[Partial hydatidiform mole in Morocco: an epidemiological and clinical study]. / Les môles hydatiformes partielles au Maroc: étude épidémiologique et clinique.

This retrospective study reviewed cases of partial hydatidiform mole (PHM) diagnosed at the University Hospital in Casablanca from 2000 to 2010 in order to examine the epidemiological, clinical, therapeutic and progressive pathological factors associated with PHM. All PHM cases confirmed clinically and sonographically at pathological examination were included. We identified 24 cases of PHM among 60 748 births and 1704 abortions, giving a frequency of 0.4 per 1000 pregnancies and 1.4% of abortions. The mean age was 26 years (range: 16-55 years). The circumstances of discovery and clinical ultrasound varied: 79.2% of patients sought consultation for bleeding; clinical thyrotoxicosis syndrome was found in 1 patient (4.2%). Physical examination showed increased uterine size in 83.3% of cases associated with a latero-uterine mass in 25%. The diagnosis was supported by an ultrasound examination combined with measurement of plasma betaHCG. Histological confirmation was made in all cases and treatment was endo-uterine aspiration. Neoplastic drift was observed in 1 case (4.2%) which went into remission with chemotherapy.

[From human andro- and parthenogenesis (hydatidiform moles and benign ovarian teratomas) to cancer]. / Des andro- et parthénogénotes humains (môles hydatiformes et tératomes ovariens) au cancer.

Genomic imprinting is a process that appeared in mammals. This phenomenon blocks the normal development of parthenogenic and androgenic conceptuses, that is to say benign ovarian teratomas and hydatidiform moles respectively. Pathological modifications of these conceptuses depend on whether the chromosomes come from the mother or father. These pathologies are associated with an accidental anomaly during gametogenesis and/or fertilizing. These reproductive anomalies are sporadic and some familial cases may exist suggesting a genetic control of such diseases. The human andro- and parthenogenetic conceptuses, but more frequently the moles, may be invasive (choriocarcinoma). An imbalance of the imprinting genes may initiate the deregulation of other genes, including oncogenes and anti-oncogenes, which can explain the cancerous modification. Immunological and environmental factors must be also considered (presence of the only paternal chromosomes in the choriocarcinoma). Numerous works on this subject are published and some recent important discoveries underline the roles of genes HOX, Tim P3, E-cad and p-16, and the recurrent chromosome anomalies 7q21+and 8p21- in the mole to choriocarcinoma processing. Although these phenomena are complex and heterogeneous, the andro- and parthenogenote conceptuses are particularly interesting models with which to understand developmental disorders and cancerous progression.

The involvement of the trans-generational effect in the high incidence of the hydatidiform mole in Africa.

INTRODUCTION: While the incidence of various chromosomal anomalies observed, including triploid partial moles is independent of the socio-economic level, higher incidences of complete hydatidiform mole "CHM" is generally associated with under developed areas. Moreover, studies have shown that some nutritional deficiencies are related to the abnormal development of oocytes and placenta. In Senegal and Morocco, the annual seasonal cycle contains one period with food shortages and the incidence of complete moles is significant. Accordingly, accurate statistical analyses have been performed in these two countries. METHODS: Each month during a one year period, we investigated the occurrence of normal conceptions, molar conceptions and the conception of the future patients in Senegal and Morocco. The comparisons of the conception dates for these three types of conception were analyzed using the Chi-squared test. RESULTS: 94% of the patients were conceived just prior to the period in the year with food shortages. Consequently, the development of the female embryos occurred under nutritional constraints, which negatively affect the recruitment of the vital factors required for the normal synthesis of DNA, proteins and placental differentiation. DISCUSSIONS: A nutritional deficiency in the mother at conception of their daughter (future patient) is implicated in the higher incidence of CHM in their daughters' filiation. These nutritional deficiencies during the first weeks of pregnancy will have repercussions on the normal development of the oocytes. Accordingly, these developmental impairments take place during the embryonic life of the future mothers of complete moles and not during the conception of the moles themselves.

Genetic, cytogenetic and physical refinement of the autosomal recessive CMT linked to 5q31-q33: exclusion of candidate genes including EGR1.

Charcot-Marie-Tooth disease is an heterogeneous group of inherited peripheral motor and sensory neuropathies with several modes of inheritance: autosomal dominant, X-linked and autosomal recessive. By homozygosity mapping, we have identified, in the 5q23-q33 region, a third locus responsible for an autosomal recessive form of demyelinating CMT. Haplotype reconstruction and determination of the minimal region of homozygosity restricted the candidate region to a 4 cM interval. A physical map of the candidate region was established by screening YACs for microsatellites used for genetic analysis. Combined genetic, cytogenetic and physical mapping restricted the locus to a less than 2 Mb interval on chromosome 5q32. Seventeen consanguineous families with demyelinating ARCMT of various origins were screened for linkage to 5q31-q33. Three of these seventeen families are probably linked to this locus, indicating that the 5q locus accounts for about 20% of demyelinating ARCMT. Several candidate genes in the region were excluded by their position on the contig and/or by sequence analysis. The most obvious candidate gene, EGR1, expressed specifically in Schwann cells, mapped outside of the candidate region and no base changes were detected in two families by sequencing of the entire coding sequence.

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human.

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.

Use of embryonic human trachea grown in nude mice to patch-repair congenital tracheal stenosis.

BACKGROUND: Long congenital tracheal stenosis is a life-threatening condition, and the available surgical treatments do not give satisfactory long-term results. METHODS: Human embryonic tracheas were implanted in the abdominal cavities of nude mice until their differentiation was completed. These differentiated tracheas were used to patch-repair surgically induced tracheal stenosis in piglets. The human, mouse, or pig origin, of all the cells in the two successive xenotransplants in the nude mouse and the pig, was determined on tissue sections by in situ hybridization with species-specific DNA probes. RESULTS: The transplanted pigs thrived and reached normal adulthood, irrespective of the administration of immunosuppressive treatment. The human tracheal tissue developed in nude mice conserved human structures, with the exception of feeding capillaries, which were of mouse origin. The tracheal patch in the adult healthy pigs comprised only pig cells organized into a fibrous scar, which was covered by normal pig epithelium. CONCLUSIONS: Results suggest that human embryonic trachea grown in nude mice can be successfully used as patch tracheoplasty for long congenital tracheal stenosis without conventional immunosuppression.

Interphasic analysis of aneuploidy in cancer cell lines using primed in situ labeling.

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific alpha-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distribution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.

[Complete hydatiforme mole in Morocco: epidemiological and clinical study]. / Les môles hydatiformes complètes au Maroc : étude épidémiologique et clinique.

OBJECTIVE: Complete hydatidiform moles (CHM) are a real public health problem, especially in the "southern countries" and Asia, because of their impact on the female reproduction and the risk to progression to either invasive mole or choriocarcinoma. PATIENTS AND METHODS: We collected the cases of CHM referred to our department over a period of ten years (2000 to 2009). We will present our results, emphasize the modalities of diagnosis, treatment and evolution, with a review of literature. RESULTS: During this study, we identified 254 cases of CHM, and recorded 57,987 births and 1627 abortions. Their incidence was 0.43% of pregnancies. The mean age of our patients is 25 years old (16 to 55). Relative risk observed was much increased among women under 20 years old (×6.8) and those over 40 years old (×15). Both of nulliparous and primiparous patients represented 52.3% of the cohort. Eighty-five percent of patients belonged to an agricultural environment associated with a low socio-economic status. Uterine bleeding was the most common symptom accounting for 93.7%. Toxic syndrome was present in 18.5% of patients. Physical examination showed a highly increased uterine size in 85% of cases associated with lateral uterine mass in 25% of cases. The diagnosis was suspected using ultrasonography in all cases associated with an elevated level of plasmatic ß-human chorionic gonadotrophin (ßhCG). All cases were confirmed histologically. Treatment used was endo-uterine aspiration in all cases. Recurrence of CHM was documented in 25 patients or 9.4%. Neoplasic progression was observed for 6.3% of cases. All of them have evolved into remission with chemotherapy. DISCUSSION AND CONCLUSION: CHM continue to be a public health problem in Morocco, their incidence is among the highest ones. In fact, this studied population corresponds to the lowest socio-economic status and generally described as population at risk. It is subject to drastic weather's conditions causing loss of fresh products. Extreme ages and degree of parity are also risk factors described in the literature. Early diagnosis, appropriate treatment, and supervision of molar pregnancies are obligatory. Despite of the unfavourable initial conditions, our study shows that relevance and continuing care can significantly reduce the morbidity of moles.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

Rapid characterization of human chromosomes in hybrid cell lines by primed in situ (PRINS) labeling.

The primed in situ (PRINS) labeling technique allows a rapid and specific labeling of human chromosomes in situ. This method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. We have developed a PRINS protocole for the cytogenetic analysis of somatic hybrid cell lines. Painting of human chromosomes is performed using Alu specific primers. Individual human chromosomes are identified using chromosome-specific alpha-satellite primers. The method was successfully tested to 3 different human-hamster hybrid cell lines. This approach provides an interesting alternative to classical cytogenetic and in situ hybridization techniques for the characterization of the human content of hybrid cell lines.

Assessment of chromosomal heterogeneity in tumoral cell lines using PRINS technique.

The primed in situ (PRINS) labeling technique has been used for the interphase cytogenetic investigation of 3 colon cancer cell lines (Caco-2, TC7 and PF1 1) derived from a same primary tumor. A panel of 10 chromosome-specific primers (for chromosomes 1, 2, 3, 7, 8, 9, 10, 13, 16 and 18) has been utilized in simple and double color PRINS reactions. Each cell line displayed a heterogeneous distribution of copy number for several chromosomes. The karyotypic heterogeneity was also significant between the 3 cell lines. These data indicate the common occurrence of chromosome heterogeneity in tumoral cell lines and demonstrate the feasibility of interphase PRINS procedure for analysis of numerical changes in tumoral cells.

Double telomeric signals on single chromatids revealed by FISH and PRINS.

FISH probes for all human telomeres and specific telomeric probes that hybridize to unique sequences on individual chromosomes have been used to characterize the telomeric hybridization pattern of human peripheral blood lymphocytes and bone-marrow cells in interphase and metaphase chromosomes. We have identified the existence of double hybridization signals on chromatids both with the (TTAGGG)n telomere repeat arrays and on non chromosome-specific subtelomeric regions as well as on chromosome-specific sequences located several kilobases from the end of chromosomes. Preliminary results using cosmid or YAC probes that hybridize to regions rich in GC sequences also revealed double fluorescent spots on a single chromatid. Double spots were detected by PRINS on terminal and interstitial telomeric sequences in avian cells. The significance of this phenomenon is discussed based on some models of chromatid and DNA organization such as uninemy, looped chromatid organization and quartet DNA structures. The occurrence of double spots should be taken into consideration for the clinical cytogenetic diagnosis of duplications.

[Tissue expression of a gene potentially implicated in some diseases with retinal and renal involvement]. / Expression tissulaire d'un gène potentiellement impliqué dans certaines maladies associant une atteinte rétinienne et rénale.

We have produced a monoclonal antibody (HA34) that specifically reveals the pigmented epithelium in the eye and the proximal convoluted tubules of the kidney, whatever the developmental stage. The results obtained with the kidney of other mammals suggest that the antigen is human specific. Its molecular weight is approximately 200 kDa. The epitope recognized by HA34 is always present on cell lines grown in vitro. This allowed us to use somatic cell interspecific hybrids to localize the gene implicated in the cytogenetic band 11q13, between microsatellites D11S1777 (AFMa046wa9) and D11S913 (AFM164zf12) in a 9 cM space. This region is involved in forms of retinitis pigmentosa, some of which can also include kidney abnormalities. We propose that this gene is possibly implicated in some of these diseases.

Generation of whole-chromosome painting probes specific to each chicken macrochromosome.

The realization of physical and genetic maps of the chicken genome is dependent on progress in cytogenetic knowledge of its karyotype. To help achieve this goal, we constructed amplified representative DNA samples of the chicken chromosomes 1, 2, 3, 4, 5, 6, 7, 8, Z, W and of the terminal heterochromatin part of Zq by chromosome microdissection and DOP-PCR amplification. These chromosome DNA samples, which represent about 75% of the chicken genome, were used to generate whole chromosome painting probes for FISH. The direct application of these chromosome specific probes is dual FISH localization and characterization of panels of chicken interspecific somatic hybrids. We discuss some aspects of the chicken genome and its repeated sequences.

Expression of fucosyltransferases in skin, conjunctiva, and cornea during human development.

During human development, type-1-precursor, sialyl-Le a, and Le x antigens were present in the periderm of skin and eye at week 6. The Le x antigen disappeared from cornea at 10 weeks and then from skin at 20 weeks. H-type-1, Le a, Le b, sialyl-Le a, H-type-2, sialyl-Le x, and Le y were found in cornea, conjunctiva, and periderm between 10 and 20 weeks. They disappear from the skin (at week 20) and progressively reappear in skin derivatives, especially in the epithelium of sweat glands. The secretory part of the sweat gland is type-1-precursor and H-type-1 positive while its excretory part is Le a, Le b, sialyl-Le a, and Le y positive. On the eye surface the disappearance of Le x at 10 weeks and of the H-type-1, sialyl-Le x, and Le y at week 35 starts in the central cornea in front of the lens. The corneal epithelium and the conjunctiva have similar antigens to those of excretory and secretory parts of the sweat gland, respectively. Invaginations and folding of the epidermis might preserve the embryonic staining. We propose that fucosylation patterns are associated with the embryonic origin and differentiation stage of tissue. The early and transient presence of Le x is associated with FUT4 or FUT9 activities, while the late appearance of Lewis antigens is related to other alpha3-fucosyltransferases.

Genetic regulation of the expression of ABH and Lewis antigens in tissues.

Sequential appearance of ABH antigens in different animal species shows a progression from tissues of endodermal to ectodermal and finally mesodermal origin, human erythrocytes being the last cells to acquire these antigens. In view of this, ABH antigens should be called tissue or histo-blood group antigens rather than blood group antigens. In addition to the glycosyltransferases encoded by the ABO genes, several alpha-2, alpha-3 and alpha-4-fucosyltransferases are needed to account for the known ABH histo-blood group antigens. The genetic polymorphism of the genes encoding each of these enzymes defines inter-individual differences. In addition, in the same individual various tissues express these antigens in a different way. For each adult epithelial tissue, antigenic expression is related to cell maturation from germinal layer to surface epithelium. Differential expression is also found at various embryonal stages of the same cells. Examples of these phenomena are presented in an effort to gain further insight into the genetic regulation of the expression of these complex oligosaccharide molecules.

Primed in situ (PRINS) labelling with Alu and satellite primers for rapid characterization of human chromosomes in hybrid cell lines.

The primed in situ (PRINS) labelling method was developed as an alternative to classical cytogenetics and fluorescence in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed by PRINS using Alu-specific primers to generate the painting of all human material associated with R-like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Using this methodology, a somatic hybrid clone was shown to be monochromosomal for the der(11) from a t(11;22) patient.

[Rapid identification of chromosomes by in situ hybridization of labelled oligonucleotides and comparison with the PRINS method]. / Identification rapide des chromosomes par hybridation in situ d'oligonucléotides marqués et comparaison avec la méthode PRINS.

We propose a simple, fast and inexpensive method of identification of human centromeres on metaphasic chromosomes and interphasic nuclei. This is based on in situ hybridization of labelled oligonucleotides. The efficiency of the methodology was demonstrated on cytogenetic preparations from human heteroploid and human x hamster hybrid cell lines and also on frozen tissue sections using an oligonucleotide specific for the alpha-satellite DNA of chromosome 1. Three versions of this oligonucleotide respectively labelled with 1, 4 and 10 fluorescein molecules were synthesized. The signal intensity provided by the oligonucleotide coupled with 4 fluoresceins allowed unambiguously the detection of the chromosome and the establishment of its ploidy using a classical cytogenetic microscope without the need for an amplification procedure. The use of different fluorochromes and possibly combination with an unlabelled elongation in 3' of the oligonucleotides which stabilize its hybridization, lead to a simple multicolour method. Preliminary quantification of the signals obtained by in situ hybridization of labelled oligonucleotides and comparison with those obtained by primed in situ labelling (PRINS) using the same nucleotides as primers, suggest that the elongation generated by PRINS may be very short compared with a PCR in solution. This limited efficiency of the in situ elongation may reflect the present difficulties of PRINS and DISC PCR (direct in situ single copy polymerase chain reaction) with primers specific for non-repetitive sequencies.

Cytogenetic characterization of interspecific somatic hybrids by PRINS.

The primed in situ (PRINS) labeling technique was developed as an alternative method to classical cytogenetics and in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed using Alu specific primers generating the painting of all human material associated with R like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Due to the use of synthetic oligonucleotide primers and of directly labeled haptens. PRINS method avoid repetitive probes preparation, eliminates secondary amplification of signals and the whole process can be performed within a timespan of 1 hour. Providing qualitative and quantitative answers, the simple PRINS method appears very well adapted to the specific problematic of somatic hybrids as for their characterization than for their periodic controls imposed by their instability. The method has been tested on 4 human-rodent hybrid cell lines. In particular, the somatic hybrid clone ALE 4 was shown to be monochromosomal for the der(11) from the reciprocal translocation t(11:22).