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.

Limb girdle muscular dystrophy 2A (MIM#253600) is an autosomal recessive disorder caused by mutations of the CAPN3 gene, which encodes for calpain-3. We describe a series of five patients from four unrelated Chilean families harbouring CAPN3 mutations. Patients 1 to 3 were female. Current age ranged from 18 to 36 years, but symptomatic onset, mainly as lower limb weakness associated with tiptoe walking, was during the first decade in four cases. Patient 3 started at the age of 25 with a proximal-distal lower limb weakness. Patient 4 aged 21, had a history of delayed motor milestones. All presented some degree of scapular winging and Achilles tendon retraction, but hyperlordosis was patent only in females. Patients 4 and 5 showed marked generalized amyotrophy predominantly involving the thighs and the anterior arm compartment. Calf hypertrophy was present only in Patient 1 and Patient 5. Patient 5 also presented some degree of rigid spine as well as elbow and ischiotibial retractions. CK levels were increased in all cases but Patient 2 (range: 5- to 65-fold increase). Muscle biopsies showed a dystrophic pattern, with eosinophilic infiltrates in Patient 5. Neither cranial muscle involvement nor cardiac or respiratory compromise was observed in none of the patients. Whole body MRI performed on Patients 1 to 4 showed variable degrees of fatty replacement, following the pattern described for LGMD2A. Genetic screening was performed on a NGS panel of 306 genes involved in neuromuscular diseases, using HaloPlex (Agilent Technologies™) enrichment method and sequenced on the NextSeq500 (IlluminaTM) by HelixioTM (Biopôle Clermont-Limagne, France). The variant p. Arg788Serfs*14 of the CAPN3 gene (NM_000070.2) was identified for Patients 2 and 3. The novel mutation p.Gly36Valfs*21 was found in homozygous state for Patient 4 and in compound heterozygous state, associated with variants p.Arg748Gln and p.Arg788Serfs*14 for Patient 1 and 5 respectively.

Distal myopathies are a heterogeneous group of muscle diseases sharing the clinical pattern of predominant weakness in the feet and/or hands. The classical approach for molecular diagnosis is based on targeted gene-by-gene analysis guided by currently existing combinatorial algorithms.1 Many patients remain undiagnosed. Within the last 5 years, next-generation sequencing (NGS) has emerged as a successful and rapid approach to simultaneously analyse multiple genes in neuromuscular disorders.2 Our objective was to evaluate the efficiency of a targeted NGS approach using a panel of neuromuscular genes on patients with distal myopathies. We first tested its validity in a control group of six index cases (IC) with known molecular diagnosis. Then, we prospectively evaluated this approach by testing a group of 17 IC without molecular diagnosis. ### Patients We prospectively included 54 patients (37 IC and 17 relatives) with a diagnosis of distal myopathy, followed at the Neuromuscular Diseases and ALS Reference Centre of La Timone Hospital, Marseille, France, between 1989 and 2014. Among these 37 IC previously explored by Sanger sequencing, 20 IC had an identified molecular diagnosis: six IC constituted the control group. The remaining 17 undiagnosed IC constituted the test group. A targeted-NGS approach was used to search mutations in 298 neuromuscular genes in both groups. Samples analysed in this study have been prepared and stored by the Center of Biological Resources, Department of Medical Genetics, La Timone Hospital, Marseille, and used following the ethical recommendations of our institution and according to the Declaration of Helsinki. All included patients gave their written consent prior to …

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.

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.

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.

Prolactin (PRL) and placental lactogens stimulate β-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 β-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 β-cell mass during both embryogenesis and the postnatal period is impaired in the PRLR−/−mouse model. PRLR−/−newborns display a 30% reduction of β-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 β-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 β-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 β-cell reserve.

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.

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. Hum Mutat 0, 1–8, 2009. © 2009 Wiley‐Liss, Inc.

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.

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.

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.

The motion of both mitral cusps and the presence of valvular regurgitation during ventricular contractions were investigated in seven experiments on dogs in which radiopaque markers had been sutured to the cusps and the valve annulus 1-32 wk before the studies. Cineangiograms of the left ventricle were obtained during ventricular ectopic beats, interposed throughout the cardiac cycle (20-99% of cycle length) and during induced variations in the P-R interval (0-200 ms). Mitral regurgitation was observed only during a) weak, early ectopic beats (peak pressure below 34 mmHg) which were incapable of closing the cusps and b) when ventricular contractions suddenly interrupted normal leaflet motion toward the ventricle, during three well-defined periods of diastole (diastolic valve opening, diastolic rebound, and atrial opening). Valve closure following sudden reversal of cusp opening was slow and the leaflets often did not arrive simultaneously at their closed positions. These findings suggest that sudden interruption of leaflet opening by ventricular contractions is an important mechanism of transient mitral regurgitation in the normal heart.

Motion and position of both mitral leaflets were studied in five normal dogs 1-11 wk after radiopaque markers were sutured on the valve cusps and on the mitral annulus. Cinefluorograms and cineangiograms (100-120 frames/s) of left atrium and left ventricle were used to study cusp motion and intraventricular flow patterns, and to detect mitral regurgitation during sinus rhythm (42-184 beats/min) and during isolated atrial or ventricular contractions. Time-motion of both leaflets was similar throughout diastole with the exception of delayed posterior cusp opening. Peak opening and closing speeds, opening and closing times, and time of complete closure, measured from the Q wave of the ECG, were not significantly affected by the variations in heart rate. Diastolic leaflet closure began immediately after opening, while the ventricular cavity was small, and was caused by flow eddies behind the cusps. Isolated ventricular contractions closed the valve leaflets completely and symmetric valve closure was ensured by the different rates of leaflet edge approximation. In contrast, atrial closure was slow, partial, and of very short duration.