Detection of Structural and Numerical Chromosome Abnormalities in Interphase Cells Using Spectral Imaging

Because cells available for PGD analysis are likely to be in interphase, we set out to develop rapid procedures for chromosome enumeration and detection of structural abnormalities based on hybridization of chromosome-specific probes and spectral imaging detection. The following describes our typical procedure to prepare case-specific probe for detection of structural chromosome alterations in interphase cells. Institutional Review Board Approval was obtained for this study. Breakpoint-spanning probes were prepared for a carrier of a balanced reciprocal translocation, 46,XY,t(3;4)(p24;p15). We selected YAC clones based on STS markers previously mapped to the approximate breakpoint regions and information provided in the database of the Whitehead Institute for Biomedical Research/MIT Center for Genome Research (Hudson et al. 1995). We prepared DNA probes from entire yeast+YAC colonies. The hybridization, washes, and detection followed our previously described protocol (Cassel et al. 1997; Fung et al. 1998b). Hybridizing selected probes to the carrier's metaphase spreads enabled us to rapidly classify clones as mapping either proximal or distal to the respective breakpoint. Repeated cycles of clone selection were guided by results from previous cycles and mapped additional clones into increasingly smaller intervals. Once the final probe sets composed of YAC clones spanning the respective breakpoints were defined, probes were optimized and tested on interphase cells. To find YAC clones spanning a breakpoint, it normally took two to five cycles of selection and cytogenetic mapping. In this case, 21 YAC clones for chromosome 3 were selected in five cycles and 14 YACs for chromosome 4 in four cycles. Three YAC clones (955b5, 958b5, and 872h5) spanned the breakpoint on chromosome 3p24 and two YAC clones (967c5 and 853c4) spanned the breakpoint on chromosome 4p15. The probe for chromosome 3 was labeled with fluorescein-12-dUTP (green) and the probe mix for chromosome 4 was labeled with digoxigenin-11-dUTP and detected with a rhodamine-conjugated anti-digoxigenin antibody (red). Hybridization results on patient lymphocytes showed one chromosomal target with green fluorescence (normal chromosome 3) while another signal domain appeared red (normal chromosome 4). At the same time, the der(3) and the der(4) were marked with red–green fusion signals. Using YAC as probes spanning or flanking translocation breakpoints, we have been able to demonstrate the utility of our technique in more than 10 additional PGD cases (Munné et al. 1998a,b). The technology can also be utilized to delineate breakpoints for the position cloning of disease-related genes.

For detection of numerical chromosome abnormalities in interphase cells using spectral imaging, we developed a 10-chromosome probe set (chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, Y). The chromosomal targets in this set were chosen to detect abnormalities responsible for more than 70% of spontaneous abortions caused by monosomies or trisomies (Hassold and Jacobs 1984). Spectral imaging analysis combining the techniques of fluorescence optical microscopy, charged-coupled device imaging, Fourier spectroscopy, and software for digital image analysis has been applied by specific staining of all 24 human chromosomes in metaphase spreads, termed “spectral karyotyping” (SKY) (Garini et al. 1996; Schröck et al. 1996). In our study, we applied the spectral imaging technique for interphase cell analysis. To simultaneously detect 10 chromosomes in interphase cells, a reference spectra library file and a combinatorial table were first built by analyzing a normal metaphase spread with known chromosome identities. Once a combinatorial table was built, we hybridized the probe set on three different types of human interphase cells. Three cells (lymphocytes, uncultured amniocytes, blastomeres) were fixed slightly differently to achieve the desired detection sensitivity and reproducibility (Fung et al. 1998a, 2000). To avoid misinterpretation by the spectral imaging system, the hybridization targets must not overlap. Therefore, the specimen preparation received special attention. Ideally, the cells are flat and in the form of a DNA monolayer. On the other hand, most interphase cells, especially uncultured amniocytes, showed elevated levels of background fluorescence compared to lymphocyte metaphases. Because the spectrum of background fluorescence differed from the reference spectra, it could easily be distinguished and subtracted using the spectral imaging software. The interphase nuclei from lymphocytes hybridized with our probes demonstrated good hybridization efficiency and were karyotyped as being normal. Overlapping signal domains became a problem in uncultured amniocytes, in which only about 20% of all cells showed interpretable spreads. All blastomeres were obtained from abnormally developing embryos donated for research. Written patient consent was obtained in all cases. The cells were fixed individually in acetic acid–methanol and spread very well. About 50% of the cells showed clearly interpretable hybridization results, and most of them were karyotyped as abnormal.

In summary, spectral imaging now allows the enumeration of 10 chromosome types in individual interphase cells. Combining the breakpoint-spanning probes with the chromosome enumeration probes will create a very efficient PGD assay for patients carrying balanced translocations. This is expected to significantly increase the success rates in IVF cycles and the chances for affected couples to conceive healthy babies.