Working with Canine Chromosomes: Current Recommendations for ...

Working with Canine Chromosomes: Current Recommendations for Karyotype Description N. Reimann, S. Bartnitzke, I. Nolte, and J. Bullerdiek

There is an increasing interest in genomic research on the domestic dog (Canis familiaris). However, these investigations are complicated by the canine karyotype comprising 76 acrocentric autosomes of similar size and shape and the metacentric sex chromosomes. None of the numerous published ideograms and karyotypes has yet been generally accepted. The present article gives a review of these descriptions of the canine karyotype. The two most recent nomenclatures and the current efforts toward a standardized canine karyotype made by the Committee for the Standardized Karyotype of the Dog are discussed in detail and recommendations for future use of a nomenclature for the canine karyotype are given.

From the Center for Human Genetics and Genetic Counselling, University of Bremen, Leobener Str. ZHG, D28359 Bremen, Germany (Reimann, Bartnitzke, and Bullerdiek) and the Clinic for Small Animals, School of Veterinary Medicine, Hannover, Germany ( Nolte). This work was supported by a grant from the Deutsche Forschungsgemeinschaft. Address correspondence to Dr. J. Bullerdiek at the address above. This paper was delivered at the International Workshop on Canine Genetics at the College of Veterinary Medicine, Cornell University, Ithaca, New York, July 12–13, 1997. q 1999 The American Genetic Association 90:31–34

Many of the genetic diseases, including cancer, known to occur in dogs (Canis familiaris) are counterparts of human genetic diseases. Comparative approaches aimed at the characterization of genetic diseases, gene mapping, and gene therapy have much to offer for the benefit of both dogs and humans. Thus there has been an increasing interest in genomic research on the dog. International projects aimed at producing a high-quality genetic map for the dog genome have been established ( Dog Genome Project, DogMap). However, this research is complicated by the dog karyotype. The canine karyotype is one of the most difficult karyotypes among the mammalian species. It consists of 76 acrocentric autosomes which are all similar in size and shape and the metacentric X and Y chromosome. The largest autosome is almost equal in length to the X chromosome and the Y chromosome is the shortest of the complement. A standardized nomenclature for the complete canine karyotype comparable to that of the human karyotype ( ISCN 1995) or the standard systems of the karyotypes of laboratory mice (Committee on Standardized Genetic Nomenclature for Mice 1972), laboratory rats (Committee for a Standardized Karyotype of Rattus norvegicus 1973), and cattle, sheep, and goat ( ISCNDA 1989) does not yet exist.

Description of the Canine Karyotype: A Historical Review During the last 100 years several reports about the description of the karyotypic

pattern of the dog have been published. In the very early reports the diploid number ranged from 50 to 78 (Ahmed 1941; Minouchi 1927; Painter 1925; Rath 1894). Because of the development of new technologies ( hypotonic treatment of the cultured cells; Hsu and Pomerat 1953) a diploid chromosome number of 78 was determined for Canis familiaris (Chiarelli 1966; Gustavsson 1964; Hare et al. 1965). Further development of various chromosomal banding techniques led to a more precise description of the canine karyotype. Table 1 summarizes the published ideograms for the canine G-banded chromosomes. The first description of the Gbanding pattern was published by Selden et al. (1975). In this report an ideogram with 331 G bands per haploid set was established. One year later Manolache et al. (1976) presented a schematic representation with 230 G bands and a different arrangement of the chromosomes. Moreover, they described the Q- and C-banding pattern of the canine karyotype. The next ideogram for canine G-banded chromosomes was proposed by Fujinaga et al. (1989). In this ideogram the arrangement of the chromosomes followed Manolache et al. (1976) and the number of G bands per haploid set was 228. This report included the N-banding pattern of the karyotype of the dog. A further ideogram for canine chromosomes was produced by Stone et al. (1991) using a cell synchronization technique. In their schematic representation 327 bands were given, while the arrangement of the chromosomes fol-

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Table 1. Published ideograms for canine G-banded chromosomes

Author (year)

Number of bands per haploid set

Chromosome arrangement according to Additional information

Selden et al. (1975) Manolache et al. (1976)

331 230

Fujinaga et al. (1989)

228

Manolache et al. (1976)

Stone et al. (1991)

327

Selden et al. (1975)

Graphodatsky et al. (1995) 460 Reimann et al. (1996)

460


Switonski et al. (1996)

235 for 21 chromosome pairs 1X1Y


lowed Selden et al. (1975). Graphodatsky et al. (1995) were the next to publish an ideogram. In this report 460 bands were numbered and characteristic landmarks were described. The alignment of the chromosomes followed none of the previous reports. In addition, ideograms for R-banded chromosomes have repeatedly been described as well, each with different chromosome arrangements and numbers of bands ( Howard-Peebles and Pryor 1980; Poulson et al. 1990). In summary, none of the published ideograms has been generally accepted. Two very recent proposals are described in detail below and recommendations concerning their future application are given.

Q banding C banding Q banding C banding N banding Q banding C banding NOR banding Landmarks Numbered bands Centromere position Landmarks Numbered bands G and R banding comparison Landmarks Numbered bands

An Extended Nomenclature of the Canine Karyotype A major disadvantage of the published ideograms and karyotypes was that the problem of the orientation of the acrocentric chromosomes was not solved. Moreover, most of the proposed ideograms were initially deduced from more than one karyotype (Graphodatsky et al. 1995; Selden et al. 1975). Consequently, the relative sizes of the chromosomes do not fit each other. In addition, in all reports a comparison between G and R bands is missing. In 1996 we published a study in which we tried to overcome these problems (Reimann et al. 1996a). A combined GTG-banding/fluorescence in situ hybridization

( FISH) approach using an alpha-satellite probe specific for canine chromosomes ( Fanning 1989) resulted in the detection of positive signals in all canine autosomes allowing us to determine the centromere position of each canine chromosome ( Figure 1). With this approach we not only solved the problem of the correct orientation of the autosomes, but we also detected polymorphisms for the centromeric regions of chromosome 12 and 37. The centromeric regions of both sex chromosomes were lacking signals with the same molecular probe, leading us to conclude that the centromeric alphoid regions of the sex chromosomes differ widely from those of the autosomes in the canine karyotype. For our proposed ideogram, no synchronization procedures or other techniques to obtain high-resolution chromosomes were applied. Cytogenetic investigations of fibroblasts of a female mixed breed revealed the best results in terms of band resolution and one of these metaphases ( Figure 2) was basically used for our ideogram with 34 characteristic landmarks and 460 numbered bands in order to maintain the correct size relations of the chromosomes. We arranged our chromosomes according to Selden et al. (1975), because it is the most commonly used nomenclature for the canine karyotype thus also facilitating the comparison between past, present, and future cytogenetic investigations in dogs. Figure 3 shows a comparison of the ideogram by Selden et al. (1975) and

Figure 1. Results of the combined GTG-banding/FISH approach with an alpha satellite probe specific for canine chromosomes: (a) prebanded GTG-banded metaphase; (b) same metaphase after FISH.

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Figure 2. Karyogram of the canine GTG-banded metaphase which was used as the basis for the proposed ideogram. To complete the karyotype a Y chromosome was taken from a different metaphase.

our schematic representation of the canine chromosomes. The ideogram was overchecked by comparing its banding pattern with several metaphases from different dogs. In previous studies we have analyzed more than 1000 metaphases from 100 different dogs (e.g., Bartnitzke et al. 1992a,b; Reimann et al. 1994, 1996b). Our study also provides a direct comparison between GTG bands and RBG bands on canine chromosomes. Not unexpectedly, when comparing two sets of karyotypes, differences in chromosome morphology and banding patterns are obvious. Nevertheless, the correspondence of the two banding patterns is approximately 80%, thus enabling the use of the

same arrangement of the chromosomes, regardless of whether R or G bands have been demonstrated.

Committee for the Standardized Karyotype of the Dog During the 11th European Colloqium on Cytogenetics of Domestic Animals, held in Copenhagen, Denmark, in 1994, the Committee for the Standardized Karyotype of the Dog, with members from six different laboratories, was established. The first approach of this group was to perform a comparison test aimed at the question how many chromosome pairs could be recognized unequivocally with the use of

G-banding techniques on mid-metaphase chromosomes. For this purpose, each laboratory sent one metaphase to the other laboratories, where it was karyotyped. The results allowed standardization of Gband patterns of the first 21 autosomes and the sex chromosomes (Switonski et al. 1996). It was foreseen that the identification of the remaining 17 autosome pairs based on this resolution of bands would require the integration of molecular cytogenetic methods. Only just recently, chromosome paints specific for canine chromosomes were developed by Langford et al. (1996). In their report the paints for the first 21 autosomes and the sex chromosomes were assigned according to the Standard Committee, for the remaining 17 autosome pairs the assignment was still missing. It was decided that the assignment of the paints to these chromosomes should be carried out by the members of the Standard Committee, thus integrating the paints into the standardization procedure. Therefore, during the second stage of the standardization of the canine karyotype, combined banding/FISH studies with chromosome paints were carried out by the different laboratories of the committee. The results of these investigations led to assignment of the remaining paints. The identified chromosomes 22–38 were arranged according to Selden et al. (1975). As for the description of characteristic landmarks and numbered bands, it was decided to adapt the ideogram described by Reimann et al. (1996a). A report about the results of the second stage of the Standard Committee for the Canine Karyotype is in preparation.

Conclusion

Figure 3. Comparison between the schematic representation for canine chromosomes by Selden et al. (1975) ( left) and by Reimann et al. (1996a) (middle). The chromosomes on the right side derived from the karyotype shown in Figure 2.

There is an increasing interest in working with canine chromosomes. On the one hand there are the international projects ( Dog Genome Project, DogMap) which include physical gene mapping, and on the other hand there are scientists analyzing the genetic background of canine diseases and cancer. However, these studies have been complicated by the rather difficult situation concerning the description of the canine karyotype. The aim of this report is to prevent this confusion. Thus, depending on the type of study and the resolution of bands on the chromosomes, basically two nomenclatures should be used in the future: the one described by the Standard Committee, and the one described by Reimann et al. (1996a). When working with mid-metaphase

Reimann et al • Working with Canine Chromosomes 33

chromosomes it is recommended that the nomenclature of the Standard Committee be used. However, in these cases the application of FISH with the painting probes specific for canine chromosomes will be essential for the identification of the smaller autosome pairs (chromosomes 22–38). A description of landmarks and bands for the small chromosomes of these metaphases is lacking from the Standard Committee. Studies dealing with chromosomes showing a higher resolution of bands, for example, tumor cytogenetic investigations and gene mapping studies, should refer to the nomenclature of Reimann et al. (1996a). These recommendations are supported by the Standard Committee. Recommendations concerning the use of a nomenclature for the description of chromosomal aberrations in the canine karyotype have not yet been made. We recommend that in these cases the ISCN (1995) should be used as a reference.

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Corresponding Editor: Gregory M. Acland