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J P Charlieu

Publications and source records attributed to J P Charlieu.

At least 19 recordsLinked to original sources
PubMed1999

A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1, which include mutations predicted to truncate the protein within the C-terminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1(tmT396), which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1(tmT396) protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Incidence of chromosome 1 disomy in human sperm estimated by the primed in situ (PRINS) labeling technique.

The primed in situ (PRINS) labeling technique was used to determine the rate of disomy of chromosomes 1 and 16 in sperm of two normal subjects. Two different but specific primers (alpha-satellite and satellite II) for chromosome 1 were used in parallel experiments to test the efficiency of PRINS labeling in sperm nuclei. A minimum of 10,000 sperm nuclei per chromosome primer was analyzed, leading to a total number of 41,651 scored spermatozoa. Similar rates of chromosome 1 disomy (mean values, 0.18% and 0.20%) were found in both donors when the alpha-satellite and satellite II primers were used, demonstrating the reliability of PRINS labeling on sperm nuclei. For chromosome 16, the disomy rate among the two donors ranged from 0.20% to 0.24%. This study confirms that PRINS provides a rapid and efficient method for in situ chromosomal screening of sperm nuclei.

Adult↗
PubMed1997

Cytogenetic analysis of meiotic segregation in sperm from two males heterozygous for reciprocal translocations using PRINS and humster techniques.

The meiotic segregation patterns of 2 reciprocal translocations t(7;9)(q33;p21) and t(7;18)(q35;q11) were analyzed in sperm of 2 heterozygote carriers. Both sperm karyotyping and in situ PRINS labeling of sperm nuclei were performed on a sperm sample from each subject. Using the humster technique, 54 and 72 sperm chromosome complements were successfully analyzed for the t(7;9) and the t(7;18) respectively. The frequencies of alternate, adjacent 1, adjacent 2 and 3:1 segregations were 44.44%, 37.04%, 12.96% and 5.56% for the t(7;9) and 33.33%, 43.05%, 19.45% and 4.17% for the t(7;18). The PRINS procedure allowed the rapid screening of large samples of spermatozoa. However, alternate and adjacent 1 segregants were not discriminated because of the generation of centromeric signals. The segregation pattern was determined on 10,658 spermatozoa for the t(7;9) and 10,462 for the t(7;18). The distributions of segregants were similar to those obtained by sperm karyotyping. These data were pooled with results from 37 reciprocal translocations previously studied by sperm karyotyping and 6 recently investigated by FISH. The analysis of these compiled data demonstrates the particularity of the production of imbalances in male gametes; independent of the predisposition for a type of imbalance at term, there is a preferential production of adjacent 1 imbalance in sperm.

Adult↗
PubMed1997

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).

Animals↗
PubMed1996

Aneuploidy detection in human sperm nuclei using PRINS technique.

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a < 2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates.

Adult↗
PubMed1996

Direct detection of disomy in human sperm by the PRINS technique.

We report the use of the PRimedIN Situ (PRINS) labelling technique for the direct estimation of disomy rates for various chromosomes in human sperm. The PRINS technique provides a rapid and reliable method for chromosome screening since specific labelling may be obtained in less than 2 h. In the present study, the disomy rates of chromosomes 2, 5, 9, 12 and 18 were investigated. The incidences of disomy are similar for all these chromosomes, ranging from 0.27% to 0.33%. These findings suggest an equal distribution of aneuploidies among autosomes in human sperm.

Aneuploidy↗
PubMed1996

Rapid chromosome detection in human gametes, zygotes, and preimplantation embryos using the PRINS technique.

PURPOSE: The analysis of the chromosomal constitution of human gametes and embryos is of particular importance for investigation of aneuploidy occurrence and diagnostic purposes. The PRINS method constitutes an alternative to FISH for in situ chromosomal identification. We have adapted this method to human gametes, zygotes, and preimplantation embryos. RESULTS: Chromosome-specific labeling was obtained in gametes, zygotes, and isolated blastomeres. Simultaneous detection of two or three chromosomes can be completed in less than 3 hr using fluorochrome-labeled nucleotides. CONCLUSIONS: The PRINS technique appears to be more efficient than FISH for detection and discrimination of alpha-satellite DNA sequences. The present study demonstrates the usefulness of PRINS for chromosomal screening and preimplantation diagnosis.

Aneuploidy↗
PubMed1996

Preimplantation embryo chromosome analysis by primed in situ labeling method.

OBJECTIVE: To present the use of primed in situ labeling method in preimplantation diagnosis. DESIGN: Double- and triple-primed in situ labeling were performed on 10 morphologically abnormal preimplantation embryos, using combinations of specific primers for chromosomes 9, 13, 16, 18, 21, X, and Y. SETTING: Embryos were obtained from patients at the Montpellier University Hospital. PATIENT(S): Seven women undergoing IVF at the Montpellier University Hospital. INTERVENTION(S): Isolated interphase nuclei from poor quality preimplantation embryos were prepared for primed in situ labeling technique. MAIN OUTCOME MEASURE(S): Numerical abnormalities assessed by primed in situ labeling analysis. RESULT(S): Using directly fluorescent-labeled nucleotides, the labeling reaction for three chromosomes did not exceed 2.30 hours. Only three analyzed embryos appeared to be chromosomally normal. Mosaicism, aneupoidy, and haploidy were observed in the seven other embryos. CONCLUSION(S): The primed in situ labeling method offers a simple and reliable screening tool for gender determination and aneuploidy detection. The use of this technique may contribute to significantly improve the procedure of preimplantation diagnosis.

Aneuploidy↗
PubMed1996

The PRINS technique: potential use for rapid preimplantation embryo chromosome screening.

The primed in-situ labelling (PRINS) method is an alternative to in-situ hybridization for chromosomal detection based on the use of chromosome-specific oligonucleotide primers. Using this process, we have developed a simple and semi-automatic method for rapid in-situ detection of human chromosomes. The reaction was performed on a programmable temperature cycler. Specific labelling was obtained in < 2 h reaction. Double PRINS techniques were performed on six morphologically abnormal preimplantation embryos using primers specific for chromosomes 9, 16, 18, 21, X and Y. The majority of these embryos displayed chromosomal abnormalities. The present results demonstrate that PRINS may be a simple and reliable technique applicable in human preimplantation diagnosis.

Blastocyst↗
PubMed1996

FISH and PRINS, a strategy for rapid chromosome screening: application to the assessment of aneuploidy in human sperm.

The co-utilization of FISH and PRINS techniques for in situ chromosome screening was tested on human sperm nuclei. We used a centromeric repeat probe specific for chromosome 4 for FISH. PRINS reactions were performed with alpha-satellite primers specific for either chromosomes 9 or chromosome 18. Double labeling was obtained and estimates of disomy rates were carried out for the three chromosomes.

Adult↗
PubMed1996

Double and triple in situ chromosomal labeling of human spermatozoa by PRINS.

The PRimed IN Situ (PRINS) labeling method allows rapid, specific detection of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for simultaneous in situ decondensation and denaturation of sperm nuclei. Using fluorochrome-labeled dNTPs in a sequential PRINS reaction, the direct detection of two or three distinct chromosomes must be performed within a timespan of 3 h. The method was tested with primers specific for chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11% to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that are indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rapid and efficient alternative to FISH for detecting nondisjunction in human sperm.

Aneuploidy↗
PubMed1996

Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in human sperm by using primed in situ labeling technique.

The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in <2 h. A total of 96,292 sperm nuclei were analyzed by two independent observers. The estimates of disomy were 0.31% for chromosome 8, 0.28% for chromosome 9, 0.28% for chromosome 13, 0.26% for chromosome 16, and 0.32% for chromosome 21. These homogeneous findings suggest an equal distribution of aneuploidies among autosomal chromosomes in males.

Adult↗
PubMed1995

Rapid in situ detection of chromosome 21 by PRINS technique.

The "PRimed IN Situ labeling" (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and interphase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

Base Sequence↗
PubMed1995

Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing.

WT1 is a tumor suppressor gene with a key role in urogenital development and the pathogenesis of Wilms' tumor. Two alternative splice sites in the WT1 transcript allow the gene to encode four proteins. These carry four Krüppel-type zinc fingers and to date have primarily been implicated in transcriptional control of genes involved in growth regulation. However, here we demonstrate colocalization of WT1 with splicing factors in the fetal kidney and testis and in expressing cell lines. Using immunoprecipitation, we show that two WT1 isoforms directly associate with one or a limited number of components in the spliceosomes and coiled bodies. Moreover, COS cell expression studies suggest that alternative splicing within the WT1 zinc finger region determines whether the protein localizes mainly with splicing factors or with DNA in transcription factor domains in the nucleus. We propose that WT1 plays roles in posttranscriptional processing of RNA as well as in transcription.

Alternative Splicing↗