Refining the phenotype associated with biallelic DNAJC21 mutations.

Inherited bone marrow failure syndromes (IBMFS) are caused by mutations in genes involved in genomic stability. Although they may be recognized by the association of typical clinical features, variable penetrance and expressivity are common, and clinical diagnosis is often challenging. DNAJC21, which is involved in ribosome biogenesis, was recently linked to bone marrow failure. However, the specific phenotype and natural history remain to be defined. We correlate molecular data, phenotype, and clinical history of 5 unreported affected children and all individuals reported in the literature. All patients present features consistent with IBMFS: bone marrow failure, growth retardation, failure to thrive, developmental delay, recurrent infections, and skin, teeth or hair abnormalities. Additional features present in some individuals include retinal abnormalities, pancreatic insufficiency, liver cirrhosis, skeletal abnormalities, congenital hip dysplasia, joint hypermobility, and cryptorchidism. We suggest that DNAJC21-related diseases constitute a distinct IBMFS, with features overlapping Shwachman-Diamond syndrome and Dyskeratosis congenita, and additional characteristics that are specific to DNAJC21 mutations. The full phenotypic spectrum, natural history, and optimal management will require more reports. Considering the aplastic anemia, the possible increased risk for leukemia, and the multisystemic features, we provide a checklist for clinical evaluation at diagnosis and regular follow-up.

Loss of the proprioception and touch sensation channel PIEZO2 in siblings with a progressive form of contractures.

Dominant mutations in PIEZO2, which codes for the principal mechanotransduction channel for proprioception and touch sensation, have been found to cause different forms of distal arthrogryposis. Some observations suggest that these dominant mutations induce a gain-of-function effect on the channel. Here, we report a consanguineous family with three siblings who showed short stature, scoliosis, gross motor impairment, and a progressive form of contractures involving the distal joints that is distinct from that found in patients with dominant mutations in PIEZO2. These siblings also displayed deficits in proprioception and touch sensation. Whole-exome sequencing performed in the three affected siblings revealed the presence of a rare homozygous variant (c.2708C>G; p.S903*) in PIEZO2. This variant is predicted to disrupt PIEZO2 function by abolishing the pore domain. Sanger sequencing confirmed that all three siblings are homozygous whereas their parents and an unaffected sibling are heterozygous for this variant. Recessive mutations in PIEZO2 thus appear to cause a progressive phenotype that overlaps with, while being mostly distinct from that associated with dominant mutations in the same gene.

Tracheotomy in children: A series of 57 consecutive cases.

OBJECTIVES: With medical improvements in pediatrics, the role of tracheotomy has evolved. The aim of this study was to specify the indications for and complications of tracheotomy performed on children in a teaching hospital containing a level-3 maternity department and pediatric intensive care unit. MATERIAL AND METHODS: A retrospective study was conducted in pediatric tracheotomies performed from 2004 to 2014. Indications, early and late complications and the number and timing of decannulations were collated. RESULTS: Fifty-seven patients were included. Tracheotomy was motivated by upper airway obstruction in 39 children (68%) (median age, 4.9 months) or the need for prolonged ventilation in 18 children (32%) (median age, 6 months). There were 4 early complications (7%) (2 decannulations, including 1 fatal; an obstructive plug, responsible for another death; and 1 pneumothorax during an EXIT procedure), and 15 secondary complications requiring further surgery (26%). Twenty-seven patients (47%) were decannulated, with a mean tracheotomy duration of 26 months. In 9 cases (33% of decannulations), persistence of tracheocutaneous fistula required surgical repair. CONCLUSION: Tracheotomy for infection is almost a thing of the past; tracheotomy for airway obstruction is also likely to decrease, thanks to medical treatment (for hemangioma) and surgical techniques (for congenital stenosis). Tracheotomy for prolonged ventilation, on the other hand, remains. Complications of tracheotomy in children are rare but potentially serious, requiring care in a specialized center within a multidisciplinary team with defined care protocols.

A homozygous mutation in SLC1A4 in siblings with severe intellectual disability and microcephaly.

We performed exome analysis in two affected siblings with severe intellectual disability (ID), microcephaly and spasticity from an Ashkenazi Jewish consanguineous family. We identified only one rare variant, a missense in SLC1A4 (c. 766G>A [p. E256K]), that is homozygous in both siblings but not in any of their 11 unaffected siblings or their parents (Logarithm of odds, LOD score: 2.6). This variant is predicted damaging. We genotyped 450 controls of Ashkenazi Jewish ancestry and identified only 5 individuals who are heterozygous for this variant (minor allele frequency: 0.0056). SLC1A4 (ASCT1) encodes a transporter for neutral aminoacids such as alanine, serine, cysteine and threonine. L-Serine is essential for neuronal survival and differentiation. Indeed, L-serine biosynthesis disorders affect brain development and cause severe ID. In the brain, L-serine is synthesized in astrocytes but not in neurons. It has been proposed that ASCT1 mediates the uptake of L-serine into neurons and the release of glia-borne L-serine to neighboring cells. SLC1A4 disruption may thus impair brain development and function by decreasing the levels of L-serine in neurons. The identification of additional families with mutations in SLC1A4 would be necessary to confirm its involvement in ID.

The cleavage of HuR interferes with its transportin-2-mediated nuclear import and promotes muscle fiber formation.

Although the function of posttranscriptional processes in regulating the expression of genes involved in muscle fiber formation (myogenesis) is well accepted, the mechanisms by which these effects are mediated remain elusive. Here, we uncover such a mechanism and show that during myogenesis, a fraction of the posttranscriptional regulator human antigen R (HuR) is cleaved in a caspase-dependent manner in both cell culture and animal models. Disruption of caspase activity in cultured myoblasts or knocking out the caspase-3 gene in mice significantly reduced HuR cleavage and the cytoplasmic accumulation of HuR in muscle fibers. The non-cleavable isoform of HuR, HuRD226A, failed to reestablish the myogenic potential of HuR-depleted myoblasts. HuR cleavage generates two fragments: HuR-cleavage product 1 (HuR-CP1) (24 kDa) and HuR-CP2 (8 kDa). Here, we show that one of these fragments (HuR-CP1) binds to the HuR import factor transportin-2 (TRN2) allowing HuR to accumulate in the cytoplasm. As this cytoplasmic accumulation is required for the promyogenic function of HuR, our data support a model, whereby during the transition phase from myoblasts to myotubes, a proportion of HuR is cleaved to generate HuR-CP1. By interfering with the TRN2-mediated import of HuR, this CP helps non-cleaved HuR accumulate in the cytoplasm thus promoting myogenesis.

SIRT1 negatively regulates HDAC1-dependent transcriptional repression by the RBP1 family of proteins.

Both RBP1 and the highly related protein BCAA play a role in the induction of growth arrest and cellular senescence via mechanisms involving transcriptional repression. While investigating the transcriptional repression activities of RBP1, we observed a genetic link between RBP1 and SIR2. Further work uncovered an interaction between RBP1 family proteins and the mammalian homologue of SIR2, SIRT1. Interestingly, the HDAC-dependent transcriptional repression domain of RBP1 proteins, termed R2, is necessary and sufficient for the interaction with SIRT1. In vitro and in vivo binding studies indicated that the p33(ING1b) and p33(ING2) subunits of the mSIN3A/HDAC1 complex are responsible for the recruitment of SIRT1 to the R2 domain. To investigate the biological relevance of this interaction, we used the sirtuin activator resveratrol and the sirtuin inhibitor sirtinol in transcriptional repression assays and demonstrated that SIRT1 activity negatively regulates R2-mediated transcriptional repression activity. We therefore propose a novel mechanism of class I HDAC regulation by a class III HDAC. Explicitly, SIRT1 is recruited by ING proteins and inhibits R2-associated mSIN3A/HDAC1 transcriptional repression activity.

Embryonic development of the Drosophila brain. I. Pattern of pioneer tracts.

The neuropile of the late embryonic Drosophila brain can be subdivided into a vertical component (cervical connective), a transverse component (supraesophageal commissure), and a horizontal component for which we propose the term protocerebral connective. The core of each neuropile component is formed by numerous axon fascicles, the trajectory of which follows an invariant pattern. In the present study we have used an antibody against the adhesion molecule Fasciclin II (FasII) that is expressed in a large number of early differentiating neurons of the Drosophila embryo to follow the development of the axon tracts of the brain. The FasII antigen appears on the surface of clusters of neuronal somata prior to axon outgrowth. These clusters, for which we propose the term fibre tract founder clusters, are laid out in a linear pattern that forms an almost uninterrupted longitudinal track reaching from the ventral nerve cord to the "tip" of the brain. After expressing FasII on their soma, neurons of the fibre tract founder clusters extend axons that grow along the surface of the founder clusters and form a simple system of pioneer tracts for each of the components of the brain neuropile. We have reconstructed the FasII-positive fibre tract founder clusters and their axons from optical sections and generated digital 3-D models that illustrate the spatial relationships of the pioneer tracts. Three fibre tract founder clusters, D/T, P1, and P3m, pioneer the cervical connective. P21 and P2m form a transverse track that pioneers the supraesophageal commissure. P4m and P41/P51/VP5m form two tracts that pioneer a medial and a lateral component of the protocerebral connective, respectively. Because FasII expression continues uninterruptedly into the larval period when the "rudiments" of many parts of the adult neuropile are readily identifiable, it was possible to assign several of the embryonic pioneer tracts to definitive neuropile components, including the median bundle, antennocerebral tract, mushroom body, and posterior optic tract.

Embryonic development of the Drosophila brain. II. Pattern of glial cells.

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion.

EGFR signaling is required for the differentiation and maintenance of neural progenitors along the dorsal midline of the Drosophila embryonic head.

EGFR signaling has been shown in recent years to be involved in the determination, differentiation and maintenance of neural and epidermal cells of the ventral midline (mesectoderm and ventromedial ectoderm). Localized activation of the TGFalpha homolog Spitz (Spi) in the mesectoderm is achieved by the products of the genes rhomboid and Star. Spi binds to its receptor, the Drosophila epidermal growth factor receptor homolog (Egfr), and triggers the Ras pathway which is needed for the survival and differentiation of ventral midline cells. The results reported here indicate that EGFR signaling is also required in a narrow medial domain of the head ectoderm (called 'head midline' in the following) that includes the anlagen of the medial brain, the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). We document that genes involved in EGFR signaling are expressed in the head midline. Loss of EGFR signaling results in an almost total absence of optic lobe and larval eye, as well as severe reduction of SNS and medial brain. The cellular mechanism by which this phenotype arises is a failure of neurectodermal cells to differentiate combined with apoptotic cell death. Overactivity of EGFR signaling, as achieved by heat-shock-driven activation of a wild-type rhomboid (rho) construct, or by loss of function of argos (aos) or yan, results in an hyperplasia and deformity of the head midline structures. We show that, beside their requirement for EGFR signaling, head and ventral midline structures share several morphogenetic and molecular properties.

The role of morphogenetic cell death during Drosophila embryonic head development.

This article addresses the role of programmed cell death (apoptosis) during embryonic head development of Drosophila. Previous studies showed that reaper (rpr) is expressed in and required by cells undergoing apoptosis. We have analyzed the correlation between the pattern of expression of rpr and morphogenetic movements affecting head development. Furthermore, we have investigated the defects in head development resulting from the absence of apoptosis in embryos deficient for rpr. Our results show that, in the head, domains of high incidence of cell death as marked by expression of rpr correlate with regions where most morphogenetic movements occur; these regions are involved in formation of mouth structures, the internalization of neural progenitors, and head involution. Cellular events driving these movements are delamination, invagination, and intercalation as well as disruption and reformation of contacts among epithelial cells. The analysis of rpr-deficient embryos demonstrates that, despite of the widespread occurrence of apoptosis during normal head morphogenesis, many aspects of this process proceed in an apparently unperturbed manner even when cell death is blocked. In particular, movements that happen early during embryonic development and that are evolutionarily more ancient (e.g., formation of the dorsal ridge and the pharynx) take place almost normally in rpr-deficient embryos. Later events which are mostly associated with head involution (e.g., retraction of the clypeolabrum, formation of the dorsal pouch, fusion of lateral gnathal lobes) are evolutionarily more recent and fail to occur normally in rpr-deficient embryos.

Early neurogenesis of the Drosophila brain.

We have studied the formation of the neuroblasts of the Drosophila brain which segregate from the procephalic neurectoderm. The expression domains of the segment polarity gene engrailed (en) allow one to subdivide the procephalic neuroectoderm into tritocerebral, deuterocerebral, and protocerebral neuromeres. Based upon the expression pattern of the proneural gene lethal of scute (l'sc), as well as the pattern of brain neuroblast segregation, the protocerebral and deuterocerebral neuromeres can be further subdivided into a central, anterior, and posterior domain. A total of 75-80 neuroblasts segregate in a stereotyped pattern from the procephalic neurectoderm of each side during stages 9-11. With respect to their position and the expression of the markers asense (ase) and seven-up (svp), 23 small groups of one to five neuroblasts each were identified. The first eight groups (Pc1-4, Dc1-3, Dp1), collectively called SI/II neuroblasts in analogy to the subpopulation of ventral neuroblasts which appear at the same stage), arise from the central domain of the protocerebral and deuterocerebral neurectoderm, respectively. Later groups form anteriorly and posteriorly from the earlier ones, leading to a centrifugal growth of the procephalic neuroblast population. SIII neuroblasts (Pa1-4, Pp1-2, Dp2) arise during stage 10, SIV neuroblasts (Pa5-6, Pp3-4, Da1, T1-2) during early stage 11, and SV neuroblasts (Pp5, Pdm) during late stage 11 and early stage 12. The dorsomedial domain of the procephalic neurectoderm represents a special case. Unlike other procephalic neuroblasts which delaminate from the surface ectoderm as individual cells, cells of the dorsomedial protocerebral domain are internalized during stage 12 as large, coherent clusters by a movement which can be best characterized as a combination of mass-delamination and invagination.