Defective prolactin signaling impairs pancreatic ß-cell development during the perinatal period.

Prolactin (PRL) and placental lactogens stimulate ß-cell replication and insulin production in pancreatic islets and insulinoma cells through binding to the PRL receptor (PRLR). However, the contribution of PRLR signaling to ß-cell ontogeny and function in perinatal life and the effects of the lactogens on adaptive islet growth are poorly understood. We provide evidence that expansion of ß-cell mass during both embryogenesis and the postnatal period is impaired in the PRLR(-/-) mouse model. PRLR(-/-) newborns display a 30% reduction of ß-cell mass, consistent with reduced proliferation index at E18.5. PRL stimulates leucine incorporation and S6 kinase phosphorylation in INS-1 cells, supporting a role for ß-cell mTOR signaling in PRL action. Interestingly, a defect in the development of acini is also observed in absence of PRLR signaling, with a sharp decline in cellular size in both endocrine and exocrine compartments. Of note, a decrease in levels of IGF-II, a PRL target, in the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes, is associated with a lack of PRL-mediated ß-cell proliferation in embryonic pancreatic buds. Reduced pancreatic IGF-II expression in both rat and mouse models suggests that this factor may constitute a molecular link between PRL signaling and cell ontogenesis. Together, these results provide evidence that PRL signaling is essential for pancreas ontogenesis during the critical perinatal window responsible for establishing functional ß-cell reserve.

Genetic Profile of Patients with Limb-Girdle Muscle Weakness in the Chilean Population.

Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy.

In Vitro studies of non poly alanine PHOX2B mutations argue against a loss-of-function mechanism for congenital central hypoventilation.

A wide range of autonomic dysfunctions, i.e. Central Hypoventilation Syndromes, Hirschsprung disease and Tumours of the Sympathetic Nervous System have been ascribed to heterozygous PHOX2B mutations in man. The PHOX2B mutations reported include polyalanine expansions in a 20 alanines tract, missense, frameshift mutations and nonsense mutation. Some genotype/phenotype correlations have been drawn, but the molecular mechanism(s) underlying them remain(s) unclear. So far, loss-of-function, gain-of-function and dominant negative effects have been proposed as disease-causing mechanisms for polyalanine expansions. Indeed, mutant with an expanded polyalanine tract result in decreased transactivation of known target genes and protein misfolding leading to oligomerisation in vitro for all expansions and to cytoplasmic protein aggregation for longer expansions. We extended the molecular studies to other non-polyalanine expansion mutations and show that most PHOX2B protein mutants oligomerize even in the absence of the normal 20 alanines tract. Conversely, a premature stop codon mutation in a CHS patient leads to the production of an N-terminally truncated protein by re-initiation of translation that does not form oligomers. Therefore, PHOX2B misfolding is not the only mechanism leading to dysfunction of the ventilatory autonomic system.

Mutational, functional, and expression studies of the TCF4 gene in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is a severe congenital encephalopathy recently ascribed to de novo heterozygous TCF4 gene mutations. We report a series of 13 novel PHS cases with a TCF4 mutation and show that EEG, brain magnetic resonance imagain (MRI), and immunological investigations provide valuable additional clues to the diagnosis. We confirm a mutational hot spot in the basic domain of the E-protein. Functional studies illustrate that heterodimerisation of mutant TCF4 proteins with a tissue-specific transcription factor is less effective than that homodimerisation in a luciferase reporter assay. We also show that the TCF4 expression pattern in human embryonic development is widespread but not ubiquitous. In summary, we further delineate an underdiagnosed mental retardation syndrome, highlighting TCF4 function during development and facilitating diagnosis within the first year of life.

Homozygous mutation of the PHOX2B gene in congenital central hypoventilation syndrome (Ondine's Curse).

Homozygosity for a dominant allele is relatively rare and preferentially observed in communities with high inbreeding. According to the definition of true dominance, similar phenotypes should be observed in patients heterozygous and homozygous for a dominant mutation. However, the homozygous phenotype usually tends to be more severe than the heterozygous one. In these cases, the wild-type and mutant alleles are semi-dominant. Here we report a patient with a Congenital Central Hypoventilation Syndrome (CCHS) phenotype and homozygosity for a PHOX2B gene mutation leading to an alanine expansion shorter than the threshold hitherto observed in CCHS patients with a heterozygous mutation. This observation adds the concept of mutational threshold per se to the discussion about dominant and recessive alleles.

Comparing targeted exome and whole exome approaches for genetic diagnosis of neuromuscular disorders.

Massively parallel sequencing is rapidly becoming a widely used method in genetic diagnostics. However, there is still no clear consensus as to which approach can most efficiently identify the pathogenic mutations carried by a given patient, while avoiding false negative and false positive results. We developed a targeted exome approach (MyoPanel2) in order to optimize genetic diagnosis of neuromuscular disorders. Using this approach, we were able to analyse 306 genes known to be mutated in myopathies as well as in related disorders, obtaining 98.8% target sequence coverage at 20 ×. Moreover, MyoPanel2 was able to detect 99.7% of 11,467 known mutations responsible for neuromuscular disorders. We have then used several quality control parameters to compare performance of the targeted exome approach with that of whole exome sequencing. The results of this pilot study of 140 DNA samples suggest that targeted exome sequencing approach is an efficient genetic diagnostic test for most neuromuscular diseases.

Exon 32 Skipping of Dysferlin Rescues Membrane Repair in Patients' Cells.

Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues.We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use of in vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients.

Contributions of PHOX2B in the pathogenesis of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital malformation of the hindgut resulting from a disruption of neural crest cell migration during embryonic development. It has a complex genetic aetiology with several genes involved in its pathogenesis. PHOX2B plays a key function in the development of neural crest derivatives, and heterozygous mutations cause a complex dysautonomia associating HSCR, Congenital Central Hypoventilation Syndrome (CCHS) and neuroblastoma (NB) in various combinations. In order to determine the role of PHOX2B in isolated HSCR, we performed a mutational screening in a cohort of 207 Spanish HSCR patients. Our most relevant finding has been the identification of a de novo and novel deletion (c.393_410del18) in a patient with HSCR. Results of in silico and functional assays support its pathogenic effect related to HSCR. Therefore our results support that PHOX2B loss-of-function is a rare cause of HSCR phenotype.

R-type anion channel activation is an essential step for ROS-dependent innate immune response in Arabidopsis suspension cells.

Plants are constantly exposed to environmental biotic and abiotic stresses. Plants cells perceive these factors and trigger early responses followed by delayed and complex adaptation processes. Using cell suspensions of Arabidopsis thaliana (L.) as a cellular model, we investigated the role of plasma membrane anion channels in Reactive Oxygen Species (ROS) production and in cell death which occurs during non-host pathogen infection. Protoplasts derived from Arabidopsis suspension cells display two anion currents with characteristics very similar to those of the slow nitrate-permeable (S-type) and rapid sulfate-permeable (R-type) channels previously characterised in hypocotyl cells and other cell types. Using seven inhibitors, we showed that the R-type channel and ROS formation in cell cultures present similar pharmacological profiles. The efficiency of anion channel blockers to inhibit ROS production was independent of the nature of the triggering signal (osmotic stress or general elicitors of plant defence), indicating that the R-type channel represents a crossroad in the signalling pathways leading to ROS production. In a second step, we show that treatment with R-type channel blockers accelerates cell death triggered by the non-specific plant pathogen Xanthomonas campestris. Finally, we discuss the hypothesis that the R-type channel is involved in innate immune response allowing cell defence via antibacterial ROS production.

Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice.

T cell development and selection require the fully mature and diverse epithelial microenvironment of the thymus. Acquisition of these characteristics is dependent on expression of the forkhead (also known as winged-helix) transcription factor FoxN1, as a lack of functional FoxN1 results in aberrant epithelial morphogenesis and an inability to attract lymphoid precursors to the thymus primordium. However, the transcriptional control of Foxn1 expression has not been elucidated. Here we report that secreted Wnt glycoproteins, expressed by thymic epithelial cells and thymocytes, regulate epithelial Foxn1 expression in both autocrine and paracrine fashions. Wnt molecules therefore provide regulatory signals critical for thymic function.