The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region.

BACKGROUND: Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/KCNQ1OT1 in Beckwith-Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain. AIM: To analyse the methylation status of various imprinted genes (IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11-13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally). RESULTS: 3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1. CONCLUSIONS: Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre-implantation development.

Evolutionary conservation of the product of human c-myc exon 1 and its inducible expression in a murine cell line.

The human c-myc proto-oncogene contains an open reading frame within its first exon which is translated into protein (MYCHEX1). While the murine c-myc exon 1 is obviously non coding, we show that in mouse cells there are polypeptides closely related to human MYCHEX1. These polypeptides share the same immunological reactivities with the human polypeptides. Furthermore, the 32 kDa polypeptide of murine cells has, like its human counterpart, the ability to dimerise in a 58 kDa form in denaturing and reducing SDS-PAGE. The human gene was introduced into a murine cell line by transfection. A cell line was studied, in which the inducible expression of the gene allows a substantial increase in the concentration of the corresponding protein. This inducible protein behaves in any respect like the murine one, either in SDS-PAGE or in a specific immunoassay. These shared properties constitute a further proof that the human and mouse MYCHEX1 proteins are encoded by the sequence overlapping the human myc exon 1 and a related murine sequence. The gene contained in the human c-myc exon 1 is not, therefore, a specific feature of human cells.

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

[Electrophysiological monitoring of epileptic patients treated with Vigabatrin]. / Surveillance électrophysiologique des patients épileptiques traités par Vigabatrin.

Vigabatrin is a GABA mimetic antiepileptic agent that has been used for 10 years in cases of epilepsy that resist other treatments. Since 1997, concentric visual field defects have been reported. Before any visual symptom complaint, they quickly become irreversible and highly disabling. To prevent this visual impairment, the monitoring protocol must be defined with reliable and well-supported tests, so that patients treated with Vigabatrin can be regularly monitored. Our purpose was to know if EOG impairments were frequent, if their severity was proportional to visual impairment, and if the Arden ratio could be a predictive criterion of Vigabatrin toxicity. Seventy-two patients treated with Vigabatrin for 2-10 years were examined, and EOG results were compared with a normal population EOG and then the patient's visual field. The monitoring protocol proposed includes EOG, which seems to be the most sensitive and specific diagnostic tool for screening Vigabatrin-treated patients.

[Ophthalmologic prevention of chloroquine and hydroxychloroquine induced retinopathy]. / Prévention ophtalmologique de l'intoxication rétinienne induite par les antipaludéens de synthèse.

INTRODUCTION: Antimalarial drugs induce severe retinal toxicity. The aims of this study were to evaluate the strategy of screening clinical and preclinical intoxication due to antimalarial agents in two centres of reference and to describe the results of ophthalmologic examination. PATIENTS AND METHODS: Patients referred for ophthalmologic evaluation in connection with antimalarial agents therapy in the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts from October 1999 to December 2000 and in the Hôpital Lariboisière from January 1995 to December 1998 were investigated. A retrospective review of results of ophthalmologic examination, electroretinogram, electro-oculogram, colour vision test and central visual field was conducted to assess retinal intoxication. RESULTS: Among 705 patients recruited in the Centre des Quinze-Vingts, 10 out of 133 who were never treated had an electrophysiological contra-indication to the treatment. Among the 572 other patients, 31 presented with preclinical intoxication (5.4 p. 100) and 8 other patients presented with clinical intoxication. Among 925 patients recruited in the Hôpital Lariboisière, 37 presented with preclinical intoxication (4 p. 100) and four patients presented with clinical intoxication. DISCUSSION: The antimalarial drugs clinical intoxication is rare but nevertheless real. Screening for preclinical intoxication can prevent the evolution toward irreversible retinal intoxication. Diagnosis of preclinical intoxication is established through the confrontation of results of different tests and their evolution. The multifocal electroretinogram remains to be evaluated. CONCLUSION: Ophthalmologic monitoring including funduscopy, should be recommended at least once a year. Visual field seems to become interesting in the screening. Electroretinogram and electro-oculogram remain useful quantitative and obvious tests. A prospective study to assess the optimal way to prevent retinal intoxication is mandatory.

cDNA cloning, tissue distribution and chromosomal localization of the human ID4 gene.

A cDNA e encoding the human Id4 protein has been isolated from an astrocytoma library. The predicted protein product shares 98% identity with the mouse Id4 protein and is markedly different from that already reported. By FISH analysis, the human ID4 gene was more precisely mapped to chromosome 6p22.3-p23. Northern blot analysis showed that ID4 is mainly expressed in thyroid, brain and fetal tissue and in some nervous system tumor cell lines.

Cloning and nucleotide sequence of the chimpanzee c-myc gene.

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.

Expression of Id2 and Id3 mRNA in human lymphocytes.

Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T-cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate.

Id gene expression during development and molecular cloning of the human Id-1 gene.

Id genes encode helix-loop-helix proteins that inhibit transcription by forming inactive heterodimers with basic helix-loop-helix (bHLH) proteins. bHLH proteins normally form either homodimers or heterodimers with other bHLH proteins and bind to a DNA sequence element activating transcription. Id-containing heterodimers are inactive because Id proteins lack the basic amino acid region necessary to form a DNA-binding domain. We have examined the relative levels of Id-1 and Id-2 mRNA during normal development and in malignant tissues. In the course of these experiments we cloned and sequenced the human Id-1 cDNA. Two related cDNA molecules encoding human Id-1 mRNAs were identified. Id-1a is a cDNA of 958 nucleotides and can encode a protein of 135 amino acids. Id-1b cDNA is 1145 nucleotides, can encode a protein of 149 amino acids, and appears to be a splice variant of Id-1a. The amino acid sequence of human Id-1 is greater than 90% homologous to that of mouse Id-1. The patterns of Id-1 and Id-2 expression during mouse development vary widely, and we detected Id-1 expression in human fetal and adult tissues from lung, liver, and brain. High Id-1 mRNA expression was found in many human tumor cell lines, including those isolated from nervous system tumors. We mapped Id-2 to human chromosome 2p25.

[Retrochiasmatic lesions in multiple sclerosis. Demonstration by visual evoked potentials. Correlation with magnetic resonance imaging]. / Lésions rétrochiasmatiques dans la sclérose en plaques. Mise en évidence par les potentiels évoqués visuels. Corrélations avec l'imagerie par résonance magnétique.

Monocular stimulation of each visual hemifield can show an interhemispheric asymmetry of VEP. Validity of this test needs a reproducibility of responses and exclusion of stimulation induced by eye movements. In a prospective study of 22 MS cases, it appeared that interhemispheric asymmetry was a criterion of dissemination is space and had a good diagnostic value: MS became clinically definite in 10/12 cases; in 10 other cases in which a correlative MRI-VEP study was possible, there were disseminated high signal areas in T2 weighted sequences on hemispheric MRI. In 7/10 cases, these areas were located on retrochiasmatic visual pathways. With MRI, VEP are the most performant tests for early diagnosis in MS. Technical progress will improve its fiability. Prospective correlative clinical, electrophysiological and MRI studies are necessary on a larger number of MS patients.

[Acute optic neuritis: clinical and MRI prognostic factors. Study of fifty patients]. / Neuropathie optique inflammatoire aiguë: facteurs pronostiques cliniques et IRM.

The objective of this study was to evaluate the risk of visual outcome after acute optic neuritis (ON) in relation to clinical and MRI findings. Fifty cases of acute ON within one month were retrospectively studied. MRI with Short Tau Inversion Recovery (STIR) sequence of the optic nerve were obtained with a median time onset of 9 days after ON. Mean age of patients was 32.8 years, mean initial visual acuity was 3/10 and orbital pain was present in 86 percent100 of patients. The STIR sequence revealed lesion in 88 percent 100 of acutely symptomatic optic nerves. An initial low visual acuity (less than 2/10), the absence of orbital pain and involvement of the intracanalicular portion of the optic nerve on STIR sequence were statistically correlated with a poorer visual outcome (respectively p=0.0041, p=0.035 and p=0.011).

[Exploration of retro-chiasmatic visual pathways in human albinism]. / Exploration des voies optiques rétro-chiasmatiques chez l'albinos.

PURPOSE: Several research studies have explored the abnormal crossing of the retinogeniculate and geniculocortical optic pathways in human albinos. This prospective study has dealt with visual evoked potentials (VEPs) of human subjects to identify the percentage of albinos with asymmetric VEPs. PATIENTS AND METHODS: A series of 16 albino patients ranging in age from 6 to 37 years were examined. They had measurable visual acuity, with or without nystagmus. Diffusion of flash stimuli not allowing selective study of the two visual pathways (direct and crossed), two stimulation patterns were used for VEP recordings: monocular full open field then hemi-field stimulation to isolate the activity of each visual pathway. ANALYSIS: In the normally pigmented subject, fibers derived from the nasal half of the retina of each eye decussate at the chiasma, while temporal retinal fibers are uncrossed and project to the ipsilateral hemisphere. In albinos, the majority of temporal retinal fibers subserving the nasal field (from fixation to an eccentricity of about 20 degrees ) anomalously cross with the nasal retinal fibers. Therefore with monocular stimulation, the evoked visual response should be obtained only in the contralateral hemisphere. The asymmetry, morphology and latency for the first major positive peak and the amplitude of the VEP were examined and compared with the normal population. CONCLUSION: We managed to demonstrate the characteristic VEP asymmetry only in 3 out of the 16 patients. The results presented herein lead to question the absolute validity of VEP abnormality in diagnosis of albinism for clinical purposes.

[Electrophysiologic diagnosis of 2 psycho-visual syndromes: Balint syndrome and cortical blindness. A propos of a case of Benson progressive posterior atrophy]. / Diagnostic électrophysiologique de deux syndromes psycho-visuels: syndrome de Balint et cécité corticale. A propos d'une observation d'atrophie postérieure progressive de Benson.

Cortical blindness and Balint's syndrome are two pathologies not well-known. It seems therefore interesting to report a typical patient case, suffering from Benson's posterior cortical atrophy, who presented successively both syndromes. The Balint's syndrome, which results from a bilateral parieto-occipital junction brain injury, and combines clinically a specified triad defects: a spatial disorder of attention, a psychic paralysis of gaze and an optic ataxia. The cortical blindness, which is caused by bilateral damage of the occipital lobes (Broadman area 17). Electrophysiologically, the abolition of short-latency components of visual evoked potentials and the presence of long-latency potentials are recorded. Visual strategy and visual evoked potentials are thus the only objective examinations allowing to diagnose and follow up these patient's evolution. In any case, an adequate visual rehabilitation has to be carried out in order to help the patient recovering his autonomy.

[Study of colour vision and visual evoked responses in multisclerosis diagnosis (author's transl)]. / Etude de la vision des couleurs et des potentiels évoqués visuels dans le diagnostic de la sclérose en plaques.

In a patient with suspected Multisclerosis (M.S.), the discovery of a lesion in the anterior optic tracks is of considerable diagnostic importance. If none of the classical clinical signs of optic neuritis can be found, the study of visual evoked responses (VER) and of colour vision is useful evidence for diagnosis. In a population of 102 patients having "possible", "probable" or "confirmed" M.S, we have compared the information provided by both these methods. 27 patients had MS with a known optic neuritis: the VER and colour vision of all of them was altered, either unilaterally or bilaterally. 75 patients had "possible" or "probable" MS without a history of optic neuritis. For 34,7%, the discovery of a dyschromatopsia showed a lesion in the optic nerve. In 68%, only the increased latency in VER demonstrated an optic neuritis. It should be noted that for all the patients with "probable" or "confirmed" MS, the VER latency was increased. The study of colour vision is therefore in our opinion, an excellent way of investigating anterior optic tracks lesions. When the study of colour vision is not sufficient, the recording of VER is a reliable technique and a very valuable acquisition in neuro-ophthalmology.

[Up-dated ophthalmological screening and follow-up management for long-term antimalarial treatment]. / Surveillance ophtalmologique de la prise des antipaludéens de synthèse au long cours: mise au point et conduite à tenir à partir de 2003.

The early detection of macular toxicity linked to long-term antimalarial treatment requires regular ophthalmological screening based on patients'classification based on their results compared to successive controls. Patients are classified as "low risk" with screening every 18 months if all of the following criteria are met: age under 65 years, no associated renal, hepatic or retinal disease, treatment for less than 5 years, dose less than or equal to 6,5mg/kg/d for hydroxychloroquine and 3mg/kg/d for chloroquine (for a lean patient's weight); "at risk, without fundus findings" with screening every 12 months if one of the following criteria is met: age over 65 years (at the start of or during treatment), antimalarial treatment for more than 5 years, daily dose higher than recommended, presence of renal and/or hepatic disease; "at risk, with fundus findings" with screening every 6 months if a retinal dysfunction has been detected and even if treatment is established or followed. Screening consists of an in-depth clinical examination and at least two complementary tests of macular function: color vision (desaturated-Panel-D15 test) and/or static macular perimetry (central 10 degrees) and/or macular electroretinography (pattern ERG/multifocal ERG). If any changes or anomalies are found between two successive check-ups, the state of the retina can be assessed by angiography and global retinal function by full-field-ERG and electro-oculogram (EOG). The progression from one check-up to the next decides whether a course of treatment will be followed.

[Functional amblyopia: a functional MRI evaluation of the visual cortex response after treatment]. / Amblyopie fonctionnelle : évaluation en IRM fonctionnelle de la réponse corticale visuelle après traitement.

PURPOSE: Functional MRI evaluation of the cortical response in treated amblyopic patients. MATERIAL AND METHODS: Clinical and functional MRI exploration of ten patients, seven men and three women aged from 21 to 59 years, with strabismus management during childhood. Functional evaluations were performed on a 1.5 Tesla MR device, with four monocular functional sessions, two stimulations per eye. Alternating rest and active phases displayed still and flickering black and white checkerboards with spatial and temporal frequencies of 1 degree/8Hz and 15'/4Hz. Anatomical realignment and statistical analysis were performed using SPM99 (Statistical Parametric Mapping) to compare the four sessions in individuals. RESULTS AND DISCUSSION: In patients presenting a visual acuity of the amblyopic eye less than 0.7, stimulation of this eye induced lower response in V1, V3, and V5 in comparison with the contralateral eye stimulation. Unexpectedly, in patients recovering normal or subnormal acuity, the amblyopic eye gave comparable or enhanced response in these areas. Additional response was found in the secondary visual cortex, the cuneus, the lingual gyrus, and in parietal, frontal, and orbitofrontal areas. These results suggest a variation in cortical response depending on the efficacy of the treatment. Recovered amblyopic eye, even with acuity less than the contralateral eye, may induce a reinforced cortical sensitivity to visual stimulus. Secondary visual areas may contribute to an attentional process in image perception and analysis. Cortical plasticity may be observed several years after amblyopia treatment. CONCLUSION: Our study substantiates the importance of an effective and early treatment of functional amblyopia, inducing cortical plasticity with reinforced attention and sensitivity to visual perception.

Organization and expression of the E4 region of adenovirus 2.

The E4 region of Adenovirus 2 is a leftward transcribed part of the viral genome. Its nucleotide sequence has already been analysed. From this sequence several open reading frames were defined, which could be used in the coding of the E4 proteins. Using S1 digestion of mRNA-DNA hybrids a precise mapping of donor and acceptor sites was done. Their use in various combinations allows the synthesis of mRNAs, able to direct the synthesis of at least 7 polypeptides, ranging in size from 9K to 34K. Comparison of the sequences of the different acceptor sites indicates that they all conform to the consensus sequence. Analysis of the ATG surrounding sequence shows that initiator ATG may be positively selected according to Kozak's rule.

Profiling of differential gene expression in Wilms tumor by cDNA expression array.

In order to identify genes or pathways involved in Wilms tumor etiology, we used the Atlas Cancer cDNA expression array to compare the gene expression profiles of five tumors, one Wilms tumor cell line (SK-NEP1), and normal mature and fetal kidneys. Of 588 genes tested, 153 had a different expression pattern in tumors compared with mature kidney. Ninety-six genes were differentially expressed in tumors compared with both normal mature and fetal kidney, and 57 genes had expression profiles similar to that of fetal kidney, which may reflect the developmental stage of the tumor cells. Comparison of the expression patterns of tumors shows that only 13% of the differentially expressed genes are constantly up- or downregulated in the five tumors tested, and this provides molecular evidence of tumor heterogeneity. We then confirmed the differential expression by an independent method, using quantitative reverse transcriptase polymerase chain reaction for two of the differentially expressed genes, MMP-14 and cyclin D2. Analysis of expression levels in a panel of 40 tumors showed that 30% overexpressed MMP-14 and 80% overexpressed cyclin D2. Profiling of gene expression using cDNA arrays in a large tumor panel will ultimately lead to the molecular classification of tumors, the identification of prognosis markers, and the design of targeted therapy.