Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings.

International audienceHoloprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis

Macroglossia: A potentially severe complication of late-onset Pompe disease

International audienceBackground: Pompe disease is a rare neuromuscular disorder caused by a deficiency of a lysosomal enzyme, acid α-glucosidase. Macroglossia is a classic clinical sign of several inherited myopathies and has also been reported to occur progressively in late-onset Pompe disease (LOPD). Methods: We describe patients with LOPD and macroglossia included in the French national Pompe disease registry. Clinical, functional, and radiological data were collected during periodic follow-up and analyzed retrospectively. These cases were compared with 15 previously reported cases. Results: Five patients, three females and two males, aged 71–88 years, were included in this study. All but one of the patients suffered from symptoms related to macroglossia before the diagnosis of Pompe disease. Three had localized tongue atrophy and one had significant localized tongue hypertrophy which led to glossectomy 10 years before diagnosis. Two patients had severe dysphagia, one of whom underwent gastrostomy for enteral nutritional support. One patient experienced the persistence of numerous sleep apneas despite nocturnal bilevel positive airway pressure (BiPAP) ventilation. All our patients had dysarthria, and two required speech therapy. Four patients had a tongue hypersignal on magnetic resonance imaging (MRI) T1 sequences. Conclusions: Detection of macroglossia should be part of the clinical diagnosis and follow-up of patients with LOPD, with a careful evaluation of its main consequences. Macroglossia can have severe functional impacts on speech, swallowing, and sleep. Whole-body MRI with facial sections may facilitate the early diagnosis of Pompe disease with the “bright tongue sign”

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

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

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

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

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

International audienceHoloprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high heterogeneity, both in clinics and genetics. We have developed a novel targeted next-generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling

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

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