Full Karyotype Interphase Cell Analysis.

Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.

Loss of CHFR in human mammary epithelial cells causes genomic instability by disrupting the mitotic spindle assembly checkpoint.

CHFR is an E3 ubiquitin ligase and an early mitotic checkpoint protein implicated in many cancers and in the maintenance of genomic stability. To analyze the role of CHFR in genomic stability, by siRNA, we decreased its expression in genomically stable MCF10A cells. Lowered CHFR expression quickly led to increased aneuploidy due to many mitotic defects. First, we confirmed that CHFR interacts with the mitotic kinase Aurora A to regulate its expression. Furthermore, we found that decreased CHFR led to disorganized multipolar mitotic spindles. This was supported by the finding that CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and the amount of acetylated alpha-tubulin, a component of the mitotic spindle. Finally, we found a novel CHFR interacting protein, the spindle checkpoint protein MAD2. Decreased CHFR expression resulted in the mislocalization of both MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation. Further evidence of a compromised spindle checkpoint was the presence of misaligned metaphase chromosomes, lagging anaphase chromosomes, and defective cytokinesis in CHFR knockdown cells. Importantly, our results suggest a novel role for CHFR regulating chromosome segregation where decreased expression, as seen in cancer cells, contributes to genomic instability by impairing the spindle assembly checkpoint.

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Aneuploidy involving chromosome 1 in failed-fertilized human oocytes is unrelated to maternal age.

PURPOSE: To study whether maternal meiotic errors in failed-fertilized oocytes involving chromosome 1 occur at frequencies similar to those involving other autosomes, and whether their frequency is affected by maternal age. METHODS: Using fluorescence in situ hybridization (FISH), frequencies of aneusomy and chromatid pre-division involving chromosomes 1, 16, 18, and 21 were determined for 273 failed-fertilized oocytes. RESULTS: The aneuploidy rate for chromosome 1 was 15.8%, and was neither age-dependent nor significantly different from that for chromosomes 16, 18 or 21. Only chromosome 16 exhibited an age-dependent increase in aneusomy rates. The frequency of chromatid pre-division was lower for chromosome 1 than for chromosome 18 (11.9% vs. 25.4%; p = 0.01), but not different from that for chromosomes 16 or 21. CONCLUSION: Aneuploidy involving chromosome 1 in failed-fertilized oocytes is unrelated to maternal age and occurs at a frequency similar to that for chromosomes 16, 18, and 21.

Recombination in men with Klinefelter syndrome.

Klinefelter syndrome (KS: 47,XXY), occurs in one in 1000 male births. Men with KS are infertile and have higher rates of aneuploidies in sperm compared with normal fertile men. In the course of analyzing recombination in a population of infertile men, we observed that four men in our study presented with KS. We examined whether these men differed in recombination parameters among themselves and relative to normal men. Even though the number of men with KS analyzed was small, we observed remarkable variation in spermatogenesis. In spite of the fact that the men had the same genetic cause for infertility, two of four KS patients had few or no spermatogenic cells that progressed through meiosis to the pachytene stage, whereas the other two men produced abundant pachytene cells that had recombination frequencies comparable with those of fertile men, although one had a significant reduction in fidelity of synapsis. Moreover, regardless of histological appearance, examination of outcomes of assisted reproduction indicated that sperm were extracted from testis biopsies in all four cases, and when used in assisted reproductive practices chromosomally normal babies were born. These results reinforce that: (i) men with the same underlying genetic cause for infertility do not present with uniform pathology, (ii) the checkpoint machinery that might arrest spermatogenesis in the face of chromosomal abnormalities does not prevent pockets of complete spermatogenesis in men with KS, and (iii) aneuploidy, in some cases, is compatible with birth of a chromosomally normal child, suggesting that sperm produced from a background of aneuploidy can be normal in men with KS.

Trisomy 21 is associated with variable defects in cytotrophoblast differentiation along the invasive pathway.

Aneuploid cells in the placenta are associated with poor pregnancy outcomes, but the mechanisms are unclear. Here, we examined the cytotrophoblast (CTB) differentiation pathway that leads to uterine invasion in pregnancies complicated by trisomy 21 (T21) as compared with their normal counterparts. Surprisingly, we observed a wide spectrum of T21 effects. Morphologically, some samples appeared near normal, while others had extensive fibrinoid deposition and apoptosis of CTBs at the maternal-fetal interface (confirmed by TUNEL labeling). At a molecular level, the cells' expression of stage-specific molecules was variably misregulated. At one end of the spectrum, samples with less apoptosis had relatively normal staining patterns. At the other end, samples with extensive apoptosis showed significantly decreased staining for these antigens. Additional studies confirmed that the effects we observed had functional consequences, because the cells exhibited marked phenotypic alterations in vitro, including a large increase in MMP-9 production, which distinguishes the effects of T21 on CTBs from those of preeclampsia. The morphologic, phenotypic, and functional differences among CTBs from pregnancies complicated by T21 illustrate the importance of the interplay between fetal/placental genotype and maternal influences on pregnancy outcome. Furthermore, our data may explain why a significant number of these pregnancies end in spontaneous abortion while others survive to term.