The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI

Complex chromosomal rearrangements are very rare events in the human population. According to our knowledge on the consequences of simple reciprocal translocations for male fertility, translocations involving three or more chromosomes are thought to lead to severe reproductive impairments in terms of meiotic disturbance or chromosomal imbalance of gametes. We report the case of a 48 year old man whose sperm count revealed either oligozoospermia (<10(3) spermatozoa/ml) or azoospermia. He was referred to the laboratory for in-vitro fertilization after intracytoplasmic sperm injection. Cytogenetic investigations showed a complex chromosomal rearrangement involving firstly a translocation between the short arm of chromosome 7 and the long arm of chromosome 13 and secondly a translocation between the short arm of the same chromosome 13 and the short arm of chromosome 9. Diagnosis was ascertained by fluorescence in-situ hybridization and staining of the nucleolar organizer regions. Theoretical study of the translocated chromosomes predicted a 'chain' configuration of the hexavalent at the pachytene stage of meiosis. In all, 32 modes of segregation were considered and only one resulted either in a normal or a balanced gamete karyotype. Genetic counselling and choice of appropriate artificial reproduction technique are discussed.      

Isolation of low-frequency class-switch variants from rat hybrid myelomas

Class-switch variants have been isolated from rat-rat hybrid myelomas by sib selection using a simple assay based on red cell-labelled antiglobulins. The variants detected are consistent with the gene order deduced from molecular cloning. They appear to arise spontaneously at a rate approximately ten-fold lower than for mouse cell lines but the rate of switching back to the parental isotype is substantial in comparison. An IgG2b variant antibody having the same specificity as CAMPATH-1 for human lymphocytes and monocytes is active in antibody-dependent cell-mediated killing (unlike the parental IgG2a) and may prove to be a valuable therapeutic antibody for immunosuppression and treatment of leukaemia and lymphoma.     

The morphological differentiation of the internal sphincter muscle of the anus in the human embryo and fetus (26.01.1990)

Conclusion The opening of the anal canal appears to be the factor which initiated the differentiation of the sphincter apparatus.The internal sphincter m. of the anus is entirely composed of smooth muscle as distinct from the striated fibers of the m. puborectalis, and the external sphincter which is a mixture of smooth and striated fibers (of skeletal type). It develops in the terminal part of the internal circular layer of the rectal m., outside which are longitudinal fibers which descend early to form the external sphincter (beginning around the third month).This study shows that the internal sphincter is scarcely evident before 12 SA. Thus continence between 10 and 12 SA (after the closure of the anal membrane) is closely related to the other components of the sphincter apparatus. On the other hand, the internal sphincter has become well formed after 28 to 30 SA and then plays a direct role in maintaining continence.     

Gonadotrophin-releasing hormone agonist dose-dependency of pituitary desensitization during controlled ovarian hyperstimulation in IVF

The aim of this study was to find the minimal effective daily s.c. dose of the gonadotrophin-releasing hormone (GnRH) agonist, triptorelin acetate, that suppresses the GnRH-induced release of luteinizing hormone (LH) at time of human chorionic gonadotrophin (HCG) injection and thereby prevents spontaneous LH surges during in-vitro fertilization (IVF) stimulation cycles. Therefore, a double-blind, prospective and randomized titration study was performed. A total of 48 IVF patients were divided into four groups of 12 patients. Each group received a different dose of triptorelin acetate, namely 5, 15, 50 or 100 microg s.c. daily. Standard ovarian stimulation was carried out using urinary follicle stimulating hormone (FSH) preparations. A 500 microg GnRH test was performed 90 min before the HCG injection in order to measure the degree of pituitary desensitization. Spontaneous LH surges were not detected in any of the groups, although three patients in the 5 microg group had ovulated at the time of ovum retrieval. The pituitary LH response to the GnRH test at time of HCG, expressed as area under the curve (AUC), appeared to be dose-dependent. Thus, a daily s.c. dose of 100 microg triptorelin acetate appears to be too high, since adequate desensitization of the pituitary (i.e. no spontaneous LH surge) can be achieved with doses as low as 15 and 50 microg.      

Episodic variations of prolactin, thyroid-stimulating hormone, luteinizing hormone, melatonin and cortisol in infertile women with subclinical hypothyroidism

Preliminary data have suggested that female infertility due to corpus luteum insufficiency may be caused by subclinical hypothyroidism [exaggerated thyroid-stimulating hormone (TSH) response to thyrotrophin- releasing hormone (TRH) stimulation]. L-Thyroxine supplementation has been recommended to achieve pregnancies in subclinical hypothyroid women. This controlled study was carried out in order to investigate the biochemical diagnosis of subclinical hypothyroidism as a possible infertility factor. Five infertile patients (aged 25-36 years) with subclinical hypothyroidism (n = 4, stimulated TSH >20 microU/ml) or primary hypothyroidism (n = 1) and five healthy controls (aged 22-39 years) with normal thyroid function (stimulated TSH <15 microU/ml), regular cycles and no history of infertility were studied in the early follicular phase. In the pre-study evaluation, eight of 23 volunteers (34.8%) had to be excluded because of subclinical hypothyroidism with stimulated TSH values (TSHs) >15 microU/ml. Cycle function of patients and controls was compared by the method of LH pulse pattern analysis. Therefore blood samples were drawn every 10 min during a 24 h period. Sleep was recorded from midnight to 7 a.m. Repetition of the TRH tests at the end of the 24 h blood sampling period confirmed the difference in stimulated TSH values of the two study groups. Pulse analysis for luteinizing hormone (LH), TSH and prolactin showed no differences between patients and controls for pulse frequency, amplitude, height, length, area under curve (AUC) and the 24 h mean. Even the hypothyroid patient had a normal LH pulse pattern. Additional measurement of melatonin in pooled sera every 30 min gave the well-documented diurnal profiles during day and night for both groups. Patients had significantly higher melatonin values at seven time points during the night. Peaks for LH, TSH, prolactin and cortisol were correlated with the sleep stages wake, rapid eye movement, 1 + 2 and 3 + 4. We concluded that corpus luteum insufficiency in female infertility cannot be explained by subclinical hypothyroidism and thus should not be treated with L-thyroxine for fertility reasons.      

Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozen-thaw transfers

In-vitro fertilization patients (n = 15) at risk of ovarian hyperstimulation syndrome (OHSS) (oestradiol > or =4500 pg/ml on the day of human chorionic gonadotrophin administration and 25 or more follicles of intermediate or large size) underwent aspiration of all follicles and cryopreservation of all fertilized oocytes at the pronuclear stage. Patients were monitored for up to 2 weeks post- retrieval. Subsequent transfer of cryopreserved-thawed embryos was performed in programmed cycles using exogenous oestrogen and progesterone for endometrial preparation. Two patients (13%) developed OHSS necessitating hospitalization and vaginal aspiration of ascitic fluid. Two other patients (13%) developed moderate OHSS requiring ascitic fluid vaginal aspiration in the office setting, with dramatic improvement of the condition. Subsequent transfer of cryopreserved- thawed embryos yielded a clinical pregnancy rate of 58% per transfer and ongoing or delivery rates of 42 and 67% per transfer and per patient respectively. By eliminating pregnancy potential with cryopreservation of all prezygotes and examining the pregnancy potential with subsequent cryopreserved-thawed transfers, it is concluded that OHSS is reduced, but not eliminated for patients at risk. Subsequent transfer of cryopreserved-thawed prezygotes in a programmed cycle with exogenous steroids yields an excellent pregnancy rate.      

Improved oocyte quality is obtained with follicle stimulating hormone alone than with follicle stimulating hormone/human menopausal gonadotrophin combination

The aim of this study was to compare the efficacy of pure follicle stimulating hormone (FSH) with that of FSH/human menopausal gonadotrophin (HMG) combination in downregulated cycles. A total of 357 patients was evaluated retrospectively. Sixty percent of patients in the FSH group and 55% in the FSH/HMG group were new; the others were repeat patients. Ovulation was suppressed with leuprolide acetate in all patients, followed by either FSH (n = 218) or FSH/HMG (n = 119). There was no difference in patients' age, infertility factors, number of ampoules used, length of stimulation, oestradiol levels on day of human chorionic gonadotrophin (HCG) administration, number of oocytes recovered or the number of embryos transferred. Also, nuclear maturity at aspiration and fertilization rates were not different between the two groups. FSH stimulation resulted in a significantly higher percentage of mature oocytes that showed the typical 'mature' morphological characteristics (P < 0.0001). The clinical pregnancy rates per transfer were 40 and 28% in patients stimulated with pure FSH and FSH/HMG respectively (P < 0.05). The significantly higher number of immature oocytes matured in vitro in the FSH/HMG group (P = 0.001) suggests a possible effect on in-vitro maturation, due to luteinizing hormone present in HMG. The difference in mature oocyte quality may be an important determinant in the higher pregnancy rates for the FSH- stimulated patients.      

Upper tibial corticocancellous flap anatomy study (19.2.93)

Summary A cortico-cancellous flap from the anterolateral aspect of the upper third of the tibia was presented. Sixty lower limbs of fresh cadavers were dissected. The vascular bundle includes the anterior tibial artery and its recurrent collateral branches and recurrent and muscllo periostal arteries. The flap is harvested with the interosseous membrane and can be used either free or pedicled. We used this flap for two patients suffering from pseudarthrosis. Long range clinical and radiological results are good.

---

Le transfert osseux vascularisé tibial superieur

Résumé Les auteurs décrivent un transfert ostéo-periosté vascularisé, prélevé sur la face antéro-latérale de l'extremité supérieure du tibia. L'étude anatomique porte sur soixante membres inférieurs, conservés au froid. La vascularisation métaphysaire du transfert provient de la branche récurrente tibiale antérieure et de ses rameaux, la vascularisation diaphysaire étant issue des branches musculo-périostiées proximales. Le transfert vascularisé est prélevé avec la membrane interosseuse et peut être pediculé ou libre. L'expérience clinique porte sur deux cas de pseudarthroses multiopérées. Les résultats cliniques et radiologiques sont bons à long terme.

     

Method of quantitative anatomical study of the dorsal neck muscles

Summary Biomechanical models of the cervical spine require knowledge of the position, size and orientation of the individual muscles that act on the cervical spine. We have developed a technique to stereometrically measure anatomical specimens. The apparatus is composed of three graduated metallic rods, which slide along a fixed support. This method is accurate to map the anatomy of individual muscles and provides quantitative data on their lines of action. Results are obtained from one specimen. The computer processing of the collected data allows formulation of a three-dimensional model of the neck muscles in man.

---

Méthode d'étude anatomique quantitative des muscles de la nuqueEtude préliminaire

Résumé Pour élaborer un modèle biomécanique de la colonne cervicale, il faut connaître la position, la taille et l'orientation des différents muscles du cou. Nous avons mis au point une méthode de mesure stéréométrique sur des sujets anatomiques. L'appareil est composé de 3 axes métalliques gradués qui coulissent sur un support fixe. Cette technique permet une étude anatomique précise de chacun des muscles de la nuque, fournissant des données quantitatives sur les différents faisceaux ou lignes d'action. Les résultats sont obtenus sur un sujet. Leur traitement informatique permettra l'élaboration d'un modèle mathématique tridimensionnel des muscles du cou chez l'homme.