Chromosome Rearrangements in Human-Chinese Hamster Somatic Cell Hybrids J

Am J Hum Genet 27:595-608, 1975

Localization of Human Gene Loci Using Spontaneous Chromosome Rearrangements in Human-Chinese Hamster Somatic Cell Hybrids J. L. HAMERTON,' T. MOHANDAS,' PHYLLIS J. MCALPINE,' AND G. R. DOUGLAS1"2

INTRODUCTION Human-rodent somatic cell hybrids have been utilized extensively for human gene assignments (see [1, 2] for review). The use of hybrid lines originating from the fusion of human cells carrying chromosome structural changes with rodent cells has allowed the assignment of gene loci to specific chromosome regions [3-5]. Regional localization of gene loci can also be achieved using spontaneously derived or induced structural alterations in the human chromosomes in human-rodent hybrid cell lines [6-9]. The use of methods similar to those used for mapping Drosophila polytene chromosomes (see [10] for review) allows hybrid cell lines carrying chromosome rearrangements to be used to assign gene loci to specific chromosome bands. We report here the regional and subregional localization of gene loci on human chromosomes 2, 12, and X using structural rearrangements of the human chromosomes which originated spontaneously in human-Chinese hamster somatic cell hybrids. These rearrangements include deletion, intraspecific translocation, and interspecific translocation.

MATERIALS AND METHODS Hybrid line 1610 was derived from fusion between a mutant Chinese hamster cell line (CHW-1102) deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) [11] and a strain of human diploid fibroblasts (DCF) derived from a male fetus. Lines 1705 and 4105 were derived from fusion of CHW-1102 with leukocytes from two different human donors. Methods for the production, propagation, cloning, and cytological analysis of hybrid lines have already been described [7, 12, 13]. The cytological and biochemical analyses of each hybrid line and clone were performed on cells at the same passage level or the closest passage levels possible. Cytological analyses were carried

Received October 29, 1974; revised February 21, 1975. This work was supported by grant MA-4061 from the Medical Research Council of Canada and by the Children's Hospital Research Foundation, Winnipeg. T. Mohandas and G. R. Douglas were the recipients of postdoctoral fellowships from the Medical Research Council. 1 Division of Genetics, Department of Paediatrics, University of Manitoba, Winnipeg, Mani- toba. Address reprint requests to J. L. Hamerton, Department of Genetics, Health Sciences Centre-Children's Centre, 685 Bannatyne Avenue, Winnipeg, Manitoba, R3E OW1, Canada. 2 Present address: Human Development Division, Department of Health and Welfare, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario. o 1975 by the American Society of Human Genetics. All rights reserved. 595 596 HAMERTON ET AL. out by T. Mohandas and G. R. Douglas and enzyme analyses by P. J. McAlpine. After both sets of results were available, the data were pooled and analyzed for gene assignments. Lysates of the lines and clones were examined electrophoretically for the following enzyme markers by the methods indicated: red cell acid phosphatase (AcP1) [14]; cytoplasmic isocitrate dehydrogenase (IDH1) [15]; peptidase B (Pep B) [16]; glucose- 6-phosphate dehydrogenase (G6PD) [17]; phosphoglycerate kinase (PGK) [18]; a galactosidase (a-gal) [19]; hypoxanthine guanine phosphoribosyltransferase (HPRT) [20]; and peptidase A (Pep A) [16]. Cellogel (Chemetron, Italy) with 0.1 M Tris- citrate pH 8.6 buffer was used for the electrophoretic examination of lactate dehydrogenase (LDH) and NAD-dependent cytoplasmic malate dehydrogenase (MDH1). Iso- zymes were visualized with slight modifications of previously published methods [21, 22]. The nomenclature used to describe chromosome rearrangements is a modification of that proposed by the Paris Conference [23]. The prefixes Hs and Cg placed before the chromosome number indicate human and Chinese hamster chromosomes, respectively.

RESULTS Cytological and Biochemical Analyses on Hybrids Carrying Rearrangements In- volving Chromosome 2 An interspecific presumptive reciprocal translocation between human chromosome 2 and Chinese hamster chromosome 9, t(Hs2;Cg9)(qi1;?), subsequently referred to as t(a), was identified in a secondary clone (1610-09-51) of hybrid line 1610. This rearrangement had presumably arisen after cloning, since cells were observed with a normal intact chromosome 2, with t(a), and with a deleted form of t(a), del[t(a)(Hs2q22)], referred to subsequently as delt(a)l (fig. 1). Sixty-three tertiary clones derived from 1610-09-51 could be separated into four phenotypic classes (table 1) on the basis of presence or absence of the genetic markers AcP1, MDH1, and IDH1 previously assigned to chromosome 2 [24-29]. Twenty-two clones representing each of the four classes were analyzed cytologically and the results correlated with their biochemical phenotypes (table 2). In addition to an intact chromosome 2, t(a), and delt(a) , a second deleted form of the translocation, del [t(a) (Hs2q24)], referred to subsequently as delt(a)2, was identified in tertiary clone 51-68. One tertiary clone (51-02) carried both products of the translocation, t(a) and del(Hs2)(ql1), and thus appeared to carry all the chromosome 2 material (fig. 1). Another tertiary clone (51-19) carried a 2p deletion, del(Hs2)(p23), and a translocation of this deleted segment to Cg9, resulting in a dicentric product tdic(Hs2;Cg9)(p23;?) referred to subsequently as tdic(b) (fig. 2). The data presented in table 2 show that in the presence of an intact human chromosome 2, the human forms of the genetic markers AcP1, IDH1, and MDH1 were expressed. In the absence of an intact human chromosome 2 or when a re- arranged chromosome 2 of type t(a), delt(a) , or delt(a)2 was present, the human form of these three markers was not expressed. Tertiary clone 51-02, which carried both t(a) and del(Hs2) (ql 1) and thus, within the limits of resolution, was balanced with respect to chromosome 2 material, expressed the human MDH1 and AcPj gene loci but not the IDH1 locus. Tertiary clone 51-19, which carried IL.

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597 598 HAMERTON ET AL. TABLE 1 BIOCHEMICAL ANALYSIS OF TERTIARY CLONES DERIVED FROM SECONDARY CLONE 1610-09-51

HUMAN MARKERS

PHENOTYPIC CLASS I DH1 MDH1 AcP1 No. SUBCLONES

I...... + + + 52 I...... - - - 9 III...... + + 1 IV ...... + - + 1

NOTE.-+ = presence of human marker; -=absence. del(Hs2) (p23) and tdic(b) and was therefore deficient for the 2pter->2p23 region, expressed the human loci IDH1 and AcP1 but not MDH1. Cytological and Biochemical Analyses on Hybrids Carrying Rearrangements In- volving Chromosomes 12 and X Hybrid line 1705 initially had human chromosomes 5, 12, 18, and X and expressed the human chromosome 12 markers LDH B and Pep B, the chromosome 18 marker Pep A, and the human X markers a-gal, PGK, HPRT, and G6PD. When this line was cloned, clone 1705-40 carried a translocation between the human X chromosome and human chromosome 12, t(X;12)(q24;q21) (fig. 3). Twenty-four secondary clones from 1705-40 could be separated into two phenotypic classes with respect to the human enzyme markers (table 3). Six secondary clones, three from each phenotypic class, were analyzed cytologically and all (table 4) carried the derivative chromosome Xpter->Xq24::12q21- >12qter. These clones had lost both the human chromosome 12 markers LDH B and Pep B and the human X chromosome marker G6PD. The remaining X chromosome loci (a-gal, PGK, and HPRT) were expressed in all secondary clones. A deleted human chromosome 12, del(I 2) (q2 1) (fig. 3), with a breakpoint in the same band as the breakpoint in the t(X;12)(q24;q21) was identified in hybrid line 4105. This line was thus deficient for the 12q2l1->12qter region of chromosome 12 and expressed the human LDH B locus but not the human Pep B locus. Hybrid line 4105 also carried a deleted human X chromosome, del(X) (p21), but expressed the human chromosome markers a-gal, PGK, HPRT, and G6PD. Three secondary clones derived from 1705-40 which expressed human Pep A had human chromosome 18, while the three that did not express human Pep A did not have human chromosome 18.

DISCUSSION Localization of Markers on Chromosome 2 The IDH1 and MDH1 human gene loci have been shown to be syntenic [24] and have been assigned to chromosome 2 [26-29]. The AcP1 locus has also been ++++++++++++IllI

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a b c FIG. 2.-Diagrams and photomicrographs of interspecific chromosome rearrangement in tertiary clone 1610-09-51-19. General description as in fig. 1. a, Normal human chromosome 2; arrow indicates breakpoint. b, Normal Chinese hamster chromosome 9. c, Deleted human chromosome 2, del(Hs2)(p23). d, Translocation between deleted human chromosome 2 and Chinese hamster chromosome 9, tdic(b) = t(Hs2 ;Cg9) (Cg9qter-- Cg9pter?: :Hs2p23-oHs2qter). assigned to chromosome 2 [25, 28, 30, 31]. On the basis of the segregation of a t(2;5)(p23;q31) translocation with the apparent hemizygous expression of the AcP1 locus in the proband carrying the der(2), Ferguson-Smith et al. [25] provided evidence that AcPj is located in the 2p23->2pter segment. The data presented here show that when a normal human chromosome 2 was present, the human loci IDH1, MDH1, and AcP1 were expressed. In the absence of chromosome 2 and, in particular, when the 2q1 -->2pter region was missing, these three loci were not expressed, indicating their assignment to the 2q1 1-2pter region. Our data therefore confirm the assignment of the AcP, locus to human chromosome 2 and provide evidence for the regional assignment of these three loci. One clone carried the t(Hs2;Cg9) (qll;?) translocation as well as a del(Hs2) (qll) chromosome and thus was effectively balanced for chromosome 2 material. This ;t50) o >,o

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601 602 HAMERTON ET AL. TABLE 3 BIOCHEMICAL ANALYSIS OF SECONDARY CLONES DERIVED FROM PRIMARY CLONE 1705-40

HUMAN MARKERS No. PHENOTYPIC CLASS a-gal PGK HPRT G6PD LDH B Pep B Pep A SUBCLONES

I ...... + + + - - - + 19 II ...... + + + - - - - 5

NOTE.- + = presence of human marker; - = absence. clone expressed the human MDH1 and AcP1 loci but not the human IDH1 locus, indicating that the MDH1 and AcP1 loci are on 2p while the IDH1 locus is on 2q, at or adjacent to the breakpoint at 2q11. Tertiary clone 51-19 carried the del (Hs2) (p23) and the tdic(Hs2; Cg9) (p23;?) and expressed human IDH1 and AcP1 loci but not the human MDH1 locus, which can thus be assigned to the 2p23->2pter region. The breakpoint at 2p23 in tertiary clone 51-19 appears to be in an identical position to the breakpoint in chromosome 2 in the t(2;5) translocation studied by Ferguson-Smith et al. [25]. The human AcP1 locus was, however, expressed in the person carrying the der(2) resulting from the t(2;5) [25]. Thus in one instance, the break at 2p23 left the AcP1 locus functional, whereas in the other, the break rendered the locus nonfunctional. These observations suggest that the human AcP1 locus lies at or adjacent to 2p23, and in some way this gene locus was affected by one chromosome break but not the other.

Localization of Markers on Chromosome 12 LDH B and Pep B have been shown to be syntenic [32, 33 ], and both loci have been assigned to chromosome 12 [34]. A recent report by Mayeda et al. [35] based on a study of a patient with a 46,XX,12p- karyotype indicates that the LDH B locus is located on 12p. Analysis of secondary clones derived from 1705-40 indicated that neither human LDH B nor Pep B were expressed in the presence of the derivative chromosome Xpter->Xp24::12q21->12pter, which suggests that the LDH B and Pep B loci can be assigned to the 12q21-> 1 2pter region. This is consistent with the results of Mayeda et al. [35]. Line 4105, which carried a long arm deletion of chromosome 12, del(12)(q21), and thus carried that part of the chromosome which is deficient in the 1705 secondary clones, expressed the human LDH B locus but not the human Pep B locus. Thus the human Pep B locus which was not expressed in either line, although between them they appear to carry the whole of chromosome 12, must have been rendered nonfunctional by breakage at 12q21, indicating that the Pep B gene locus must be located at or adjacent to this point. The expression of human LDH B in line 4105 and its absence in 1705-40 secondary clones is consistent with the assignment of this locus to the short arm of chromosome 12, as suggested by Mayeda et al. [35]. + +++ I

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603 604 HAMERTON ET AL. Localization of Markers on X Chromosome The G6PD, HPRT, PGK, and a-gal loci have all been assigned to the X chromosome by segregation analysis in families [36]. X linkage of HPRT, PGK, G6PD, and a-gal has also been demonstrated using somatic cell hybrids [37-40]. Grzeschik et al. [3], studying human-mouse hybrids carrying a human t(X; 14) translocation, postulated that HPRT and G6PD were on the short arm while PGK was on the long arm of the X chromosome. Ricciuti and Ruddle [4], however, using other human-mouse hybrids carrying the same t(X;14) translocation, found PGK, HPRT, and G6PD were on the long arm of the X chromosome. The results presented here from the clones of 1705-40 show that when there was a deficiency of the Xq24->qter region, the human loci PGK, HPRT, and a-gal were expressed whereas G6PD was not. Thus G6PD must be located distal to Xq24 and the other three loci, PGK, HPRT, and a-gal, proximal to that band or on the short arm. Line 4105, which carried a deleted X chromosome, del(X)(p21), expressed the human X chromosome markers a-gal, PGK, HPRT, and G6PD; combining these results allows the human gene loci a-gal, PGK, and HPRT to be localized to the Xp2l1-Xq24 region. Our results for these X chromosome loci are consistent with all observations reported to date [4, 5, 41] except those of Grzeschik et al. [3]. Assignment of Pep A to Chromosome 18 Since the human Pep A gene locus was expressed in the tertiary clones derived from 1705-40 only when human chromosome 18 was present (table 4), these data are in agreement with earlier work [42, 43] assigning this gene locus to chromosome 18. The methods described here for the localization of human gene loci to specific bands are analogous to those which are well established for the mapping of Drosophila polytene chromosomes (reviewed in [10, 44]). In Drosophila it has been shown in the case of Notch that "breaks not only within, but also imme- diately to the right as well as to the left of the band involved can be associated with inactivation of the unit in question" [10]. Furthermore Beerman [10] points out that "in view of the relatively gross structural effect that can be caused by chromosome breakage, especially by X-rays, this type of experiment does not carry beyond the statement that a given band (including both adjacent bands) is most likely to be the site of the genetic function under study." The methods used here for the assignment of gene loci to specific bands are based upon the following: (1) the expression and the nonexpression of a gene locus in independent hybrid clones carrying apparently identical deletions of chromosome material; or (2) the nonexpression of a gene locus which has been assigned to a chromosome, even in the presence of effectively all of that chromosome, when it has been involved in a structural rearrangement with no detectable loss of chromosome material. These methods imply that in one instance a chromosome break has resulted either in the deletion of the locus in question or in its being rendered nonfunctional, whereas in the alternative situation an apparently iden- LINKAGE IN SOMATIC CELL HYBRIDS 605 tical break has left the locus functional, thus implying its location at or adjacent to the breakpoint. Owing to the crudity of the present banding techniques, break- points located in the same band may and, in fact, are likely to be at different points in the DNA helix, so that we may be dealing with the excision of all or part of the gene locus or operon under study. Regional localization of gene loci to a chromosome band can also be made by correlating the loss of a specific gene function with the deletion of a chromosome band. German and Chaganti [45] have recently assigned the MN blood group locus to 2q14 in a patient who carried a derivative chromosome 2 in which band 2q14 had been deleted and who was hemizygous at the MN locus. In contrast, there is no evidence of any loss of chromosome material in the structural rearrangements described here. The localization of human loci IDH1 to 2q11, AcP1 to 2p23, Pep B to 12q21 (fig. 4), and the regional assignment of MDH1 to 2p23->2pter should be con-

}LDH B PGK ~~~fl.uPeP B6P~~~~ PRT

2 12 X FIG. 4.-Regional localization of gene loci on human chromosomes 2, 12, and X. sidered provisional since evidence for each of these assignments comes from a single independent clone or line. The presence of the relevant chromosome structural alterations in only one clone or line in each case demonstrates the limitations of using spontaneously derived chromosome mutations in somatic cell hybrids. The provisional assignment of AcP1 to 2p23 illustrates that combining data from patients carrying chromosome rearrangements in vivo with in vitro studies of somatic cell hybrids can provide more information than could otherwise be ob- tained by either approach.

SUMMARY Analysis of human-Chinese hamster somatic cell hybrids with spontaneously derived chromosome structural changes has provided data for the regional and subregional localization of gene loci which have previously been assigned to human chromosomes 2, 12, and X. Correlation of the expression of human gene loci with 606 HAMERTON ET AL. the human chromosome complements present in somatic cell hybrids indicates that the cytoplasmic malate dehydrogenase (MDH1) locus is in the 2p23-+2pter region, and red cell acid phosphatase (AcP,) is at or adjacent to 2p23. The cytoplasmic isocitrate dehydrogenase (IDH1) locus is at or adjacent to 2q11, peptidase B (Pep B) is at or adjacent to 12q21, lactate dehydrogenase B (LDH B) is in the 12q2 1-12pter region, glucose-6-phosphate dehydrogenase (G6PD) is in the Xq24-*Xqter region, and the gene loci for phosphoglycerate kinase (PGK), a-galactosidase (a-gal), and hypoxanthine guanine phosphoribosyltransferase (HPRT) are in the Xp2l->Xq24 region. 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