Am J Hum Genet 28:417-419, 1976
Cytogenetic Darkroom Magic: Now You See Them, Now You Don't KATHLEEN M. OVERTON,1 R. ELLEN MAGENIS, THOMAS BRADY, JAN CHAMBERLIN, AND MAUREEN PARKS INTRODUCTION
We have undertaken a long term study of the no. 21 chromosomes in Down syndrome to determine the stage of meiosis at which the error occurs and to elicit the parental origin of the three chromosomes. There have been a number of papers on this subject [1-5], although some report difficulties due to lack of chromosome 21 "markers." Knowing the frustration involved in this type of study, we decided to report our darkroom technique. Using this technique, 22 out of 24 families were informative. METHODS
Modified standard techniques were utilized for culturing and harvesting the cells [6]. The fluorochrome, quinacrine mustard, was used for chromosome banding and characterization of the variable short arm and satellite regions of the no. 21 chromosomes [7]. Kodak TriX pan 35 mm film was used; cells were photographed with a Zeiss photomicroscope II equipped with a fluorescent illuminator (HBO 200), a BG 12 exciter filter, and a barrier filter 53. Reflected light was utilized with a darkfield condenser with a 1.2/1.4 aperture. The automatic exposure control was set at 400 ASA and a din of 27. To obtain optimal information from the quinacrine-stained short arm and the satellite regions, serial prints were made of each 21 in each metaphase. To make prints we used a Durst M35 enlarger (with the lens aperture, in most cases, set at f 5.6) and AgfaGevaert FPI contrast 4 enlarging paper covered by a card in which a small slit had been cut (a 1 cm X 5 cm, depending on the desired enlargement). Using a red filter, the chromosome was centered in this slit on one end of the photographic paper. The filter was removed, and the paper exposed for approximately 2 seconds. The paper was then shifted about 1.5 cm under the card to uncover an unexposed section; this section was exposed for 3 seconds. The paper was moved again, followed by a 4 second exposure. This process was repeated with successive exposures increasing usually by 1 second increments until the last strip of paper had been exposed for 9 seconds. The actual exposure time will vary with the density of the negative. It is important, however, that the series cover a range from underexposed to overexposed. This is similar to the process employed in obtaining test prints to determine proper exposures. After the photographic paper was exposed, it was developed in an Agfa-Gevaert rapidoprint LD37. Utilizing this procedure, Received January 23, 1976. This work was supported in part by grants from the National Institute of Child Health and Human Development (HD 07997) and Maternal and Child Health Services (920). 1A11 authors: Child Development and Rehabilitation Center, Division of Medical Genetics, Department of Pediatrics, University of Oregon Health Sciences Center, Portland, Oregon. i 1976 by the American Society of Human Genetics. All rights reserved.
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the entire chromosome can be examined rather than only the banding pattern, the short arm, or the satellite region. Generally, the printing exposure best for the banding pattern of the long arm will be inadequate for analyzing the short arm or satellite. Examples of this are shown in figure 1 in which the exposures are arranged from left to right in order of decreasing exposure times (i.e., longest to shortest). Figure 1A exemplifies a chromosome 21 with bright "marker" satellites which obviously did not need this technique to expose them. Figure lB represents the few individuals found with no. 21
I
on~~~~~~~~~~
FIG. 1.-A, A chromosome 21 with bright satellites at eight different exposures (decreasing from left to right); B, eight different exposures of a chromosome 21 with no satellites; C, eight different exposures of a chromosome 21 with satellites visible only at shorter exposure times; D, eight different exposures of a chromosome 21 with satellites visible only at shorter exposure times; E, eight different exposures of a chromosome 21 with visible but less discretely defined satellites, at shorter exposure times; F, eight different exposures of a chromosome 21 with visible but less discretely defined satellites, at shorter exposure times.
CYTOGENETIC DARKROOM MAGIC
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chromosomes showing no satellite development at any exposure; this proved to be a very useful, heritable marker. Most of the "markers" that we worked with fell somewhere between these two extremes. Figures 1C and 1D illustrate the lack of satellite development until the 4th or 5th exposure. The intensity necessary to bring out these satellites was not satisfactory for evaluating the banding pattern in the long arm of these no. 21 chromosomes; in fact, at this intensity the chromosome 21 banding pattern could not be distinguished from that of chromosome 22. Therefore, the exposure for chromosomes in a karyotype (9-10 seconds) will be quite different from that needed to analyze the short arm, satellite region (2-3 seconds). Satellites from different individuals with a similar morphology may also need different exposures (i.e., 1-2 second exposures may be good for one, while another may take 5-6 seconds). This variability was useful when both parents had satellites of similar morphology, because the exposure time needed to demonstrate the satellites (1-2 vs. 5-6 second exposures) was inherited by the child. So, although the morphology is similar, the intensity is not. Figure 1E and F show other types of satellite development which are enhanced by this sequential printing. These are not well-defined satellites but appear "fuzzy." This variation was useful in designating the parental origin of these chromosomes in the families studied here. SUMMARY
Customary procedures used to determine chromosomal inheritance do not disclose many of the chromosomal polymorphisms known to be present, resulting in uninformative families. The sequential printing of individual chromosomes presented here is a technique that has increased the number of informative families in our studies. This technique has revealed previously unseen heritable chromosome differences and allowed the designation of parental origin. ACKNOWLEDGMENTS We would like to thank Patricia Evans for her assistance in the typing and editing of this article, and Douglas Hepburn for his invaluable help in preparation of the illustration. 1. 2. 3. 4.
5. 6. 7.
REFERENCES ROBINSON JA: Origin of extra chromosome in trisomy 21. Lancet 1:131-133, 1973 SMITH GF, SHANTA S: Origin of extra chromosome in trisomy 21. Lancet 1:487, 1973 PUNNETT HH, KISTENMACHER ML: The origin of the extra chromosome in trisomy 21. Genetics (Suppl. 2) 74:S222, 1973 SCHMIDT R, DAR H, NITOWSKY HM: Origin of extra 21 chromosome in patients with Down syndrome. Pediatr Res 9:318, 1975 BOTT CE, SEKHON GS, LUBs HA: Unexpected high frequency of paternal origin of trisomy 21. Am J Hum Genet 27:20A, 1975 MOORHEAD PS, NOWELL PC, MELLMAN WJ, BATTIPS DM, HUNGERFORD DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613-616, 1960 CASPERSON T, LOMAKKA G, ZECH L: The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability. Hereditas 67:89-102, 1971
Cytogenetic Darkroom Magic: Now You See Them, Now You Don't KATHLEEN M. OVERTON,1 R. ELLEN MAGENIS, THOMAS BRADY, JAN CHAMBERLIN, AND MAUREEN PARKS INTRODUCTION
We have undertaken a long term study of the no. 21 chromosomes in Down syndrome to determine the stage of meiosis at which the error occurs and to elicit the parental origin of the three chromosomes. There have been a number of papers on this subject [1-5], although some report difficulties due to lack of chromosome 21 "markers." Knowing the frustration involved in this type of study, we decided to report our darkroom technique. Using this technique, 22 out of 24 families were informative. METHODS
Modified standard techniques were utilized for culturing and harvesting the cells [6]. The fluorochrome, quinacrine mustard, was used for chromosome banding and characterization of the variable short arm and satellite regions of the no. 21 chromosomes [7]. Kodak TriX pan 35 mm film was used; cells were photographed with a Zeiss photomicroscope II equipped with a fluorescent illuminator (HBO 200), a BG 12 exciter filter, and a barrier filter 53. Reflected light was utilized with a darkfield condenser with a 1.2/1.4 aperture. The automatic exposure control was set at 400 ASA and a din of 27. To obtain optimal information from the quinacrine-stained short arm and the satellite regions, serial prints were made of each 21 in each metaphase. To make prints we used a Durst M35 enlarger (with the lens aperture, in most cases, set at f 5.6) and AgfaGevaert FPI contrast 4 enlarging paper covered by a card in which a small slit had been cut (a 1 cm X 5 cm, depending on the desired enlargement). Using a red filter, the chromosome was centered in this slit on one end of the photographic paper. The filter was removed, and the paper exposed for approximately 2 seconds. The paper was then shifted about 1.5 cm under the card to uncover an unexposed section; this section was exposed for 3 seconds. The paper was moved again, followed by a 4 second exposure. This process was repeated with successive exposures increasing usually by 1 second increments until the last strip of paper had been exposed for 9 seconds. The actual exposure time will vary with the density of the negative. It is important, however, that the series cover a range from underexposed to overexposed. This is similar to the process employed in obtaining test prints to determine proper exposures. After the photographic paper was exposed, it was developed in an Agfa-Gevaert rapidoprint LD37. Utilizing this procedure, Received January 23, 1976. This work was supported in part by grants from the National Institute of Child Health and Human Development (HD 07997) and Maternal and Child Health Services (920). 1A11 authors: Child Development and Rehabilitation Center, Division of Medical Genetics, Department of Pediatrics, University of Oregon Health Sciences Center, Portland, Oregon. i 1976 by the American Society of Human Genetics. All rights reserved.
417
418
OVERTON ET AL.
the entire chromosome can be examined rather than only the banding pattern, the short arm, or the satellite region. Generally, the printing exposure best for the banding pattern of the long arm will be inadequate for analyzing the short arm or satellite. Examples of this are shown in figure 1 in which the exposures are arranged from left to right in order of decreasing exposure times (i.e., longest to shortest). Figure 1A exemplifies a chromosome 21 with bright "marker" satellites which obviously did not need this technique to expose them. Figure lB represents the few individuals found with no. 21
I
on~~~~~~~~~~
FIG. 1.-A, A chromosome 21 with bright satellites at eight different exposures (decreasing from left to right); B, eight different exposures of a chromosome 21 with no satellites; C, eight different exposures of a chromosome 21 with satellites visible only at shorter exposure times; D, eight different exposures of a chromosome 21 with satellites visible only at shorter exposure times; E, eight different exposures of a chromosome 21 with visible but less discretely defined satellites, at shorter exposure times; F, eight different exposures of a chromosome 21 with visible but less discretely defined satellites, at shorter exposure times.
CYTOGENETIC DARKROOM MAGIC
419
chromosomes showing no satellite development at any exposure; this proved to be a very useful, heritable marker. Most of the "markers" that we worked with fell somewhere between these two extremes. Figures 1C and 1D illustrate the lack of satellite development until the 4th or 5th exposure. The intensity necessary to bring out these satellites was not satisfactory for evaluating the banding pattern in the long arm of these no. 21 chromosomes; in fact, at this intensity the chromosome 21 banding pattern could not be distinguished from that of chromosome 22. Therefore, the exposure for chromosomes in a karyotype (9-10 seconds) will be quite different from that needed to analyze the short arm, satellite region (2-3 seconds). Satellites from different individuals with a similar morphology may also need different exposures (i.e., 1-2 second exposures may be good for one, while another may take 5-6 seconds). This variability was useful when both parents had satellites of similar morphology, because the exposure time needed to demonstrate the satellites (1-2 vs. 5-6 second exposures) was inherited by the child. So, although the morphology is similar, the intensity is not. Figure 1E and F show other types of satellite development which are enhanced by this sequential printing. These are not well-defined satellites but appear "fuzzy." This variation was useful in designating the parental origin of these chromosomes in the families studied here. SUMMARY
Customary procedures used to determine chromosomal inheritance do not disclose many of the chromosomal polymorphisms known to be present, resulting in uninformative families. The sequential printing of individual chromosomes presented here is a technique that has increased the number of informative families in our studies. This technique has revealed previously unseen heritable chromosome differences and allowed the designation of parental origin. ACKNOWLEDGMENTS We would like to thank Patricia Evans for her assistance in the typing and editing of this article, and Douglas Hepburn for his invaluable help in preparation of the illustration. 1. 2. 3. 4.
5. 6. 7.
REFERENCES ROBINSON JA: Origin of extra chromosome in trisomy 21. Lancet 1:131-133, 1973 SMITH GF, SHANTA S: Origin of extra chromosome in trisomy 21. Lancet 1:487, 1973 PUNNETT HH, KISTENMACHER ML: The origin of the extra chromosome in trisomy 21. Genetics (Suppl. 2) 74:S222, 1973 SCHMIDT R, DAR H, NITOWSKY HM: Origin of extra 21 chromosome in patients with Down syndrome. Pediatr Res 9:318, 1975 BOTT CE, SEKHON GS, LUBs HA: Unexpected high frequency of paternal origin of trisomy 21. Am J Hum Genet 27:20A, 1975 MOORHEAD PS, NOWELL PC, MELLMAN WJ, BATTIPS DM, HUNGERFORD DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613-616, 1960 CASPERSON T, LOMAKKA G, ZECH L: The 24 fluorescence patterns of the human metaphase chromosomes-distinguishing characters and variability. Hereditas 67:89-102, 1971
