obesity. Metabolism investigations showed abnormal glucose tolerance tests and low HDL values in some patients, and these could be risk factors for the development of diabetes mellitus and/or cardiovascular complications. To understand the mechanisms involved in CS fat storage, we used two models of adipogenesis differentiation: (i) SGBS pre-adipocytes with VPS13B invalidation thanks to siRNA delivery and (ii) CS primary fibroblasts. In both models, VPS13B invalidation led to accelerated differentiation into fat cells, which was confirmed by the earlier and increased expression of specific adipogenic genes, consequent to the increased response of cells to insulin stimulation. At the end of the differentiation protocol, these fat cells exhibited decreased AKT2 phosphorylation after insulin stimulation, which suggests insulin resistance. This study, in association with the in-depth analysis of the metabolic status of the patients, thus allowed us to recommend appropriate nutritional education to prevent the occurrence of diabetes mellitus and to put forward recommendations for the follow-up of CS patients, in particular with regard to the development of metabolic syndrome. We also suggest replacing the term obesity by abnormal fat distribution in CS, which should reduce the number of inappropriate diagnoses in patients who are referred only on the basis of intellectual deficiency associated with obesity.

The hematopoietic system declines with age. Myeloid-biased differentiation and increased incidence of myeloid malignancies feature aging of hematopoietic stem cells (HSCs), but the mechanisms involved remain uncertain. Here, we report that 4-mo-old mice deleted for transcription intermediary factor 1γ (Tif1γ) in HSCs developed an accelerated aging phenotype. To reinforce this result, we also show that Tif1γ is down-regulated in HSCs during aging in 20-mo-old wild-type mice. We established that Tif1γ controls TGF-β1 receptor (Tgfbr1) turnover. Compared with young HSCs, Tif1γ(-/-) and old HSCs are more sensitive to TGF-β signaling. Importantly, we identified two populations of HSCs specifically discriminated by Tgfbr1 expression level and provided evidence of the capture of myeloid-biased (Tgfbr1(hi)) and myeloid-lymphoid-balanced (Tgfbr1(lo)) HSCs. In conclusion, our data provide a new paradigm for Tif1γ in regulating the balance between lymphoid- and myeloid-derived HSCs through TGF-β signaling, leading to HSC aging.

International audience; Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS–PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal–lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal–lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

International audience; Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85 alpha, p55 alpha, and p50 alpha regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.

International audience; Shprintzen-Goldberg syndrome (SGS) is characterized by severe marfanoid habitus, intellectual disability, camptodactyly, typical facial dysmorphism, and craniosynostosis. Using family-based exome sequencing, we identified a dominantly inherited heterozygous in-frame deletion in exon 1 of SKI. Direct sequencing of SKI further identified one overlapping heterozygous in-frame deletion and ten heterozygous missense mutations affecting recurrent residues in 18 of the 19 individuals screened for SGS; these individuals included one family affected by somatic mosaicism. All mutations were located in a restricted area of exon 1, within the R-SMAD binding domain of SKI. No mutation was found in a cohort of 11 individuals with other marfanoid-craniosynostosis phenotypes. The interaction between SKI and Smad2/3 and Smad 4 regulates TGF-β signaling, and the pattern of anomalies in Ski-deficient mice corresponds to the clinical manifestations of SGS. These findings define SGS as a member of the family of diseases associated with the TGF-β-signaling pathway.

Speech sound disorders are heterogeneous conditions, and sporadic and familial cases have been described. However, monogenic inheritance explains only a small proportion of such disorders, in particular in cases with childhood apraxia of speech (CAS). Deletions of

Background Non-progressive congenital ataxias (NPCA) with or without intellectual disability (ID) are clinically and genetically heterogeneous conditions. As a consequence, the identification of the genes responsible for these phenotypes remained limited. Objective Identification of a new gene responsible for NPCA and ID. Methods Following the discovery of three familial or sporadic cases with an intragenic calmodulin-binding transcription activator 1 ( CAMTA1 ) rearrangement identified by an array-CGH and recruited from a national collaboration, the authors defined the clinical and molecular characteristics of such rearrangements, and searched for patients with point mutations by direct sequencing. Results Intragenic copy number variations of CAMTA1 were all located in the CG-1 domain of the gene. It segregated with autosomal dominant ID with non-progressive congenital cerebellar ataxia (NPCA) in two unrelated families, and was de novo deletion located in the same domain in a child presenting with NPCA. In the patients with ID, the deletion led to a frameshift, producing a truncated protein, while this was not the case for the patient with isolated childhood ataxia. Brain MRI of the patients revealed a pattern of progressive atrophy of cerebellum medium lobes and superior vermis, parietal lobes and hippocampi. DNA sequencing of the CG-1 domain in 197 patients with sporadic or familial non-syndromic intellectual deficiency, extended to full DNA sequencing in 50 patients with ID and 47 additional patients with childhood ataxia, identified no pathogenic mutation. Conclusion The authors have evidence that loss-of-function of CAMTA1 , a brain-specific calcium responsive transcription factor, is responsible for NPCA with or without ID. Accession numbers CAMTA1 reference sequence used was ENST00000303635. Protein sequence was ENSP00000306522.

BACKGROUND: The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation. METHODOLOGY/PRINCIPAL FINDINGS: We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice. CONCLUSIONS/SIGNIFICANCE: These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.

Successful tissue engineering with the aid of a polymer scaffold offers the possibility to produce a larger construct and to mould the shape after the defect. We investigated the use of cryogelation to form protein-based scaffolds through different types of formation mechanisms; enzymatic crosslinking, chemical crosslinking, and non-covalent interactions. Casein was found to best suited for enzymatic crosslinking, gelatin for chemical crosslinking, and ovalbumin for non-covalent interactions. Fibroblasts and myoblasts were used to evaluate the cryogels for tissue engineering purposes. The stability of the cryogels over time in culture differed depending on formation mechanism. Casein cryogels showed best potential to be used in skeletal tissue engineering, whereas gelatin cryogels would be more suitable for compliable soft tissues even though it also seemed to support a myogenic phenotype. Ovalbumin cryogels would be better suited for elastic tissues with faster regeneration properties due to its faster degradation time. Overall, the cryogelation technique offers a fast, cheap and reproducible way of creating porous scaffolds from proteins without the use of toxic compounds.

The extracellular matrix (ECM) provides a solid scaffold and signals to cells through ECM receptors. The cell-matrix interactions are crucial for normal biological processes and when disrupted they may lead to pathological processes. In particular, the biological importance of ECM-cell membrane-cytoskeleton interactions in skeletal muscle is accentuated by the number of inherited muscle diseases caused by mutations in proteins conferring these interactions. In this review we introduce laminins, collagens, dystroglycan, integrins, dystrophin and sarcoglycans. Mutations in corresponding genes cause various forms of muscular dystrophy. The muscle disorders are presented as well as advances toward the development of treatment.

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.