Loss of function of Ywhah in mice induces deafness and cochlear outer hair cells' degeneration.

In vertebrates, 14-3-3 proteins form a family of seven highly conserved isoforms with chaperone activity, which bind phosphorylated substrates mostly involved in regulatory and checkpoint pathways. 14-3-3 proteins are the most abundant protein in the brain and are abundantly found in the cerebrospinal fluid in neurodegenerative diseases, suggesting a critical role in neuron physiology and death. Here we show that 14-3-3eta-deficient mice displayed auditory impairment accompanied by cochlear hair cells' degeneration. We show that 14-3-3eta is highly expressed in the outer and inner hair cells, spiral ganglion neurons of cochlea and retinal ganglion cells. Screening of YWHAH, the gene encoding the 14-3-3eta isoform, in non-syndromic and syndromic deafness, revealed seven non-synonymous variants never reported before. Among them, two were predicted to be damaging in families with syndromic deafness. In vitro, variants of YWHAH induce mild mitochondrial fragmentation and severe susceptibility to apoptosis, in agreement with a reduced capacity of mutated 14-3-3eta to bind the pro-apoptotic Bad protein. This study demonstrates that YWHAH variants can have a substantial effect on 14-3-3eta function and that 14-3-3eta could be a critical factor in the survival of outer hair cells.

The retinal pigment epithelium undergoes massive apoptosis during early differentiation and pigmentation of the optic cup.

PURPOSE: The aim of our work was to study apoptosis during the development of the retinal pigment epithelium (RPE) in mice between embryonic day (E) 10.5 and E12.5 and to examine a possible link between apoptosis and pigmentation. METHODS: We collected mouse embryos at E10.5, E11.5, and E12.5 and labeled apoptotic cells in 5-µm paraffin sections, using the terminal deoxynucleotidyl transferase dUTP nick end labeling technique. We counted the total number of cells and the number of apoptotic cells in the early developing RPE and calculated the percentage of apoptosis at each stage. RESULTS: In the C57BL/6J mouse, 17% of the RPE cells were apoptotic at E10.5 compared to 0.9% at E12.5. At E11.5, three-quarters of the RPE cells began to pigment, and apoptotic cells were located mostly in the nonpigmented part. In contrast, in the BALB/c mouse (tyrosinase-deficient) and pJ mouse (carrying mutations in the p gene) hypopigmented strains, the RPE contained significantly fewer apoptotic cells (7.5% and 10.1%, respectively) at E10.5 than controls. Subsequently at E11.5 and E12.5, the two hypopigmented strains displayed different apoptotic patterns; the BALB/c RPE had a similar percentage of apoptotic cells to controls (1.5% and 1.1%, respectively, for BALB/c versus 3.0% and 0.9%, respectively, for C57BL/6J), whereas the pJ RPE contained significantly more apoptosis (7.5% and 3.5%, respectively). Overall we observed differences in the evolution of the relative total number of RPE cells between the three strains. CONCLUSIONS: Apoptosis is a main event during the first stages of normal RPE development, indicating an essential role during RPE differentiation. Moreover, the early apoptotic pattern and possibly the whole early development of the RPE is different between hypopigmented and pigmented strains, as well as between BALB/c and pJ mice. This suggests the existence of regulatory and developmental differences with a more complex origin than just differing pigmentation levels.

Major role of BAX in apoptosis during retinal development and in establishment of a functional postnatal retina.

Apoptosis plays a major role in the development of the central nervous system. Previous studies of apoptosis induction during retinal development are difficult to interpret, however, because they explored different mouse strains, different developmental periods, and used different assays. Here, we first established a comprehensive sequential pattern of cell death during the whole development of the C57BL/6J mouse retina, from E10.5 to postnatal day (P) 21 by using the terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-biotinylated nick end labeling (TUNEL) assay. We confirmed the existence of three previously described apoptotic peaks and identified another, later peak at P15, in both the outer nuclear layer, in which the photoreceptors differentiate, and the ganglion cell layer. Comparison of wild-type C57BL/6 mice, gld mice, defective in the death ligand fasL, and bax-/- mice, defective in the pro-apoptotic BAX protein, revealed a minor role for FAS ligand but a crucial role for BAX in both apoptosis and normal retinal development. The lack of BAX resulted in thicker than normal inner neuroblastic and ganglion cell layers in adults, with larger numbers of cells and an impaired electroretinogram response related to a decreased number of responsive cells. Our findings indicate that cell death during normal retinal development is important for the modeling of a functional vision organ and showed that the pro-apoptotic BAX protein plays a crucial role in this process.

Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome C oxidase deficiency.

Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.

Mutation analysis of the tyrosinase gene in oculocutaneous albinism.

Type I oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by the reduction or the absence of tyrosinase (TYR) activity in melanocytes of the skin, hair and eyes. Here we report an analysis of 45 patients with OCA. We found five novel mutations in the tyrosinase gene involved in the pathogenesis of oculocutaneous albinism type IA or type IB (OCA-1A/B) in five unrelated patients. Three mutations are missense mutations (G109R, P205T and H256Y) and two are nucleotide deletions (336-337delCA and 678-680delAGG). One patient is homozygous for the previously known V275F mutation but has an extremely mild OCA phenotype and has no eye features typical of OCA. In several patients we discovered only one or even no mutation in the coding sequence of the TYR gene. Thus, this disease may also result from mutations in non coding regions of the gene or in another gene involved in the biosynthesis of melanin. Hum Mutat 17:352, 2001.

Identification of novel VMD2 gene mutations in patients with best vitelliform macular dystrophy.

ABSTRACT We report five novel VMD2 mutations in Best's macular dystrophy patients (S16F, I73N, R92H, V235L, and N296S). An SSCP analysis of the VMD2 11 exons revealed electrophoretic mobility shifts exclusively in exons 2, 3, 4, 6 and 8. Direct sequencing indicated that these shifts are caused by mono-allelic transition in exons 2, 4, 6, 8 and transversion in exons 3 and 6. Five novel "silent" polymorphisms are also reported: 213T>C, 323C>A, 1514A>G, 1661C>T, and 1712T>C. Hum Mutat 17:235, 2001.

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis.

The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties.

[High frequency jet ventilation in heated gas saturated in water vapor without a reserve of medical gas under pressure. An original method]. / Jet ventilation à haute fréquence en gaz réchauffés et saturés en vapeur d'eau sans réserve de gaz médical sous pression. Une méthode originale.

A prototype of high frequency jet ventilator is compared with a classic device: Gambro Soxijet ventilator. The advantages of the prototype are: no need of pressured medical gas; warming and saturated moisture of the gas. A powerful compressor (2 M3.H-1 flow--3 bar pressure) draws up the moistened and warmed gases and injects them into a double pneumatic capacity. The first capacity is pressure limited by a relief valve (3 bar). Exhausted gases flow back to the pump. A miniature pressure regulator, placed between the two capacities, rules the driving pressure. Gas mixture is injected through a solenoid valve controlled by an electronic twin-timer. Results of both devices are similar. However, our prototype seems to be very convenient for developing countries where medical gases under high pressure are not often available.

[An analgesic strategy for tympanoplasties]. / Stratégie analgésique au cours des tympanoplasties.

UNLABELLED: After induction with vecuronium, etomidate and then isoflurane or enflurane, nitrous oxide, useful at the beginning of tympanoplasty is washed out before the end of operation. So barotrauma on the graft is avoided. In the expectation of analgesia insufficiency, alfentanil is infused intravenously all over the operation period following two modes: constant flow mode (1.25 micrograms.kg-1.min-1) after a bolus (25 micrograms.kg-1): 33 patients; decreasing hyperbolic flow mode (H): cumulative dose = 10.8 x t0.5 (where t = minutes of infusion) = 47 patients. In this mode, plasma concentration is measured by 12 patients. RESULTS: the mean plasmatic level of alfentanil is steady during the 120 minutes duration of anaesthesia: standard deviation is higher than 30%. After high quality anaesthesia in both technics, recovery time was shorter with H mode than with constant flow one (extubation time = 46 +/- 31 min. versus 92 +/- 54 min). H. mode seems to be safer. Though, individual reactivity, drug interaction and genetic polymorphism must make us cautious! Two patients presented apnea 20 and 60 minutes after an efficient awakening.

[Two-years' experience of ambulatory anesthesia in otorhinolaryngology in children]. / Expérience de deux années d'anesthésie ambulatoire en ORL chez l'enfant.

Three hundred children were anaesthetized for E.N.T. operations. The anaesthetic consultation took place one week prior to operation. The anaesthetic comprises premedication, inhalation of halothane for infants, or propofol drip for the others with tracheal tubing for 10% of them. Recovery in every case is supervised in the recovery room. There are few setbacks with this method. Eleven children were not accepted for operation for sociological or medical reasons (Willebrand diseases) and only 3 had to remain in hospital until the following day due to slow recovery from anaesthetic.

[Prevention of early respiratory complications in esophageal surgery by ventilation of the independent lungs]. / Prévention des complications respiratoires précoces en chirurgie oesophagienne par la ventilation à poumons séparés.

Independent lung controlled ventilation by a double lumen tube has a beneficial effect in oesophagus surgery. Use of a tidal volume and an end-expiratory pressure different for each lung produces a drastic reduction of chest-X-ray abnormalities. In an homogen group of ten patients studied before and treated with conventional respiratory support, chest-X-ray abnormalities were seen in 80% cases. In this group of 34 patients treated with independent lung ventilation the rate of abnormalities is only of 20%. Independent lung ventilation decreases early pulmonary complications in dependent regions of the lungs, and late pulmonary complications in non dependent regions. This form of mechanical ventilation is performed with a "prototype" ventilator of small size, which permits synchronized or independent ventilation of the lungs.