Fetal phenotypes in otopalatodigital spectrum disorders.

Otopalatodigital spectrum disorders (OPDSD) include OPD syndromes types 1 and type 2 (OPD1, OPD2), Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). These conditions are clinically characterized by variable skeletal dysplasia associated in males, with extra-skeletal features including brain malformations, cleft palate, cardiac anomalies, omphalocele and obstructive uropathy. Mutations in the FLNA gene have been reported in most FMD and OPD2 cases and in all instances of typical OPD1 and MNS. Here, we report a series of 10 fetuses and a neonatally deceased newborn displaying a multiple congenital anomalies syndrome suggestive of OPDSD and in whom we performed FLNA analysis. We found a global mutation rate of 44%. This series allows expanding the clinical and FLNA mutational spectrum in OPDSD. However, we emphasize difficulties to correctly discriminate OPDSD based on clinical criteria in fetuses due to the major overlap between these conditions. Molecular analyses may help pathologists to refine clinical diagnosis according to the type and the location of FLNA mutations. Discriminating the type of OPDSD is of importance in order to improve the genetic counseling to provide to families.

Autosomal dominant familial spastic paraplegia is genetically heterogeneous and one locus maps to chromosome 14q.

Autosomal dominant familial spastic paraplegia (FSP) is a degenerative disorder of unknown aetiology characterized by a progressive spasticity of the legs. Three families with autosomal dominant FSP of early onset were analysed in linkage studies using highly polymorphic microsatellite markers. Close linkage to a group of markers on chromosome 14q (maximum multipoint lodscore z = 10) was observed in one family. This chromosome 14q candidate region was entirely excluded in the two other families, providing evidence of genetic heterogeneity within a homogeneous clinical form of FSP.

Correlation between severity and SMN protein level in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord. Three different forms of childhood SMA have been recognized on the basis of age at onset and clinical course: Werdnig-Hoffmann disease (type-1), the intermediate form (type-II) and Kugelberg-Welander disease (type-III). A gene termed 'survival of motor neuron' (SMN) has been recognized as the disease-causing gene in SMA. SMN encodes a protein located within a novel nuclear structure and interacts with RNA-binding proteins. To elucidate the molecular mechanism underlying the pathogenesis of the disease, we examined the expression of the SMN gene in both controls and SMA patients by western blot and immunohistochemical analyses using antibodies raised against the SMN protein. The present study shows a marked deficiency of the SMN protein in SMA.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been recently reported as a cause of stroke. It is characterized, in the absence of hypertension, by recurrent subcortical ischaemic strokes, starting in early or midadulthood and leading in some patients to dementia. Magnetic resonance imaging and pathological examination show numerous small subcortical infarcts and a diffuse leukoencephalopathy underlaid by a non-arteriosclerotic, non-amyloid angiopathy. We performed genetic linkage analysis in two unrelated families and assigned the disease locus to chromosome 19q12. Multilocus analysis with the location scores method established the best estimate for the location of the affected gene within a 14 centimorgan interval bracketed by D19S221 and D19S222 loci.

Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes. FRDA is caused by severely reduced levels of frataxin, a mitochondrial protein of unknown function. Yeast knockout models as well as histological and biochemical data from heart biopsies or autopsies of FRDA patients have shown that frataxin defects cause a specific iron-sulfur protein deficiency and intramitochondrial iron accumulation. We have recently shown that complete absence of frataxin in the mouse leads to early embryonic lethality, demonstrating an important role for frataxin during mouse development. Through a conditional gene-targeting approach, we have generated in parallel a striated muscle frataxin-deficient line and a neuron/cardiac muscle frataxin-deficient line, which together reproduce important progressive pathophysiological and biochemical features of the human disease: cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I-III of the respiratory chain and of the aconitases. Our models demonstrate time-dependent intramitochondrial iron accumulation in a frataxin-deficient mammal, which occurs after onset of the pathology and after inactivation of the Fe-S-dependent enzymes. These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.

Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy.

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.

De novo and inherited deletions of the 5q13 region in spinal muscular atrophies.

Spinal muscular atrophies (SMAs) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood spinal muscular atrophies are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a yeast artificial chromosome contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in nine unrelated SMA patients. Moreover, deletions were strongly suggested in at least 18 percent of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of spinal muscular atrophy.

[Stretching and chylothorax]. / Stretching et chylothorax.

INTRODUCTION: Chylothorax is a rare cause of pleural effusion. The most common causes are iatrogenic or medical. We report an unusual and rare cause of bilateral chylothorax. CASE REPORT: A 73-year-old woman with no past history was admitted to the emergency department for sudden onset of dyspnoea. Chest X-ray and thoracic CT scan revealed large bilateral pleural effusions. Analysis of the fluid revealed a chylothorax. The patient was treated by chest tube drainage and a fat free (medium chain triglyceride) diet. This led to drying up of the effusions and rapid discharge. Complementary imaging examinations with chest-abdomen-pelvis CT, PET CT and pelvic MRI did not reveal any underlying cause. The final diagnosis was bilateral traumatic chylothorax caused by tearing of the thoracic duct during stretching exercises. CONCLUSION: Following a literature review, similar cases with the same clinical presentation were found. Combined treatment with thoracic drainage and medium chain triglyceride diet was effective in drying up the effusions. Our diagnosis was a diagnosis of exclusion. It is important to exclude a medical cause by thorough investigation.

Survival motor neuron gene deletion in the arthrogryposis multiplex congenita-spinal muscular atrophy association.

The survival motor neuron (SMN) gene was lacking in 6/12 patients with arthrogryposis multiplex congenita (AMC) associated with spinal muscular atrophy (SMA). Neither point mutation in the SMN gene nor evidence for linkage to chromosome 5q13 were found in the other patients. Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA. Our data strongly suggest that AMC of neurogenic origin is genetically heterogeneous, with a subgroup being allelic to SMA. Absence or interruption of the SMN gene in the AMC-SMA association will make the diagnosis easier and genetic counselling will now become feasible.

[Thymomectomy by minimally invasive surgery. Comparative study videosurgery versus robot-assisted surgery]. / Traitement des thymomes par chirurgie mini-invasive. Étude comparative vidéochirurgie versus voie robot-assistée.

OBJECTIVES: To report the results of minimally invasive surgery in patients with stage I or II thymoma in the Masaoka classification. The reference technique is partial or complete thymectomy by sternotonomy. METHODS: A retrospective single-center study of a prospective database including all cases of thymoma operated from April 2009 to February 2015 by minimally invasive techniques: either videosurgery (VATS) or robot-assisted surgery (RATS). The surgical technique, type of resection, length of hospital stay, postoperative complications and recurrences were analysed. RESULTS: Our series consisted of 22 patients (15 women and 7 men). The average age was 53 years. Myasthenia gravis was present in 12 patients. Eight patients were operated on by VATS and 14 patiens by RATS. There were no conversions to sternotomy and no perioperative deaths. The mean operating time was 92min for VATS and 137min for RATS (P<0.001). The average hospital stay was 5 days. The mean weight of the specimen for the VATS group was 13.2 and 45.7mg for the RATS group. Twelve patients were classified Masaoka stage I and 10 were stage II. According to the WHO classification there were 7 patients type A, 5 type AB, 4 type B1, 4 type B2 4 and 2 type B3. As proposed by the Group ITMIG-IASLC in 2015 all patients corresponded to group I. The mean follow-up period was 36 months. We noted 3 major perioperative complications according to the Clavien-Dindo classification: one pneumonia, one phrenic nerve paralysis and one recurrent laryngeal nerve palsy. We observed one case of local recurrence at 22 months. Following surgery 4 patients were treated with radiotherapy and 2 patients with chemotherapy. CONCLUSIONS: The minimally invasive route is safe, relatively atraumatic and may be incorporated in the therapeutic arsenal for the treatment of Masaoka stage I and II thymoma as an alternative to conventional sternotomy. RATS and VATS are two minimally invasive techniques and the results in the short and medium term are acceptable. The clinical advantages of one over the other are sifficult to establish. RATS could handle larger and more complex lesions in view of the weight and size of the operating instrument.

Gene targeting restricted to mouse striated muscle lineage.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/LoxP recombination system of bacteriophage P1. In order to develop gene knockouts restricted to striated muscle, we generated a transgenic mouse line expressing Cre recombinase under the control of the human alpha-skeletal actin promoter. Specific excision of a loxP-flanked gene was demonstrated in striated muscle, heart and skeletal muscle, in a pattern very similar to the expression of the endogenous alpha-skeletal actin gene. Therefore, the reported transgenic line can be used to target inactivation or activation of a given gene to the skeletal muscle lineage.

Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia.

Hypermethylation in cancer often occurs in CpG islands that span the promoter regions of tumor suppressor genes. However, it is not clear if hypermethylation is limited to single target genes or if multiple genes are simultaneously methylated. To understand the extent of aberrant de novo methylation, we have analyzed the methylation pattern of a number of tumor-related genes in leukemia from the same cohort of patients. We used bisulfite genomic sequencing to characterize the methylation pattern of the CpG islands associated with the calcitonin, estrogen receptor, E-cadherin, p15, p16, Rb, GST-Pi, and HIC1 genes in the bone marrow from 9 normal and 20 patients with acute myeloid leukaemia (AML). All of the normal control samples were essentially unmethylated for each of the eight tumor-related genes studied. In contrast, 19 of 20 (95%) of the AML patients had an abnormal methylation pattern in at least one gene, and 15 of 20 (75%) had abnormal methylation patterns in two or more of the target genes. We conclude that there is a general deregulation of CpG island methylation in leukemia and that hypermethylation is not limited to single genes, but a number of genes are methylated concurrently. Moreover, the subset of genes that are commonly methylated in leukemia appear to be cancer type specific.

Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells.

Inactivation of p16INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M0). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16INK4 expression in postselection HMECs. In contrast, p16INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16INK4 CpG island and loss of p16INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

[Implementation of a robotic video-assisted thoracic surgical program]. / Mise en place d'un programme de chirurgie thoracique mini-invasive robot-assistée.

AIMS: Recent publications from North America have shown the benefits of robot-assisted thoracic surgery. We report here the process of setting up such a program in a French university centre and early results in a unit with an average treatment volume. METHODS: Retrospective review of a single institution database. The program was launched after a 6-month preparation period. RESULTS: From January 2012 to January 2013, totally endoscopic, full robot-assisted procedures were performed on 30 patients (17 males). Median age was 54 [Q1-Q3, 48-63] years and ASA score 2 [1,2]. Operative procedures included thymectomy (9 ; 30%), lobectomy with nodes resection (11 ; 38%), segmentectomy (4 ; 14%), lymphadenectomy (3 ; 10%), Bronchogenic cyst (2, 5%) and posterior mediastinal mass resection (1 ; 3%). No conversion was required. Median blood loss was 50mL [10-100]. Median operating time was 135 min (105-165) including 30 min [20-40] for docking, 90min for robot-assisted operating [70-120] and 15 min [10-15] for lesion extraction. CO2 insufflation was used in 28 cases (93%). Hospital stay was 4 days [4-6] with 6 minor complications (20%) (Grade 1 according to the Clavien-Dindo classification). After a median 4 months follow-up [2-7], all patients were alive and demonstrated a good quality of life. CONCLUSION: This series suggests that full robotic thoracic procedures are safe and effective treatment for various pathologies, with low morbidity and without a significant learning curve, even in a lower volume centre. This technology should accompany the development of minimally invasive thoracic surgery. The importance of robotic training should be emphasized to optimize procedures and costs.

Mutation and methylation analysis of TP53 in adrenal carcinogenesis.

AIM: To investigate the role of coding region mutation and promoter hypermethylation of TP53 in adrenocortical cancer formation. METHODS: Twenty sporadic adrenocortical cancers (ACCs) and five normal adrenal tissue samples were available for analysis. Coding region mutation of TP53 in 20 ACCs was examined by polymerase chain amplification using intronic primers for exons 2-11 and direct sequencing of the product. In 10 ACCs and five normal adrenal tissue specimens, methylation of the 16 CpG sites within the TP53 promoter was examined using bisulphite methylation sequencing. RESULTS: Coding region mutation in TP53 was demonstrated in 5 of 20 ACCs. There were four mis-sense mutations and one frameshift mutation. Four of 5 patients with a TP53 mutation had metastases at diagnosis or detected soon thereafter and 3 of 4 died of disease within 12 months of surgical resection. No methylation was seen in the TP53 promoter in 10 ACC and the five normal adrenal tissues examined. CONCLUSION: Coding region mutation in TP53 occurs in 25% of ACCs with a trend toward a poorer prognosis. Promoter methylation of TP53 is not present in ACC as a mechanism for tumour suppressor gene (TSG) inactivation and, therefore, other genes in the 17p13 region are implicated in adrenal carcinogenesis.

Increased DNA methyltransferase expression in leukaemia.

Aberrant DNA methylation has been observed consistently in many human tumours, in particular in the CpG islands of tumour suppressor genes, but the underlying mechanism of these changes remains unclear. To determine whether DNA methyltransferase expression is increased in leukaemia, we developed a standardised competitive RT-PCR assay to measure the level of DNA methyltransferase transcripts. Using this assay on bone marrow RNA samples from 12 patients with acute leukaemia, we observed a 4.4-fold mean increase in the level of DNA methyltransferase mRNA compared with normal bone marrow. These results support but do not prove the hypothesis that an increase in DNA methyltransferase activity is associated with malignant haematological diseases and may constitute a key step in carcinogenesis.

Cancer-specific region of hypermethylation identified within the HIC1 putative tumour suppressor gene in acute myeloid leukaemia.

Abnormal DNA methylation has been found to be a common feature in cancer cells, although the mechanism of this alteration remains poorly understood. HIC1 is a putative tumour suppressor gene on chromosome 17p13.3 and is hypermethylated in a number of cancers including leukaemia. In this study, using bisulphite genomic sequencing, we have identified a 'boundary' sequence within the HIC1 CpG island that shows a marked junction between methylated and unmethylated DNA in normal haematopoietic cells. Surprisingly, this boundary of differential methylation lies exactly between the intron 2 and exon 3 junction. In contrast to normal haematopoietic cells, hypermethylation extends past this boundary at a high frequency (83%) in newly diagnosed acute myeloid leukaemias (AML). Identification of the hypermethylated boundary sequence not only provides the first step in understanding the mechanisms that normally protect CpG islands from de novo methylation but also may prove to be a useful cancer-specific marker.

Allelic heterogeneity of SMARD1 at the IGHMBP2 locus.

Spinal Muscular Atrophy with Respiratory Distress (SMARD) is an autosomal recessive disorder characterized by neurogenic muscular atrophy due to progressive anterior horn cell degeneration and early life-threatening respiratory failure ascribed to diaphragmatic dysfunction. SMARD is clinically and genetically heterogeneous. SMARD type 1 is characterized by onset of respiratory failure within the first weeks of life and has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We report here the identification of nine novel IGHMBP2 mutations in five SMARD1 patients, including seven missense [ c.587A>G (p.Gln196Arg), c.647C>T (p.Pro216Leu), c.752T>C (p.Leu251Pro), c.1693G>A (p.Asp565Asn), c.1730T>C (p.Leu577Pro), c.1807C>T (p.Arg603Cys), c.1909C>T (p.Arg637Cys)] and two nonsense mutations [ c.1488C>A (p.Cys496X), c.2368C>T (p.Arg790X)]. Interestingly, 7 of 9 mutations occurred at highly conserved residues of the putative DNA helicase domain. The identification of novel IGHMBP2 variants will hopefully help diagnosing SMARD1 and contribute to a better functional characterization of IGHMBP2 gene product.

Rett phenotype with X/autosome translocation: possible mapping to the short arm of chromosome X.

Rett syndrome (RS) was diagnosed in a girl with a t(X;22) (p11.22;p11). This translocation was also present in her unaffected mother and her sister affected by a neurological disorder compatible with a "forme fruste" of RS. Different etiological mechanisms are considered: gene disruption, X inactivation disturbance, metabolic interference. Whatever this may be, the localization of a RS related gene to the short arm of chromosome X is likely.

Generalized molecular defects of the neuromuscular junction in skeletal muscle of the wobbler mutant mouse.

Available models of motorneuron disease that occur naturally in animals provide a useful approach to study human motorneuron disease. The wobbler mutant mouse displays a hereditary lower motorneuron disease which leads to progressive partial denervation of skeletal muscle and, at the same time, axonal regeneration with attempted reinnervation. In order to determine the consequences of these processes at the neuromuscular level, we undertook a study of key molecular components of the neuromuscular junction in wobbler mice. Increased levels of acetylcholine-receptor (AChR) and neural cell adhesion molecule (N-CAM) in wobbler muscle, together with an intense axon sprouting, suggest a complex denervation-reinnervation phenomenon. Furthermore, the appearance of ectopic clusters of AChR, spatially related with regrowth of axons, suggests ectopic formation of new synaptic areas, while, at the same time, some old synaptic sites fail to be reinnervated. Finally, 90% of wobbler neuromuscular contacts present a reduced acetylcholinesterase activity and a lack of N-CAM, which suggests a generalized defect of the mutant neuromuscular junction. These observed abnormalities may well be the consequence of a specific motorneuron defect.