PubMed Health⌕ Search

Biomedical subjects

F Pellestor

Publications and source records attributed to F Pellestor.

At least 19 recordsLinked to original sources

Rare Robertsonian translocations and meiotic behaviour: sperm FISH analysis of t(13;15) and t(14;15) translocations: a case report.

t(13;15) and t(14;15) are two rare Robertsonian translocations. Meiotic segregation was studied in four males heterozygous for the rare Robertsonian translocations t(13;15) and t(14;15). Both locus-specific probes (LSPs) and whole chromosome painting (WCP) probes, specific to chromosomes 13, 14 and 15, were used in this study. The number of spermatozoa scored for each carrier ranged from 891 to 5000. The frequencies of normal and balanced sperm resulting from the alternate mode of segregation ranged from 77.6 to 92.8%, confirming the prevalence of alternate segregation over other segregation modes in all Robertsonian translocations. The incidences of unbalanced complements ranged from 6.7 to 20.4%, with a significant excess of disomy rates over the complementary frequencies of nullisomy. This variability might reflect differences in the location of breakpoints in translocated chromosomes, leading to the variable production of unbalanced gametes and the variable alterations of semen parameters in Robertsonian translocation carriers.

Adult↗

Meiotic segregation of rare Robertsonian translocations: sperm analysis of three t(14q;22q) cases.

BACKGROUND: The t(14;22) remains one of the rare Robertsonian translocations observed in human, with an occurrence estimated at 1.2%. Three cases of rare Robertsonian translocation t(14;22) were investigated for meiotic segregation in sperm samples from male carriers using the fluorescent in situ hybridization (FISH) procedure. The three carriers included two men with an abnormal semen analysis and one with normal semen parameters. METHODS: Both locus-specific probes and whole-chromosome painting probes, specific for chromosomes 14 and 22, were used in this study. The number of spermatozoa scored for each probe set ranged from 3279 to 10,024. RESULTS: In the three carriers, similar frequencies, ranging from 78.53 to 81.76%, were found for normal and balanced spermatozoa resulting from alternate segregation. The total proportion of unbalanced spermatozoa resulting from adjacent modes of segregation ranged from 17.59 to 20.94%. CONCLUSION: This finding confirmed the predominance of alternate segregation over other segregation types in all Robertsonian translocations and indicates a higher production of imbalances in the t(14;22) than in most of the Robertsonian translocations previously analysed. This could be related to the variable location of breakpoints in Robertsonian translocations. This breakpoint diversity could also play a role in the differences in reproductive status observed in male carriers of Robertsonian translocations.

Adult↗

Multicolor PRINS and multicolor PNA.

Both PRimed IN Situ (PRINS) and Peptide Nucleic Acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures now constitute efficient alternatives to the conventional fluorescence in situ hybridization (FISH) procedure for in situ chromosome identification and aneuploidy detection. They present several advantages (specificity, speed, discriminating ability) that make them very attractive for a number of cytogenetic purposes. Multicolor PRINS and PNA protocols have been described for the specific identification of human chromosomes. Various applications have already been developed in human genetics and new adaptations are ongoing.

Chromosome Mapping↗

Comparison of male and female meiotic segregation patterns in translocation heterozygotes: a case study in an animal model (Sus scrofa domestica L.).

BACKGROUND: The comparison of male and female meiotic segregation patterns for individuals carrying identical reciprocal translocations has been rarely reported in mammalian species. The main comparative study involving males and females with comparable genetic background has been performed in the mouse. Swine is another relevant animal model species for meiotic studies. Here we present the segregation patterns determined for sows carrying one of the two following reciprocal translocations: 38, XX, rcp(3;15)(q27;q13), and 38, XX, rcp(12;14)(q13;q21). These segregation data were compared to those previously obtained for closely related boars carrying the same balanced chromosomal rearrangements. METHODS: Dual colour in situ hybridization of whole chromosome painting probes was carried out on metaphases of in vitro-matured oocytes II. Segregation results were obtained for 118 and 206 metaphases II respectively for the two translocations. RESULTS: Significant differences between sexes were demonstrated for both rearrangements. For instance, for the 3/15 translocation, the chromosomally unbalanced gametes were of different origin: preponderance of the adjacent-I segregation in the male (31.4%), and of the adjacent-II (14.3%) and 3:1 (14.3%) segregations in females. For the 12/14 translocation, the proportion of balanced gametes was greater in males than in females (75.9 and 59.4% respectively). CONCLUSION: This study is a new scientific contribution to compare the segregation patterns of male and female carriers of identical chromosomal rearrangements. The results obtained are consistent with those previously reported in mice. Hypotheses to interpret the observed differences between the two translocations, as well as between the male and female segregation patterns, are formulated and discussed.

Animals↗

Sperm segregation analysis of a (13;22) Robertsonian translocation carrier by FISH: a comparison of locus-specific probe and whole chromosome painting.

BACKGROUND: The t(13;22) Robertsonian translocation constitutes a rare form of rearrangement between acrocentric human chromosomes. Most of the meiotic segregation studies of human Robertsonian translocations have been performed on common t(13;14) and t(14;21) translocations. Analysis of the chromosomal constitution in sperm of Robertsonian translocation carriers is of great interest for assessing the risk of unbalanced forms and adapting genetic counselling. In the present study, we present the first meiotic segregation study of a t(13;22) Robertsonian translocation in human sperm. METHODS: A total of 11 787 sperm nuclei were scored using two distinct FISH labelling techniques, i.e. the locus-specific probes (LSI) method and the whole chromosome painting (WCP) technique. RESULTS: The frequency of normal or balanced sperm resulting from alternate meiotic segregation was 86%. Incidences of unbalanced complements resulting from adjacent segregation modes were 12.79% and 14.36% in LSI and WCP assays, respectively. No significant excess of nullisomy or disomy for the affected chromosomes was observed. CONCLUSIONS: Similar results in segregation were obtained with the two techniques, demonstrating the efficiency of the two strategies for the direct segregation analysis of Roberstsonian translocations. The results obtained indicated a moderate meiotic production of imbalance. This study shows that the rare Robertsonian translocation (13;22) displays a similar distribution of balanced and unbalanced sperm patterns as the common Robertsonian translocations previously studied. This suggests that the behaviour of acrocentric chromosomes was similar in all cases of centric fusion.

Adult↗

[The cytogenetics of human oocytes: 40 years of progress].

Chromosomal abnormalities account for the majority of pre- and post- implantation embryo wastage in humans. Most of these abnormalities result from maternal meiotic errors, which preferentially occur during the first meiotic division. Consequently, the cytogenetic analysis of human oocytes has then been considered as a highly valuable source of data for the investigation of both the occurrence and the origin of chromosomal abnormalities in human. During the last 4 decades, the cytogenetic analysis of human oocytes has never stopped progressing, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome non-disjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidy in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies.

Cell Division↗

Effect of maternal age on the frequency of cytogenetic abnormalities in human oocytes.

The cytogenetic investigation of human oocytes was initiated in the Sixties, and for the last four decades, this field of research has never stopped progressing as new technologies appear. Numerous karyotyping studies and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes, but also displaying a great variability in results, which may be essentially attributable to the technical limitations of these in situ methods when applied to human oocytes. Essentially, the most relevant analyses have led to the estimate that 15-20% of human oocytes display chromosome abnormalities, and they have emphasized the implication of both whole chromosome nondisjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidies has also been investigated in human oocytes. Most previous studies have failed to confirm any relationship between maternal age and aneuploidy frequency in human oocytes, whereas the more recent reports based on large samples of oocytes or polar bodies have provided evidence for a direct correlation between increased aneuploidy frequency and advanced maternal age, and have clarified the contribution of the various types of malsegregation in the maternal age-dependent aneuploidies.

Adult↗

The chromosomal analysis of human oocytes. An overview of established procedures.

The cytogenetic survey of mature human oocytes has been and remains a subject of great interest because of the prevalence of aneuploidy of maternal origin in abnormal human conceptuses, and the lack of understanding about the non-disjunction processes in human meiosis. The first attempts to analyse the chromosomal content of human female gametes were made in the early 1970s, and led to limited data because of the paucity of materials and the inadequacy of the procedure used. The years to follow brought a resurgence of interest in this field, because of the development of human IVF techniques which made oocytes unfertilized in vitro available for cytogenetic analysis. Numerous studies have since been performed. However, the difficulties in obtaining good chromosome preparations and of performing accurate chromosome identification have reduced the viability of these studies, resulting in large variations in the reported incidences of chromosomal abnormalities. The further introduction of new procedures for oocyte fixation and the screening of large oocyte samples have allowed more reliable data to be obtained and to identify premature chromatid separation as a major mechanism in aneuploidy occurrence. The last decade has been privileged to witness the adaptation of molecular cytogenetic techniques to human oocytes, and thus various powerful procedures have been tried not only on female gametes, but also on polar bodies, involving sequential and multicolour fluorescent in situ hybridization (FISH) labelling, comparative genomic hybridization (CGH), spectral karyotyping and alternative methods such as primed in situ labelling (PRINS) and peptide nucleic acid (PNA) techniques. A large body of data has been obtained, but these studies also display a great variability in the frequency of abnormalities, which may be essentially attributable to the technical limitations of these in situ methods when applied to human oocytes. However, molecular cytogenetic approaches have also evidenced the co-existence of both whole chromosome non-disjunction and chromatid separation in maternal aneuploidy. In addition, the extension of these techniques to oocyte polar body materials has provided additional data on the mechanism of meiotic malsegregation. Improvements of some of these techniques have already been reported. The further development of new approaches for the in situ analysis of human meiosis will increase the impact of cytogenetic investigation of human oocytes in the understanding of aneuploidy processes in humans.

Aneuploidy↗

The peptide nucleic acids: a new way for chromosomal investigation on isolated cells?

The development of nucleic acid analogues has become an important feature due to the potential use of this new biomolecular tool in genetic diagnostics and investigations. Among all the synthetic oligonucleotides designed, the peptide nucleic acids (PNA) constitute a remarkable class of nucleic acid mimics, with important physico-chemical properties which have been exploited to develop a wide range of powerful biomolecular tools, including molecular probes, biosensors and anti-gene agents. New applications of PNA involve their use as hybridization probes, and consequently the PNA technology is now developing within the field of in situ hybridization techniques. Recent studies have reported the successful use of centromeric PNA probes on human lymphocytes, sperm as well as on isolated oocytes and blastomeres. Muticolour PNA protocols have been described for the specific identification of several human chromosomes. These data show that PNA could become a powerful complement to fluorescence in situ hybridization (FISH) for in situ chromosomal investigation, especially on isolated cells. The present paper gives an overview of the properties of PNA and the assays exploiting PNA technology in molecular genetics and cytogenetics.

Animals↗

The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos.

Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we tested multicolour labelling PNA reaction on 27 oocytes and 23 blastomeres. Sequential PNA hybridization was performed on five oocytes and combined PNA and fluorescence in situ hybridization (FISH) reactions on two oocytes. Both the rates and the types of abnormalities observed are in agreement with results from previous FISH studies. This preliminary study indicates that PNA probes allow a reliable chromosomal analysis in isolated human oocytes and blastomeres and consequently might provide an interesting adjunct to FISH for diagnostic analysis.

Blastocyst↗

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting.

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.

Centromere↗

Ultra-rapid multicolor PRINS protocol for chromosome detection in human sperm.

We report a new multicolor PRINS procedure for chromosome identification on human sperm. Based on the direct in-situ mixing of the colors of the fluorochromes (FITC, TRITC, Cascade Blue) incorporated in sequential PRINS reactions, this method facilitates rapid distinct labeling of 3 or 4 chromosomes. Each PRINS reaction consists of a unique 4 minute step for annealing and elongation. The method was successfully tested on lymphocytes and spermatozoa. Estimates of disomy were performed for chromosomes 7, 9 and 16 on sperm samples from 2 healthy donors. There was no significant difference between the disomy rates obtained with the conventional two-color PRINS technique and this new three-color procedure. By simplifying the multicolor PRINS protocol, this new protocol should facilitate the use and adaptation of PRINS to various cytogenetic applications.

Chromosomes, Human, Pair 16↗

6p abnormalities and TNF-alpha over-expression in retinoblastoma cell line.

Retinoblastoma is the most common primary intra-ocular tumor in childhood. It has been established that recurrent cytogenetic abnormalities are the hallmark of mostly all malignant tumors. Recurrent atypical cytogenetic abnormalities of the short arm of chromosome 6 were reported in several cases of retinoblastoma, mainly in association with reciprocal translocations. In the present study, alterations of chromosome 6 associated with retinoblastoma were studies in the Y-79 and the WERI-Rb-1 cell lines established from highly malignant retinoblastomas. Fluorescence in situ hybridization (FISH) technique was used to identify the chromosomal breakpoint on 6p in this cell line. To perform this detection, yeast artificial chromosome (YAC) clones from p21 to p22 bands were used and the breakpoint was localized on 6p21.3. Previous studies had suggested that activation of some genes on 6p could be dependent on a translocation mechanism. Expression of a candidate gene localized near the chromosomal breakpoint was measured by immunocytochemistry and flow cytometry techniques. An enhancement of the tumor necrosis factor-alpha (TNF-alpha) protein expression in Y79 cells was detected by this approach. The relationship between TNF-alpha over-expression and the malignancy of retinoblastoma is discussed.

Blotting, Southern↗

Study of the occurrence of interchromosomal effect in spermatozoa of chromosomal rearrangement carriers by fluorescence in-situ hybridization and primed in-situ labelling techniques.

The possibility that a chromosomal rearrangement might disturb the meiotic behaviour of chromosomes not involved in the rearrangement and favour non-disjunction is a controversial issue in human cytogenetics. Using two-colour fluorescence in-situ hybridization and primed in-situ labelling techniques, we have investigated the segregation pattern of 10 chromosomes (chromosomes 1, 4, 9, 13, 15, 16, 20, 21, X and Y) in spermatozoa from nine carriers of balanced structural rearrangements and three normal men. The patients were divided into two groups according to their semen parameters. In rearrangement carriers and normal subjects, sex chromosomes and chromosome 21 displayed a higher rate of disomy than the other chromosomes. No evidence for the occurrence of interchromosomal effect was found in the spermatozoa of fertile rearrangement carriers, but significant variations were observed for all chromosomes tested in the group of infertile translocation carriers, suggesting a direct correlation between poor quality spermatozoa and increased aneuploidy rate in this group. In fertile carriers of chromosomal rearrangements, the occurrence of non-disjunction of chromosomes not involved in the rearrangement might therefore be considered as fortuitous, whereas in infertile carriers, the risk for interchromosomal effect appears to be real and should be taken into consideration in the genetic counselling of infertile couples with a male partner carrying a chromosomal rearrangement.

Adult↗

Spontaneous occurrence of a Robertsonian fusion involving chromosome 19 by single whole-arm reciprocal translocation (WART) in wild-derived house mice.

Chromosomal races of the house mouse (Mus musculus domesticus) bear Robertsonian (Rb) fusions, which consist of centric translocations between two non-homologous acrocentric chromosomes. The high level of diversity of these fusions in house mice is generated by de-novo formation of Rb fusions and subsequent whole-arm reciprocal exchanges (WARTs). This paper describes the spontaneous occurrence of a new Rb fusion, Rb(4.19), in progeny of wild-derived house mice segregating for Rb(4.12). The chromosomal mutation was traced to a female which exhibited germline and somatic mosaicism indicating an early embryonic origin of the mutation. FISH analysis of centromerically-located ribosomal genes suggested that no modification was observed on chromosomes 12 and 19 prior to or following the occurrence of Rb(4.19). Distribution of telomeric sequences showed that both Rb fusions lacked telomeres in their centromeric regions. It is argued that this spontaneous mutation most likely originated by single whole-arm reciprocal translocation (WART) between Rb(4.12) and an acrocentric chromosome 19, resulting in Rb(4.19) and a neo-acrocentric chromosome 12. Sequences required for centromeric function and proximal telomeres would have been transferred to the neo-chromosome 12 from chromosome 19 during the translocation. The existence of such WARTs which generate derived acrocentric chromosomes has several implications for chromosomal evolution in house mice.

Animals↗

Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single-sperm typing.

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.

Adult↗

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.

Aneuploidy↗