Molecular Cytogenetics - Most accessed articles

Human interphase chromosomes: a review of available molecular cytogenetic technologies

Svetlana Vorsanova — 2010-01-11T00:00:00Z

Human karyotype is usually studied by classical cytogenetic (banding) techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB). Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions) being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations). Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB) allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.

On the paternal origin of trisomy 21 Down syndrome

Maj Hulten — 2010-02-23T00:00:00Z

Background: Down syndrome (DS), characterized by an extra free chromosome 21 is the most common genetic cause for congenital malformations and learning disability. It is well known that the extra chromosome 21 originates from the mother in more than 90% of cases, the incidence increases with maternal age and there is a high recurrence in young women. In a previous report we have presented data to indicate that maternal trisomy 21 (T21) ovarian mosaicism might provide the major causative factor underlying these patterns of DS inheritance. One important outstanding question concerns the reason why the extra chromosome 21 in DS rarely originates from the father, i.e. in less than 10% of T21 DS cases. We here report data indicating that one reason for this parental sex difference is a very much lower degree of fetal testicular in comparison to ovarian T21 mosaicism. Results: We used fluorescence in situ hybridisation (FISH) with two chromosome 21-specific probes to determine the copy number of chromosome 21 in fetal testicular cell nuclei from four male fetuses, following termination of pregnancy for a non-medical/social reason at gestational age 14-19 weeks. The cells studied were selected on the basis of their morphology alone, pending immunological specification of the relevant cell types. We could not detect any indication of testicular T21 mosaicism in any of these four male fetuses, when analysing at least 2000 cells per case (range 2038-3971, total 11.842). This result is highly statistically significant (p<0.001) in comparison to the average of 0.54% ovarian T21 mosaicism (range 0.20-0.88%) that we identified in eight female fetuses analysing a total of 12.634 cells, as documented in a previous report in this journal. Conclusion: Based on these observations we suggest that there is a significant sex difference in degrees of fetal germ line T21 mosaicism. Thus, it would appear that most female fetuses are T21 ovarian mosaics, while in sharp contrast most male fetuses may be either very low grade T21 testicular mosaics or they may be non-mosaics. We further propose that this sex difference in germ line T21 mosaicism may explain the much less frequent paternal origin of T21 DS than maternal. The mechanisms underlying the DS cases, where the extra chromosome 21 does originate from the father, remains unknown and further studies in this respect are required.

8p23.1 duplication syndrome differentiated from copy number variation of the defensin cluster at prenatal diagnosis in four new families

John Barber — 2010-02-18T00:00:00Z

Background: The 8p23.1 duplication syndrome and copy number variation of the 8p23.1 defensin gene cluster are cytogenetically indistinguishable but distinct at the molecular level. To our knowledge, the 8p23.1 duplication syndrome has been described at prenatal diagnosis only once and we report our experience with four further apparent duplications ascertained at prenatal diagnosis. Methods: Additional material at band 8p23.1 was detected using conventional G-banded cytogenetics in each case. Multiplex Ligation-dependent Probe Amplification (MLPA) or Fluorescence In Situ Hybridisation (FISH) were used depending on whether only DNA (Cases 1 and 4) or cytogenetic preparations (Cases 2 and 3) were available from the laboratory of origin. The extent of the duplication in Case 1 was retrospectively determined using array Comparative Genomic Hybridisation (array CGH). Results: Three cases of 8p23.1 duplication syndrome were found (Cases 1 to 3). Two were de novo and continued to term and the third, a paternally transmitted duplication, was terminated because of a previous child with psychomotor delay and 8p23.1 duplication syndrome. Case 1 was ascertained with a hypoplastic left heart but the ventricular septal and interventricular defects, in Cases 2 and 3 respectively, were found after ascertainment for advanced maternal age. By contrast, case 4 was a maternally transmitted copy number variation of the defensin cluster with normal outcome. Conclusions: Our data underline the need to differentiate 8p23.1 duplications from copy number variation of the defensin cluster using FISH, MLPA or array CGH. Cardiac defects were ascertained by ultrasound in only one of the three duplication 8p23.1 pregnancies but were visible in two of the three at 21 to 22 weeks gestation. Our results provide further evidence that both deletion and duplication of the GATA4 transcription factor can give rise to a variety of conotruncal heart defects with variable penetrance and expressivity.

Detailed molecular and clinical investigation of a child with a partial deletion of chromosome 11 (Jacobsen syndrome)

Emmanouil Manolakos — 2009-12-09T00:00:00Z

Background: Jacobsen syndrome (JBS) is a rare chromosomal disorder leading to multiple physical and mental impairment. This syndrome is caused by a partial deletion of chromosome 11, especially subband 11q24.1 has been proven to be involved. Clinical cases may easily escape diagnosis, however pancytopenia or thrombocytopenia may be indicative for JBS. Results: We report a 7.5 years old boy presenting with speech development delay, hearing impairment and abnormal platelet function. High resolution SNP oligonucleotide microarray analysis revealed a terminal deletion of 11.4 Mb in size, in the area 11q24.1-11qter. This specific deletion encompasses around 170 genes. Other molecular techniques such as fluorescence in situ hybridization and multiplex ligation-dependent probe amplification were used to confirm the array-result.DiscussionOur results suggest that the identification and detailed analysis of similar patients with abnormal platelet function and otherwise mild clinical features will contribute to identification of more patients with 11q deletion and JBS.

Two siblings with immunodeficiency, facial abnormalities and chromosomal instability without mutation in DNMT3B gene but liability towards malignancy; a new chromatin disorder delineation?

Anna Polityko — 2010-03-08T00:00:00Z

Background: ICF syndrome (standing for Immunodeficiency, Centromere instability and Facial anomalies syndrome) is a very rare autosomal recessive immune disorder caused by mutations of the gene de novo DNA-methyltransferase 3B (DNMT3B). However, in the literature similar clinical cases without such mutations are reported, as well. Results: We report on a family in which the unrelated spouses had two female siblings sharing similar phenotypic features resembling ICF-syndrome, i.e. congenital abnormalities, immunodeficiency, developmental delay and high level of chromosomal instability, including high frequency of centromeric/pericentromeric rearrangements and breaks, chromosomal fragments despiralization or pulverization. However, mutations in DNMT3B could not be detected. Conclusion: The discovery of a new so-called "chromatin disorder" is suggested. Clinical, molecular genetic and cytogenetic characteristics are reported and compared to other "chromatin disorders".

Autistic disorder associated with a paternally derived unbalanced translocation leading to duplication of chromosome 15pter-q13.2: a case report

David Wu — 2009-12-18T00:00:00Z

Autism spectrum disorders have been associated with maternally derived duplications that involve the imprinted region on the proximal long arm of chromosome 15. Here we describe a boy with a chromosome 15 duplication arising from a 3:1 segregation error of a paternally derived translocation between chromosome 15q13.2 and chromosome 9q34.12, which led to trisomy of chromosome 15pter-q13.2 and 9q34.12-qter. Using array comparative genome hybridization, we localized the breakpoints on both chromosomes and sequence homology suggests that the translocation arose from non-allelic homologous recombination involving the low copy repeats on chromosome 15. The child manifests many characteristics of the maternally-derived duplication chromosome 15 phenotype including developmental delays with cognitive impairment, autism, hypotonia and facial dysmorphisms with nominal overlap of the most general symptoms found in duplications of chromosome 9q34. This case suggests that biallelically expressed genes on proximal 15q contribute to the idic(15) autism phenotype.

Male and female meiotic behaviour of an intrachromosomal insertion determined by preimplantation genetic diagnosis

Leoni Xanthopoulou — 2010-02-08T00:00:00Z

Background: Two related family members, a female and a male balanced carrier of an intrachromosomal insertion on chromosome 7 were referred to our centre for preimplantation genetic diagnosis. This presented a rare opportunity to investigate the behaviour of the insertion chromosome during meiosis in two related carriers. The aim of this study was to carry out a detailed genetic analysis of the preimplantation embryos that were generated from the three treatment cycles for the male and two for the female carrier.Patients underwent in vitro fertilization and on day 3, 22 embryos from the female carrier and 19 embryos from the male carrier were biopsied and cells analysed by fluorescent in situ hybridization. Follow up analysis of 29 untransferred embryos was also performed for confirmation of the diagnosis and to obtain information on meiotic and mitotic outcome. Results: In this study, the female carrier produced more than twice as many chromosomally balanced embryos as the male (76.5% vs. 36%), and two pregnancies were achieved for her. Follow up analysis showed that the male carrier had produced more highly abnormal embryos than the female (25% and 15% respectively) and no pregnancies occurred for the male carrier and his partner. Conclusion: This study compares how an intrachromosomal insertion has behaved in the meiotic and preimplantation stages of development in sibling male and female carriers. It confirms that PGD is an appropriate treatment in such cases. Reasons for the differing outcome for the two carriers are discussed.

Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy

Lucia Moraes — 2009-10-07T00:00:00Z

Background: Pentasomy X (49,XXXXX) has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent) cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype. Results: 5-bromodeoxyuridine (BrdU)-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P), indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the HUMANDREC region of the androgen receptor (AR) gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 AR allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT) allowed for the identification of three maternal X haplotypes (M1, M2 and MR) and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation. Conclusion: Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype) were likely to be functionally active in the proband's cells, a finding indicating that disruption of X inactivation was associated to her severe phenotype.

On the origin of trisomy 21 Down syndrome

Maj Hulten — 2008-09-18T00:00:00Z

Background: Down syndrome, characterized by an extra chromosome 21 is the most common genetic cause for congenital malformations and learning disability. It is well known that the extra chromosome 21 most often originates from the mother, the incidence increases with maternal age, there may be aberrant maternal chromosome 21 recombination and there is a higher recurrence in young women. In spite of intensive efforts to understand the underlying reason(s) for these characteristics, the origin still remains unknown. We hypothesize that maternal trisomy 21 ovarian mosaicism might provide the major causative factor. Results: We used fluorescence in situ hybridization (FISH) with two chromosome 21-specific probes to determine the copy number of chromosome 21 in ovarian cells from eight female foetuses at gestational age 14–22 weeks. All eight phenotypically normal female foetuses were found to be mosaics, containing ovarian cells with an extra chromosome 21. Trisomy 21 occurred with about the same frequency in cells that had entered meiosis as in pre-meiotic and ovarian mesenchymal stroma cells. Conclusion: We suggest that most normal female foetuses are trisomy 21 ovarian mosaics and the maternal age effect is caused by differential selection of these cells during foetal and postnatal development until ovulation. The exceptional occurrence of high-grade ovarian mosaicism may explain why some women have a child with Down syndrome already at young age as well as the associated increased incidence at subsequent conceptions. We also propose that our findings may explain the aberrant maternal recombination patterns previously found by family linkage analysis.

FISH mapping of Philadelphia negative BCR/ABL1 positive CML

Anna Virgili — 2008-07-18T00:00:00Z

Background: Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder, almost always characterized by the presence of the Philadelphia chromosome (Ph), usually due to t(9;22)(q34;q11) or its variants. The Ph results in the formation of the BCR/ABL1 fusion gene, which is a constitutively activated tyrosine kinase. Around 1% of CML patients appear to have a Ph negative karyotype but carry a cryptic BCR/ABL1 fusion that can be located by fluorescence in situ hybridisation (FISH) at chromosome 22q11, 9q34 or a third chromosome. Here we present FISH mapping data of BCR and ABL1 flanking regions and associated chromosomal rearrangements in 9 Ph negative BCR/ABL1 positive CML patients plus the cell line CML-T1. Results: BCR/ABL1 was located at 9q34 in 3 patients, 22q11 in 5 patients and CML-T1 and 22p11 in 1 patient. In 3 of 6 cases with the fusion at 22q11 a distal breakpoint cluster was found within a 280 Kb region containing the RAPGEF1 gene, while in another patient and the CML-T1 the distal breakpoint fell within a single BAC clone containing the 3' RXRA gene. Two cases had a duplication of the masked Ph while genomic deletions of the flanking regions were identified in 3 cases. Even more complex rearrangements were found in 3 further cases. Conclusion: BCR/ABL1 formation resulted from a direct insertion (one step mechanism) in 6 patients and CML-T1, while in 3 patients the fusion gene originated from a sequence of rearrangements (multiple steps). The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in Ph negative BCR/ABL1 positive CML.