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Proc. Nat. Acad. Sci. USAVol. 72, No. 1, pp. 263-267, January
1975
Tay-Sachs' and Sandhoffs Diseases: The Assignment of Genes
forHexosaminidase 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 HumanGenetics, Yale
University School of Medicine, New Haven, Connecticut 06510
Communicated by Victor A. McKusick, June 6, 1974
ABSTRACT The techniques of somatic cell geneticshave been used
to establish the linkage relationshipsof loci coding for two forms
(A and B) of hexosaminidase(EC 3.2.1.30;
2-acetamido-2-deoxy-B-D-glucoside acet-amidodeoxyglucohydrolase)
and to determine whether astructural relationship exists between
these forms. In aseries of human-mouse hybrid cell lines,
hexosaminidaseA and B segregated independently. Our results and
thosereported by other investigators are used to analyze
theproposed structural models for hexosaminidase. We havealso been
able to establish a syntenic relationship betweenthe gene locus
responsible for the expression of hexosami-nidase A and those
responsible for mannosephosphateisomerase and pyruvate kinase-3 and
to assign the genefor hexosaminidase B to chromosome 5 in man.
Tbere isthus a linkage between specific human autosomes andenzymes
implicated in the production of lipid storagediseases.
The lipid storage diseases are a family of inherited
disorderscharacterized by the excessive accumulation of
sphingolipidsin the body's tissues. In each, the metabolic
derangement ap-pears to be the result of a deficiency of a specific
lysosomalhydrolase which is involved in the catabolism of these
complexlipids (1). One of these enzymes,
P-N-acetylglucosaminidase(Hex; EC 3.2.1.30) is thought to be
responsible for at least twolipodystrophies, Tay-Sachs' disease
(TSD; GM2 gangliosidosis,type I) and Sandhoff's disease (SD; GM2
gangliosidosis, typeII). When examined electrophoretically, this
enzyme is foundto exist in multiple forms, two of which (Hex A and
B) havebeen well characterized biochemically (2). A third form of
theenzyme (Hex C), about which relatively little is known,
hasrecently been described (3). TSD is associated with a
defi-ciency of Hex A and an increased activity of Hex B, and SDis
associated with a deficiency of both Hex A and B (4, 5).No
individual has yet been reported in whom Hex A is presentin the
absence of Hex B.Biochemical, genetic, and immunological evidence
suggests
that a structural relationship exists between Hex A and B.Two
theories concerning this relationship have recently beenadvanced
(2, 6). The first proposes that Hex A is a conversionproduct of Hex
B (2). TSD would then result from the de-ficiency of a functional
conversion enzyme, and SD would re-
Abbreviations: Hex, hexosaminidase; TSD, Tay-Sachs' disease;SD,
Sandhoff's disease.§ Present address: Department of Human Genetics,
Universityof Pennsylvania School of Medicine, Philadelphia, Pa.
19104.t To whom reprint requests should be addressed.
sult from a defect in the gene coding for the basic Hex
protein.The second theory proposes that Hex A and B are
eachcomposed of multiple subunits, one of which is common toboth
forms (6). In this hypothesis, TSD would result from thedeficiency
of the Hex A-specific subunit and SD from thedeficiency of the
common subunit. It is also possible that thetwo forms of Hex are
not structurally related. Hex A and Bmay be controlled by two
independent genes. TSD would thenresult from an effective
deficiency of the normal Hex A struc-tural gene product and SD
might result from a mutation in alocus controlling expression of
both enzymes or required fortheir activation.A series of
human-mouse hybrid cell lines were examined for
the expression of Hex activity to determine whether a
struc-tural relationship does in fact exist between Hex A and
B.Such interspecific hybrid cell lines, which
preferentiallysegregate the chromosomes of one parent in the cross
(in thisinstance, the human), have already proved useful in the
as-signment of genes for specific enzymes to individual
chromo-somes in man. However, their potential value as a tool for
thestudy of enzyme structure has not yet been fully appreciated.We
have found that, in this series of hybrid cells, human HexA and B
are expressed independently.The hybrid cells were also used to
establish the linkage
relationships of genes coding for Hex A and B. The
humanchromosome complements and patterns of expression of aseries
of isozymes with known chromosome assignments werecompared with the
retention of Hex A and B activity in theseclones. On the basis of
these studies we were able to assign thelocus involved in the
expression of Hex B to human chromo-some 5 and to establish a
syntenic relationship between genescoding for Hex A,
mannosephosphate isomerase, and pyruvatekinase-3.
MATERIALS AND METHODS
Hexosaminidase Assay. The assay for hexosaminidase activ-ity is
a modification of the published procedures of Okadaand O'Brien, and
van Someren and van Henegouwen (4, 8).The cell homogenates were
prepared as described (9). Electro-phoresis was performed (at 40)
on cellulose acetate gel (Cello-gel: Chemetron, Milan, Italy) in a
citrate phosphate buffer(25 mM, pH 5.6) for 3 hr at 250 V. The gel
was incubatedwith the artificial substrate, 4-methyl
umbelliferyl-N-acetyl-,3-glucosaminide [Pierce Chemical Co.; 1.5 mM
in 0.5 M Nacitrate (pH 4.0) and 1% agarose] for 1-2 hr and then
exposed
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Proc. Nat. Acad. Sci. USA 72 (1975)
(4)
-4*- C
-_- A
-B
0
1 2 3 4 5 6 (H)
FIG. 1. Hexosaminidase activity on cellulose acetate.
Cellextracts were applied to cellulose acetate and assayed as
de-scribed in the text. The development time of the gel was
chosento allow optimal visualization of the Hex A and B bands.
Fourbands are evident-corresponding to human Hex A, Hex B, andto
two mouse activities, one at the origin and a second
migratingcoincidentally with human Hex C. Slot 1, hybrid with
humanHex A and B; slot 2, hybrid with Hex B alone; slot 3,
hybridwith Hex A alone; slot 4, hybrid with no human Hex; slot
5,mouse parent; slot 6, human parent. The differences in
intensityof Hex A and B between cell lines possibly reflect
quantitativedifferences in the amounts of material applied to each
slot. HexC activity (mouse and/or human) was clearly apparent in
allsamples, though in some (slots 2, 3, and 6) it was faint andthus
was reproduced poorly in the photograph. Some variationin the
intensity of the Hex C in each cell line has been notedbetween
gels. The slight difference in mobility of Hex C inslots 4 and 5
possibly reflects differences in the age of each sample.
to ammonia vapor before it was made visible under ultra-violet
light.
Other Enzymes. Twenty enzymes with established mouseand human
electrophoretic differences and known humanchromosome assignments
(Table 1) were also assayed bymethods detailed by Nichols and
Ruddle (9).
Production of Hybrids. Series A: A number of primaryhybrid
clones were established, using techniques outlined,after the fusion
of a mouse cell line (A9) lacking the enzymehypoxanthine-guanine
phosphoribosyltransferase (HGPRT)with two diploid human fibroblast
lines (GM 17 and Yoder,obtained from the Institute for Medical
Research, Camden,N.J. and Dr. D. Borgaonkar, Johns Hopkins
UniversitySchool of Medicine, respectively) and isolation in the
HATselection system (10).
Series B: This represents a number of primary and second-ary
hybrid clones resulting from the fusion of several differenthuman
diploid fibroblast and leukocyte lines with two HGP-RT- mouse cell
lines (A9 and RAG) and established throughthe use of the methods
described above. The human chromo-some complements were analyzed as
the cell pellets wereprepared (R. Creagan and F. H. Ruddle,
unpublished data).An average of over 25 metaphases per clone was
examined.
TABLE 1. Enzymes studied and their human
chromosomeasszgnments*
HumanEnzyme chromosome
Dipeptidase-1 (PEP C) 1Malate dehydrogenase (NAD +) (MDH-1)
2Isocitrate dehydrogenase (NADP+) (IDH-1) 2Malic enzyme (NADP+)
(MOD) 6Indophenol oxidase (SOD-2) 6Mannosephosphate isomerase (MPI)
15Pyruvate kinase (PK) 15Glutamate-oxaloacetate transaminase (GOT)
10Esterase A4 (Es-A4) 11Lactate dehydrogenase-A (LDH-A) 11Lactate
dehydrogenase-B (LDH-B) 12Tripeptidase-1 (PEP B) 12Nucleoside
phosphorylase (NP) 14Adenine phosphoribosyltransferase (APRT)
16Dipeptidase-2 (PEP A) 18Glucosephosphate isomerase (GPI)
19Adenosine deaminase (ADA) 20Indophenol oxidase (SOD-1)
21Phosphoglycerate kinase (PGK) XGlucose 6-phosphate dehydrogenase
(G6PD) X
* See ref. 9.
Heat Inactivation. Cell homogenates of the human andmouse
fibroblast parents and of hybrid lines from both seriesA and B were
heated for three hours at 500. The heat-in-activated samples as
well as untreated controls were thenanalyzed for hexosaminidase
activity by cellulose acetate gelelectrophoresis. Hex A activity
has been reported to be thermo-labile while Hex B activity is
thermostable.
Toxin Treatment. Human, mouse, and hybrid somatic cellswere
subjected to various concentrations of diphtheria toxin asdescribed
(R. P. Creagan, S. Chen, and F. H. Ruddle, manu-script in
preparation).
RESULTSThe electrophoretic patterns of the multiple forms of Hex
inthe human and mouse parental cell lines and in selectedhybrid
clones are illustrated in Fig. 1. The human cell linesdemonstrate
the previously described three-band pattern cor-responding to Hex
A, B, and C. The mouse cell line has onedistinct band which
migrates to a position coincident with thehuman Hex C and a second
band at the origin. A mixture ofhomogenates of the parent lines
(human and mouse), whenassayed for Hex, demonstrates all four
bands.Two series of human-mouse hybrid clones (series A and B)
were examined for the presence of Hex A and B. All of theclones
retained enzymatic activity in the Hex C region thatcould be of
human and/or mouse origin. Cell homogenates ofthe mouse and human
fibroblast parents and of several hybridclones from both series A
and B (three of which were HexA+/B+, one that was Hex-/B+, and
three that were HexA+/B+) were subjected to heat inactivation and
analyzed forhexosaminidase activity by gel assay. Heat inactivation
re-sulted in the loss of Hex A activity in all of the lines in
which itwas present prior to treatment. Hex B activity was
unaffectedby the heat treatment. The distribution of clones
retainingboth Hex A and B, either Hex A or B alone, or neither,
is
264 Genetics: Gilbert et al.
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Tay-Sachs' and Sandhoff's Diseases 265
TABLE 2. Hexosaminidase activity in human-mousehybrid clones
Hexosaminidase activity (no. of clones)
Hex A Hex A Hex Band B alone alone Neither
Series A 12 2 8 7Series B 0 4 6 5
Two series of human-mouse hybrid clones were established
asdescribed in the text and analyzed for expression of Hex
activity.
given in Table 2. The results indicate that human Hex Aand B
segregate independently.The hybrid clones in series A were also
analyzed for twenty
other enzymes whose human chromosome assignments havealready
been established (see Table 1). The only concordancenoted between
the multiple forms of Hex and the other en-zymes was between Hex A
and the human forms of mannose-phosphate isomerase (EC 5.3.1.8) and
pyruvate kinase-3 (EC2.7.1.40) (Table 3). There was no positive
correlation evidentbetween the expression of Hex B activity and the
otherenzymes studied. Chromosome analysis of hybrid clones inseries
B revealed that Hex B activity was correlated with thepresence of
chromosome 5 (Table 4).
In order to further test the independent expression of Hex Aand
B and to confirm our assignment of the gene for Hex B tochromosome
5, we treated three cell lines, AIM-15a, JFA-14a-13, and JFA-16a-8,
with various concentrations ofdiphtheria toxin. Mouse cells are
resistant to the toxinwhereas human cells are sensitive. Hybrid
cells that retainhuman chromosome 5 are as sensitive as human
cells, whereasother human chromosomes leave them toxin-resistant
(R. P.Creagan, S. Chen, and F. H. Ruddle, manuscript in
prepara-tion). The results of toxin treatment, in terms of their
Hexexpression, are presented in Table 5. These results confirmour
assignment of Hex B to chromosome 5 and the inde-pendence of Hex A
and B expression.
DISCUSSIONThe lysosomal enzyme, Hex, implicated in the
production ofthe GM2 gangliosidoses, is found to exist in at least
two elec-trophoretic forms, A and B, between which a structural
rela-tionship has been presumed to exist. We have used the
tech-niques of somatic cell genetics to gain insight into this
relation-ship and to establish the chromosomal assignments of
genesinvolved in the expression of this enzyme.
TABLE 3. Segregation ofHex with mannosephosphate isomeraseand
pyruvate kinase
MPI PK
+ - + -+ 12 1 + 5 2
Hex A Hex A- 1 15 - 0 10
Human-mouse hybrid clones (series A) were analyzed for
Hexactivity and for the enzymes listed in Table 1. Synteny
wasevident only between Hex A and mannosephosphate isomerase(MPI)
and pyruvate kinase (PK).
TABLE 4. Segregation of Hex B with specific humanchromosome
Chromosome 5
+ 7 0Hex B
- 0 9
Human-mouse hybrid clones (series B) whose human karyo-types are
known were analyzed for Hex activity. Synteny wasevident between
Hex B and chromosome 5. In clones positive forHex B, chromosome 5
was found in 30-90% of the metaphasesexamined. In clones negative
for Hex B, this specific chromosomewas absent in all of the
metaphases examined.
In a series of human-mouse somatic cell hybrid clones, HexA was
found to segregate concordantly with the human formsof
mannosephosphate isomerase and pyruvate kinase-3.This synteny has
been independently confirmed by anothergroup of investigators (13,
16). The gene for mannosephos-phate isomerase has been assigned to
chromosome 7(11).Van Heyningen et al. (17) in a recent study
assigned the genesfor mannosephosphate isomerase and pyruvate
kinase-3 tochromosome 15. Ruddle and McMorris (18), on the basis
ofan extensive study, retracted their original assignment
ofmannosephosphate isomerase to chromosome 7. It is
thereforepresumed that a genetic locus, most probably the
structurallocus, for Hex A can be assigned to chromosome 15 in
man.Hex B was found to segregate independently of Hex A. On
thebasis of an analysis of a series of hybrid clones with
definedhuman chromosome complements, we were able to assign alocus,
again probably the structural locus, for Hex B to chro-mosome 5.
The independent segregation of Hex A and B andthe assignment of Hex
B to chromosome 5 have been con-firmed by our studies on the
effects of diphtheria toxin treat-ment of three hybrid cell
lines.Hypotheses have been advanced that seek to explain mecha-
nisms governing the expression of Hex A and B in man.
Thesehypotheses are divisible into two major categories: (A)
modelsbased on structural interrelationships between Hex A and
B,and (B) a model that proposes complete structural indepen-dence
of the Hex A and Hex B gene products. The two majortheories under
consideration in category A are: (1) the enzymeconversion model and
(2) the common subunit model. Thefirst would require there be at
least two loci involved in Hexexpression, one coding for the basic
structural protein, pre-sumed to be Hex B, and a second coding for
the enzyme re-sponsible for the conversion of this protein to Hex
A. Thesecond hypothesis, advanced by Beutler and illustrated in
TABLE 5. Expression of hexosaminidase in three hybrid celllines
before and after treatment of the cells with diphtheria toxin
Hex expression Chromosome 5
Cell line No toxin Toxin No toxin Toxin
AIM-15a A+B+ A+B- + n.d.JFA-14a-13 A-B+ A-B- +JFA-16a-8 A-B-
A-B-
n.d. = not done.
Proc. Nat. Acad. Sci. USA 72 (1975)
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Proc. Nat. Acad. Sci. USA 72 (1976)
HexA: AB HexA: AXHexB: BB HexB: BX
I II
FIG. 2. Models of subunit structure of hexosaminidase A andB
dimers. (I) Two-subunit model: B = common subunit, A =Hex
A-specific subunit. (II) Three-subunit model: X = commonsubunit, A
= Hex A-specific subunit, B = Hex B-specific subunit.
Fig. 2, would require two or three loci, each coding for a
dis-tinct subunit type (6). In the two-subunit model, each Hex
Adimer would be formed from two subunit types, one of whichis
common to both forms and one of which is Hex A-specific.Each Hex B
dimer would consist exclusively of the commonsubunits. The
three-subunit model would require three loci,one coding for the
subunit common to both forms and twoothers coding for the Hex A-
and Hex B-specific subunits.The Hex A and B dimers would each be
composed of onecommon and one specific subunit. It has recently
been pro-posed that Hex C may be the Hex A-specific subunit
(14).
In a study of 60 human-mouse hybrid clones analyzed forHex
activity and for a number of other enzymes, Lalley et al.reported
no clones in which Hex A was expressed in the ab-sence of Hex B
(13, 16). They concluded that the expressionof Hex B is a necessary
prerequisite for the expression of HexA. If this were so, and one
assumed random segregation ofhuman chromosomes in the hybrid
clones, it would be expectedthat a significant number of clones
that retained mannose-phosphate isomerase and pyruvate kinase-3
activity would notexpress Hex A since they have lost the chromosome
responsi-ble for Hex B (chromosome 5). They found, however, onlyone
of 35 clones in which mannosephosphate isomerase waspresent and Hex
A was absent. Thus, their data, while con-sistent with a model
requiring the presence of Hex B forexpression of Hex A, do not
provide conclusive proof for sucha scheme.
In our series of hybrid clones we found all four possible
com-binations of Hex A and B-those with both, those withneither,
and those with Hex A or B alone. The studies by vanSomeren and van
Henegouwen (8) of 105 Chinese hamster-human hybrids also show that
the two enzymic forms segre-gate independently. The finding that
human Hex A activitycan exist in the absence of demonstrable Hex B
in these studiesmakes it doubtful that the former is a conversion
product ofthe latter.
If it were possible, in the hybrid situation, for a
hypotheticalmouse conversion enzyme to substitute for the
correspondinghuman form and change Hex B to Hex A, one would
expectto find hybrid clones that have chromosome 5 and thus HexB as
well as Hex A activity but that lack mannosephosphateisomerase and
pyruvate kinase-3 activity as a consequenceof the loss of human Hex
A, mannosephosphate isomerase,and pyruvate kinase-3 linkage group.
Such clones were notobserved. It is also conceivable, though
unlikely, that ahuman conversion enzyme, present in the hybrids
which re-tained mannosephosphate isomerase expression but
notchromosome 5, could alter the mouse structural protein suchthat
it migrates coincidentally with human Hex A. However,it would seem
unlikelv that such a conversion product wouldhave an
electrophoretic mobility precisely that of humanHex A.Based on
immunochemical data, Beutler (6) proposed a
unit. This would require two or three genetic loci, each
codingfor a different subunit. If the three-subunit model were
cor-rect, one would expect to correlate the presence of Hex A
andHex B with two chromosomes each. Our karyotypic data in-dicate
that only one chromosome each is required for the ex-pression of
these enzymes. The two-subunit model predictsdependent segregation
of Hex A or Hex B, depending uponwhich of these enzymes is the
heteropolymer. If we assumethat there is no interaction between the
mouse and humanforms of the enzyme, our results do not support this
hy-pothesis.In clones that express Hex A alone, a situation that
has not
yet been reported in vivo, the possibility that a
heteropolymermay have been formed between human HexA or B
componentand a component contributed by the mouse cell has to be
con-sidered. This heteropolymer then could migrate to a
positionsimilar to that of normal human Hex A. Heteropolymers
havebeen reported to occur in somatic cell hybrids (see, for
ex-ample, ref. 15). If we assume that this hypothesis is
correct,our results might be considered to be consistent with
Beutler'stwo-subunit model. However, one might expect to see
somedifferences in the mobilities of the heteropolymer and
thenormal human Hex A. On repeated enzyme analyses we foundthat Hex
A migration in all hybrid clones was identical andthat there was no
reduction in the intensity of mouse or humanbands. This observation
makes it less likely that the two-sub-unit model of Hex structure
is correct, but does not exclude itentirely.Our data strongly
support the hypothesis that there is no
structural relationship between the two human specific en-zymes.
The independent segregation of the two enzymic formsin hybrid cell
lines reported here and by van Someren and vanHenegouwen (8) are
consistent with this model. On this basis,TSD can be explained by a
mutation at a locus responsible forHex A expression (possibly on
chromosome 15) and SD to bethe result of either two mutations
involving Hex A and Bstructural proteins (involving chromosome 5 in
addition tochromosome 15), or more likely a single mutation at a
locusthat regulates the expression of these genes, or is necessary
forthe proper packaging of the enzymes within the lysosome, oris
required for enzyme activation.We have thus been able to assign the
locus involved in the
expression of Hex B to chromosome 5 and establish
syntenicrelationship between the genes for Hex A,
mannosephosphateisomerase, and pyruvate kinase-3.
We are grateful to Ms. Elizabeth A. Nichols, Ms. Susie Chen,and
Mrs. Mae Reger for their expert assistance. This investigationwas
supported by N.I.H. Grant USPHS 5-RO1-GM-09966, andNSF-GB 34303.
R.K. is a Damon Runyon Memorial CancerResearch Fellow.
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