Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 263-267, January 1975

Tay-Sachs' and Sandhoffs Diseases: The Assignment of Genes for Hexosaminidase A and B to Individual Human Chromosomes (human genetics/somatic cell genetics/lipid storage diseases/GM2 gangliosidoses) F. GILBERT* t §, R. KUCHERLAPATI*, R. P. CREAGAN*, M. J. MURNANE*, G. J. DARLINGTON*, AND F. H. RUDDLE* t t * Department of Biology, Yale University, Kline Biology Tower, New Haven, Connecticut 06520; and t Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Victor A. McKusick, June 6, 1974

ABSTRACT The techniques of somatic cell genetics sult from a defect in the gene coding for the basic Hex protein. have been used to establish the linkage relationships The second theory proposes that Hex A and B are each of loci coding for two forms (A and B) of hexosaminidase (EC 3.2.1.30; 2-acetamido-2-deoxy-B-D-glucoside acet- composed of multiple subunits, one of which is common to amidodeoxyglucohydrolase) and to determine whether a both forms (6). In this hypothesis, TSD would result from the structural relationship exists between these forms. In a deficiency of the Hex A-specific subunit and SD from the series of human-mouse hybrid cell lines, hexosaminidase deficiency of the common subunit. It is also possible that the A and B segregated independently. Our results and those A and B reported by other investigators are used to analyze the two forms of Hex are not structurally related. Hex proposed structural models for hexosaminidase. We have may be controlled by two independent genes. TSD would then also been able to establish a syntenic relationship between result from an effective deficiency of the normal Hex A struc- the gene locus responsible for the expression of hexosami- tural gene product and SD might result from a mutation in a nidase A and those responsible for mannosephosphate locus controlling expression of both enzymes or required for isomerase and pyruvate kinase-3 and to assign the gene for hexosaminidase B to chromosome 5 in man. Tbere is their activation. thus a linkage between specific human autosomes and A series of human-mouse hybrid cell lines were examined for enzymes implicated in the production of lipid storage the expression of Hex activity to determine whether a struc- diseases. tural relationship does in fact exist between Hex A and B. The lipid storage diseases are a family of inherited disorders Such interspecific hybrid cell lines, which preferentially characterized by the excessive accumulation of sphingolipids segregate the chromosomes of one parent in the cross (in this in the body's tissues. In each, the metabolic derangement ap- instance, the human), have already proved useful in the as- pears to be the result of a deficiency of a specific lysosomal signment of genes for specific enzymes to individual chromo- hydrolase which is involved in the catabolism of these complex somes in man. However, their potential value as a tool for the lipids (1). One of these enzymes, P-N-acetylglucosaminidase study of enzyme structure has not yet been fully appreciated. (Hex; EC 3.2.1.30) is thought to be responsible for at least two We have found that, in this series of hybrid cells, human Hex lipodystrophies, Tay-Sachs' disease (TSD; GM2 gangliosidosis, A and B are expressed independently. type I) and Sandhoff's disease (SD; GM2 gangliosidosis, type The hybrid cells were also used to establish the linkage II). When examined electrophoretically, this enzyme is found relationships of genes coding for Hex A and B. The human to exist in multiple forms, two of which (Hex A and B) have chromosome complements and patterns of expression of a been well characterized biochemically (2). A third form of the series of isozymes with known chromosome assignments were enzyme (Hex C), about which relatively little is known, has compared with the retention of Hex A and B activity in these recently been described (3). TSD is associated with a defi- clones. On the basis of these studies we were able to assign the ciency of Hex A and an increased activity of Hex B, and SD locus involved in the expression of Hex B to human chromo- is associated with a deficiency of both Hex A and B (4, 5). some 5 and to establish a syntenic relationship between genes No individual has yet been reported in whom Hex A is present coding for Hex A, mannosephosphate isomerase, and pyruvate in the absence of Hex B. kinase-3. Biochemical, genetic, and immunological evidence suggests MATERIALS AND METHODS that a structural relationship exists between Hex A and B. Two theories concerning this relationship have recently been Hexosaminidase Assay. The assay for hexosaminidase activ- advanced (2, 6). The first proposes that Hex A is a conversion ity is a modification of the published procedures of Okada product of Hex B (2). TSD would then result from the de- and O'Brien, and van Someren and van Henegouwen (4, 8). ficiency of a functional conversion enzyme, and SD would re- The cell homogenates were prepared as described (9). Electro- phoresis was performed (at 40) on cellulose acetate gel (Cello- Abbreviations: Hex, hexosaminidase; TSD, Tay-Sachs' disease; gel: Chemetron, Milan, Italy) in a citrate phosphate buffer SD, Sandhoff's disease. (25 mM, pH 5.6) for 3 hr at 250 V. The gel was incubated § Present address: Department of Human Genetics, University with the artificial substrate, 4-methyl umbelliferyl-N-acetyl-,3- of Pennsylvania School of Medicine, Philadelphia, Pa. 19104. glucosaminide [Pierce Chemical Co.; 1.5 mM in 0.5 M Na t To whom reprint requests should be addressed. citrate (pH 4.0) and 1% agarose] for 1-2 hr and then exposed 263 Downloaded by guest on September 28, 2021 264 Genetics: Gilbert et al. Proc. Nat. Acad. Sci. USA 72 (1975)

(4) TABLE 1. Enzymes studied and their human chromosome asszgnments* Human Enzyme chromosome

-4*- C Dipeptidase-1 (PEP C) 1 Malate dehydrogenase (NAD +) (MDH-1) 2 Isocitrate dehydrogenase (NADP+) (IDH-1) 2 Malic enzyme (NADP+) (MOD) 6 Indophenol oxidase (SOD-2) 6 -_- A Mannosephosphate isomerase (MPI) 15 Pyruvate kinase (PK) 15 Glutamate-oxaloacetate transaminase (GOT) 10 Esterase A4 (Es-A4) 11 0 Lactate dehydrogenase-A (LDH-A) 11 Lactate dehydrogenase-B (LDH-B) 12 -B Tripeptidase-1 (PEP B) 12 Nucleoside phosphorylase (NP) 14 Adenine phosphoribosyltransferase (APRT) 16 18 1 2 3 4 5 6 (H) Dipeptidase-2 (PEP A) Glucosephosphate isomerase (GPI) 19 FIG. 1. Hexosaminidase activity on cellulose acetate. Cell Adenosine deaminase (ADA) 20 extracts were applied to cellulose acetate and assayed as de- Indophenol oxidase (SOD-1) 21 scribed in the text. The development time of the gel was chosen Phosphoglycerate kinase (PGK) X to allow optimal visualization of the Hex A and B bands. Four Glucose 6-phosphate dehydrogenase (G6PD) X bands are evident-corresponding to human Hex A, Hex B, and to two mouse activities, one at the origin and a second migrating * See ref. 9. coincidentally with human Hex C. Slot 1, hybrid with human Hex A and B; slot 2, hybrid with Hex B alone; slot 3, hybrid Heat Inactivation. Cell homogenates of the human and with Hex A alone; slot 4, hybrid with no human Hex; slot 5, mouse fibroblast parents and of hybrid lines from both series mouse parent; slot 6, human parent. The differences in intensity A and B were heated for three hours at 500. The heat-in- of Hex A and B between cell lines possibly reflect quantitative differences in the amounts of material applied to each slot. Hex activated samples as well as untreated controls were then C activity (mouse and/or human) was clearly apparent in all analyzed for hexosaminidase activity by cellulose acetate gel samples, though in some (slots 2, 3, and 6) it was faint and electrophoresis. Hex A activity has been reported to be thermo- thus was reproduced poorly in the photograph. Some variation labile while Hex B activity is thermostable. in the intensity of the Hex C in each cell line has been noted The slight difference in mobility of Hex C in Toxin Treatment. Human, mouse, and hybrid somatic cells between gels. as slots 4 and 5 possibly reflects differences in the age of each sample. were subjected to various concentrations of diphtheria toxin described (R. P. Creagan, S. Chen, and F. H. Ruddle, manu- script in preparation). to ammonia vapor before it was made visible under ultra- violet light. RESULTS The electrophoretic patterns of the multiple forms of Hex in Other Enzymes. Twenty enzymes with established mouse the human and mouse parental cell lines and in selected and human electrophoretic differences and known human hybrid clones are illustrated in Fig. 1. The human cell lines chromosome assignments (Table 1) were also assayed by demonstrate the previously described three-band pattern cor- methods detailed by Nichols and Ruddle (9). responding to Hex A, B, and C. The mouse cell line has one Production of Hybrids. Series A: A number of primary distinct band which migrates to a position coincident with the hybrid clones were established, using techniques outlined, human Hex C and a second band at the origin. A mixture of after the fusion of a mouse cell line (A9) lacking the enzyme homogenates of the parent lines (human and mouse), when hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assayed for Hex, demonstrates all four bands. with two diploid human fibroblast lines (GM 17 and Yoder, Two series of human-mouse hybrid clones (series A and B) obtained from the Institute for Medical Research, Camden, were examined for the presence of Hex A and B. All of the N.J. and Dr. D. Borgaonkar, Johns Hopkins University clones retained enzymatic activity in the Hex C region that School of Medicine, respectively) and isolation in the HAT could be of human and/or mouse origin. Cell homogenates of selection system (10). the mouse and human fibroblast parents and of several hybrid Series B: This represents a number of primary and second- clones from both series A and B (three of which were Hex ary hybrid clones resulting from the fusion of several different A+/B+, one that was Hex-/B+, and three that were Hex human diploid fibroblast and leukocyte lines with two HGP- A+/B+) were subjected to heat inactivation and analyzed for RT- mouse cell lines (A9 and RAG) and established through hexosaminidase activity by gel assay. Heat inactivation re- the use of the methods described above. The human chromo- sulted in the loss of Hex A activity in all of the lines in which it some complements were analyzed as the cell pellets were was present prior to treatment. Hex B activity was unaffected prepared (R. Creagan and F. H. Ruddle, unpublished data). by the heat treatment. The distribution of clones retaining An average of over 25 metaphases per clone was examined. both Hex A and B, either Hex A or B alone, or neither, is Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Tay-Sachs' and Sandhoff's Diseases 265 TABLE 2. Hexosaminidase activity in human-mouse TABLE 4. Segregation of Hex B with specific human hybrid clones chromosome

Hexosaminidase activity (no. of clones) Chromosome 5 Hex A Hex A Hex B and B alone alone Neither + 7 0 Hex B Series A 12 2 8 7 - 0 9 Series B 0 4 6 5 Human-mouse hybrid clones (series B) whose human karyo- Two series of human-mouse hybrid clones were established as types are known were analyzed for Hex activity. Synteny was described in the text and analyzed for expression of Hex activity. evident between Hex B and chromosome 5. In clones positive for Hex B, chromosome 5 was found in 30-90% of the metaphases given in Table 2. The results indicate that human Hex A examined. In clones negative for Hex B, this specific chromosome and B segregate independently. was absent in all of the metaphases examined. The hybrid clones in series A were also analyzed for twenty other enzymes whose human chromosome assignments have In a series of human-mouse somatic cell hybrid clones, Hex already been established (see Table 1). The only concordance A was found to segregate concordantly with the human forms noted between the multiple forms of Hex and the other en- of mannosephosphate isomerase and pyruvate kinase-3. zymes was between Hex A and the human forms of mannose- This synteny has been independently confirmed by another phosphate isomerase (EC 5.3.1.8) and pyruvate kinase-3 (EC group of investigators (13, 16). The gene for mannosephos- 2.7.1.40) (Table 3). There was no positive correlation evident phate isomerase has been assigned to chromosome 7(11). between the expression of Hex B activity and the other Van Heyningen et al. (17) in a recent study assigned the genes enzymes studied. Chromosome analysis of hybrid clones in for mannosephosphate isomerase and pyruvate kinase-3 to series B revealed that Hex B activity was correlated with the chromosome 15. Ruddle and McMorris (18), on the basis of presence of chromosome 5 (Table 4). an extensive study, retracted their original assignment of In order to further test the independent expression of Hex A mannosephosphate isomerase to chromosome 7. It is therefore and B and to confirm our assignment of the gene for Hex B to presumed that a genetic locus, most probably the structural chromosome 5, we treated three cell lines, AIM-15a, JFA- locus, for Hex A can be assigned to chromosome 15 in man. 14a-13, and JFA-16a-8, with various concentrations of Hex B was found to segregate independently of Hex A. On the diphtheria toxin. Mouse cells are resistant to the toxin basis of an analysis of a series of hybrid clones with defined whereas human cells are sensitive. Hybrid cells that retain human chromosome complements, we were able to assign a human chromosome 5 are as sensitive as human cells, whereas locus, again probably the structural locus, for Hex B to chro- other human chromosomes leave them toxin-resistant (R. P. mosome 5. The independent segregation of Hex A and B and Creagan, S. Chen, and F. H. Ruddle, manuscript in prepara- the assignment of Hex B to chromosome 5 have been con- tion). The results of toxin treatment, in terms of their Hex firmed by our studies on the effects of diphtheria toxin treat- expression, are presented in Table 5. These results confirm ment of three hybrid cell lines. our assignment of Hex B to chromosome 5 and the inde- Hypotheses have been advanced that seek to explain mecha- pendence of Hex A and B expression. nisms governing the expression of Hex A and B in man. These hypotheses are divisible into two major categories: (A) models DISCUSSION based on structural interrelationships between Hex A and B, The lysosomal enzyme, Hex, implicated in the production of and (B) a model that proposes complete structural indepen- the GM2 gangliosidoses, is found to exist in at least two elec- dence of the Hex A and Hex B gene products. The two major trophoretic forms, A and B, between which a structural rela- theories under consideration in category A are: (1) the enzyme tionship has been presumed to exist. We have used the tech- conversion model and (2) the common subunit model. The niques of somatic cell genetics to gain insight into this relation- first would require there be at least two loci involved in Hex ship and to establish the chromosomal assignments of genes expression, one coding for the basic structural protein, pre- involved in the expression of this enzyme. sumed to be Hex B, and a second coding for the enzyme re- sponsible for the conversion of this protein to Hex A. The TABLE 3. Segregation ofHex with mannosephosphate isomerase second hypothesis, advanced by Beutler and illustrated in and pyruvate kinase TABLE 5. Expression of hexosaminidase in three hybrid cell MPI PK lines before and after treatment of the cells with diphtheria toxin + - + - + 12 1 + 5 2 Hex expression Chromosome 5 Hex A Hex A Cell line No toxin - 1 15 - 0 10 Toxin No toxin Toxin AIM-15a A+B+ A+B- + n.d. Human-mouse hybrid clones (series A) were analyzed for Hex JFA-14a-13 A-B+ A-B- + activity and for the enzymes listed in Table 1. Synteny was JFA-16a-8 A-B- A-B- evident only between Hex A and mannosephosphate isomerase (MPI) and pyruvate kinase (PK). n.d. = not done. Downloaded by guest on September 28, 2021 266 Genetics: -Gilbertet al. Proc. Nat. Acad. Sci. USA 72 (1976)

HexA: AB HexA: AX unit. This would require two or three genetic loci, each coding HexB: BB HexB: BX for a different subunit. If the three-subunit model were cor- I II rect, one would expect to correlate the presence of Hex A and FIG. 2. Models of subunit structure of hexosaminidase A and Hex B with two chromosomes each. Our karyotypic data in- B dimers. (I) Two-subunit model: B = common subunit, A = dicate that only one chromosome each is required for the ex- Hex A-specific subunit. (II) Three-subunit model: X = common pression of these enzymes. The two-subunit model predicts subunit, A = Hex A-specific subunit, B = Hex B-specific subunit. dependent segregation of Hex A or Hex B, depending upon which of these enzymes is the heteropolymer. If we assume Fig. 2, would require two or three loci, each coding for a dis- that there is no interaction between the mouse and human tinct subunit type (6). In the two-subunit model, each Hex A forms of the enzyme, our results do not support this hy- dimer would be formed from two subunit types, one of which pothesis. is common to both forms and one of which is Hex A-specific. In clones that express Hex A alone, a situation that has not Each Hex B dimer would consist exclusively of the common yet been reported in vivo, the possibility that a heteropolymer subunits. The three-subunit model would require three loci, may have been formed between human Hex A or B component one coding for the subunit common to both forms and two and a component contributed by the mouse cell has to be con- others coding for the Hex A- and Hex B-specific subunits. sidered. This heteropolymer then could migrate to a position The Hex A and B dimers would each be composed of one similar to that of normal human Hex A. Heteropolymers have common and one specific subunit. It has recently been pro- been reported to occur in somatic cell hybrids (see, for ex- posed that Hex C may be the Hex A-specific subunit (14). ample, ref. 15). If we assume that this hypothesis is correct, In a study of 60 human-mouse hybrid clones analyzed for our results might be considered to be consistent with Beutler's Hex activity and for a number of other enzymes, Lalley et al. two-subunit model. However, one might expect to see some reported no clones in which Hex A was expressed in the ab- differences in the mobilities of the heteropolymer and the sence of Hex B (13, 16). They concluded that the expression normal human Hex A. On repeated enzyme analyses we found of Hex B is a necessary prerequisite for the expression of Hex that Hex A migration in all hybrid clones was identical and A. If this were so, and one assumed random segregation of that there was no reduction in the intensity of mouse or human human chromosomes in the hybrid clones, it would be expected bands. This observation makes it less likely that the two-sub- that a significant number of clones that retained mannose- unit model of Hex structure is correct, but does not exclude it phosphate isomerase and pyruvate kinase-3 activity would not entirely. express Hex A since they have lost the chromosome responsi- Our data strongly support the hypothesis that there is no ble for Hex B (chromosome 5). They found, however, only structural relationship between the two human specific en- one of 35 clones in which mannosephosphate isomerase was zymes. The independent segregation of the two enzymic forms present and Hex A was absent. Thus, their data, while con- in hybrid cell lines reported here and by van Someren and van sistent with a model requiring the presence of Hex B for Henegouwen (8) are consistent with this model. On this basis, expression of Hex A, do not provide conclusive proof for such TSD can be explained by a mutation at a locus responsible for a scheme. Hex A expression (possibly on chromosome 15) and SD to be In our series of hybrid clones we found all four possible com- the result of either two mutations involving Hex A and B binations of Hex A and B-those with both, those with structural proteins (involving chromosome 5 in addition to neither, and those with Hex A or B alone. The studies by van chromosome 15), or more likely a single mutation at a locus Someren and van Henegouwen (8) of 105 Chinese hamster- that regulates the expression of these genes, or is necessary for human hybrids also show that the two enzymic forms segre- the proper packaging of the enzymes within the lysosome, or gate independently. The finding that human Hex A activity is required for enzyme activation. can exist in the absence of demonstrable Hex B in these studies We have thus been able to assign the locus involved in the makes it doubtful that the former is a conversion product of expression of Hex B to chromosome 5 and establish syntenic the latter. relationship between the genes for Hex A, mannosephosphate If it were possible, in the hybrid situation, for a hypothetical isomerase, and pyruvate kinase-3. mouse conversion enzyme to substitute for the corresponding We are grateful to Ms. Elizabeth A. Nichols, Ms. Susie Chen, human form and change Hex B to Hex A, one would expect and Mrs. Mae Reger for their expert assistance. This investigation to find hybrid clones that have chromosome 5 and thus Hex was supported by N.I.H. Grant USPHS 5-RO1-GM-09966, and B as well as Hex A activity but that lack mannosephosphate NSF-GB 34303. R.K. is a Damon Runyon Memorial Cancer isomerase and pyruvate kinase-3 activity as a consequence Research Fellow. of the loss of human Hex A, mannosephosphate isomerase, 1. Brady, R. 0. (1973) Fed. Proc. 32, 1660-1667. and pyruvate kinase-3 linkage group. Such clones were not 2. Robinson, D. & Stirling, J. L. (1968) Biochem. J. 107, observed. It is also conceivable, though unlikely, that a 321-327. human conversion enzyme, present in the hybrids which re- 3. Hooghwinkel, G. J. M., Veltkamp, W. A., Overdijlc, B. & tained mannosephosphate isomerase expression but not Lisman, J. W. (1972) Z. Physiol. Chem. 353, 839-841. 4. Okada, S. & O'Brien, J. S. (1969) Science 165, 698-700. chromosome 5, could alter the mouse structural protein such 5. Sandhoff, K., Andrae, U. & Jatzkewitz, H. (1968) Life Sci. that it migrates coincidentally with human Hex A. However, 7, 283-288. it would seem unlikelv that such a conversion product would 6. Srivastava, S. K. & Beutler, E. (1973) Nature 241, 463. have an electrophoretic mobility precisely that of human 7. Ruddle, F. H. (1973) Nature 242, 165-169. A. 8. van Someren, H. & van Henegouwen, H. (1973) Human- Hex genetik 18, 171-174. Based on immunochemical data, Beutler (6) proposed a 9. Nichols, E. A. & Ruddle, F. H. (1973) J. Histochem. Cyto- model in which each of the enzymic forms has a common sub- chem. 21, 1066-1081. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Tay-Sachs' and Sandhoff's Diseases 267

10. Klebe, R. J., Chen, T. R. & Ruddle, F. H. (1970) J. Cell 15. Shows, T. B. & Ruddle, F. H. (1968) Science 160,1356-1357. Biol. 45, 74-82. 16. Lalley, P. A., Rattazzi, M. C. & Shows, T. B. (1974) Proc. 11. McMorris, F. A., Chen, T. R., Ricciuti, F., Tischfield, J., Nat. Acad. Sci. USA 71, 1569-1573. Creagan, R. & Ruddle, F. H. (1973) Science 179, 1129-1131. 17. van Heyningen, V., Bobrow, M. & Bodmer, W. F. (1974). 12. Shows, T. B. (1972) Amer. J. Hum. Genet. 24, 13 abstr. In Rotterdam Conference (1974). Second International 13. Lalley, P. A., Rattazzi, M. C. &' Shows, T. B. (1973) New Workshop on Humran Gene Mapping. Birth Defects: Original Haven Conference, First International Workshop on Human Article Series, in press. Gene Mapping. Birth Defects: Original Article Series, ed. 18. Ruddle, F. H. & McMorris, F. A. (1974). In Rotterdam Con- Bergsma, D., Vol. X, no. 3. ference (1974). Second International Workshop on Human 14. Ropers, H. H. & Schwantes, U. (1973) Humangenetik 20, Gene Mapping. Birth Defects: Original Article Series, in 167-170. press. Downloaded by guest on September 28, 2021