273

A n n . Hum. Genet., L o n d . (1979), 42, 273 Printed in Great Britain

Assignment of the human acid u-glucosidase gene (uGLU) to chromosome 17 using somatic cell hybrids BY ELLEN SOLOMON,* DALLAS SWALLOW,? SUZANNE BURGESS* AND LORRAINE EVANS?

* Genetics Laboratory, Department

of Biochemistry, University of Oxford, South Parks Road, Oxford and ? M.R.C. H u m a n Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolfson House, 4, Stephenson W a y , London N W1 2HE INTRODUCTION

Human acid a-glucosidase shows a genetically determined polymorphism detectable by starch gel electrophoresis but not by other standard electrophoretic methods. It is determined by two common alleles at an autosomal gene locus now designated aGLU with gene frequencies of 0.97 (aGLU1) and 0.03 (aGLU2)in the European population (Swallow et al. 1975). Deficiency of a-glucosidase, presumably due to homozygosity of a null allele at the aGLU locus is the primary defect in type 2 glycogen storage disease (Pompe’s disease). Assignment of aGLU to a particular human chromosome using somatic cell hybrids has been hindered by the fact that the more common human enzyme (aGLU 1) and the rodent enzyme have not been separable by conventional electrophoretic techniques or iso-electrofocusing (unpublished results). The separation of the two human aGLU allele products by starch gel electrophoresis has been attributed to a difference in their affinity for the starch gel. Mouse aGLU resembles the human aGLU 1 enzyme in having a high affinity for the starch gel, but can clearly be distinguished from human aGLU 2. We have therefore exploited this difference and made human/mouse hybrids using, as the human parent, fibroblasts from an individual who had the rare clGLU 2 phenotype. We have also demonstrated a difference in thermostability between the human and mouse enzymes. Making use of this difference we have been able to score a wide range of other human/ mouse hybrids for the presence of human aGLU.

METHODS

MOG hybrids The human parent (HM) is a primary fibroblast, grown from a skin biopsy, with the aGLU 2 phenotype. These cells were fused with the mouse line RAG (6-thioguanine-resistant), using inactivated Sendai virus. Hybrids were selected in medium containing hypoxanthine (10-4 M), thymidine (1.6 x M), methotrexate (loF5M) and oubain (5 x lo4 M). Fifteen independent primary hybrid clones were picked, each from a separate dish (MOG hybrids). Subclones were generated by plating a particular hybrid clone a t low density, and again picking independent colonies. Nineteen subclones were picked from two different primary hybrids. Subcloning was repeated on one of these subclones (MOG 3.4A) and 13 independent colonies picked. 0003-4S00/79/0000-4202 $02.00 0 1979 University College London

ELLENSOLOMON AND

274

OTHERS

Table 1. Hybrid details Parents , -A- - (

No. of clones analysed

Hybrids

Human

Mouse

DFP

Primary fibroblast

PG19 (6-TGresistant melanoma)

CTP

T lymphocyte

PG19

DUR

Primary fibroblast

IR (6-TGresistant L cell)

I

Solomon et al. 1976

ZWI-I

Lymphocyte

IR

I

Nabholz, Miggiano & Bodmer, 1969

HORL 9

Lymphocyte

IR

I

van Heyningen, Craig & Bodmer, 1973

P7A/z

Primary fibroblast

Clone ID (BUdR-resistant L cell)

I

Fellous et aE. 1973

9

I0

Reference

D. Buck, personal communication

Jones et al. 1976

Cells were routinely grown in RPMI 1640 medium supplemented with lo(% foetal calf serum, penicillin and streptomycin.

Other hybrids The origins of the 23 hybrid clones used for thermostability studies are presented in Table I . All of the clones from each fusion are independent. All of the hybrids, except P7A/2 were produced in the Genetics Laboratory, Oxford. P7A/2R was derived from P7A/2 by selection in BUdR (30 pglml). Chromosome analysis of hybrids Karyotypes of the MOG hybrids were done by a combination of G-1I and quinacrine banding (Bobrow & Cross, 1974). Electrophoresis and detection of aGLU Starch gel electrophoresis was conducted using a citrate/phosphate buffer system a t pH 6.4 (Swallow et al. 1975). The acid a-glucosidase isozymes were located after electrophoresis using 4-methylumbelliferyl a-D-glucopyranosideas substrate as described previously (Swallow et al. 1975), but using 0.2 M citrate buffer at pH 3.5 rather than pH 4.0 in order to minimize the staining of the neutral a-glucosidase isozymes. Electrophoresis and detection of marker enzymes Analysis of enzymes determined by genes located on known chromosomes (Winnipeg Conference, 1977) was done using routine methods (Harris & Hopkinson, 1976, 19771, except that galactokinase was stained as follows : The staining mixture contained 3-36 mlO.2 M Tris/HCl, 0-14 mlO.2 M MgCl,, 8 mg ATP and 0.15 ml (containing 2 pCi) [14C]galactoseat pH 7.2. This was applied to the cut surface of the gel on Whatman DE8l paper and incubated at 37 "C for 2 h. The papers were then washed with approximately 151 distilled water and dried overnight. The dried papers were then autoradiographed with X-ray film for 1 week.

I2 I2 I2 I2

I3 I4 I5

16

I0 I1 I2

I0 I1 I1 I1

9

I2 I1 I2 I2

++

9

5 6 7

I 2

Chromosome

2 I

3

2

-

2

I I

3

--

\

r

-

-

I 2 2 I I

2

2

3

I

+-

Primary clones

I 2

-

I

3

I

-

2 I I I I 2

-+

16 18

I7 I7

18

I7 15

6 5

16

I7

18 16

++

-

-

I

-

I

-

-

I I

I

I1

6 12

22

X

-

I

-

**

-

-

-

2 2 -

18

3

9

I I

I

16

**

-

-

-

3

**

I

-

I

I3

18 18

I2

I

I I*

-

1 1 - 1 1

28 18

+

3 4 30

'7 I

6 28

-

2

I*

2

-

3

4

2

3

20

32 33

23

31

32 30

31

29 29 29

I

I

-

-

~

29

5 6 4 5 4

5 6

28

17

3

Discordant

3 6

31

Concordant

--

+

PGM,, phosphoglucomutase I ; ENOI, enolase I ; PEPC, peptidase C ; MDH,, malate dehydrogenase (soluble); ICD,, isocitrate dehydrogenase (soluble); HEXfi, N-acetyl B-D-hexosaminidase 8; ME,, malic enzyme (soluble); SODB, superoxide dismutase B ; BGUS, B-glucuronidase; AK,, adenylate kinase I ; AK,, adenylate kinase 3 ; GOT,, glutamate oxaloacetate transaminase (soluble); LDH,, lactate dehydrogenase A; PEPB, peptidase B ; ESD, esterase D ; NP, purine nucleoside phosphorylase ; MPI, mannose phosphate isomerase ; APRT, adenine phosphoribosyl transferase; GALK, galactokinase; PEPA, peptidase A; GPI, glucose phosphate isomerase; ADA, adenosine deaminase; SODA, superoxide dismutase A; ACONM, aconitase (mitochrondrial); Gd, glucose-6-phosphate dehydrogenase. + + , aGLU + , marker + ; , aGLU - ,marker - ; + - ,aGLU + , marker - ; - , aGLU - ,marker . The complete set of enzymes was not tested in every clone. * GPI only weak in this hybrid. ** Expression of ADA is unusual in these hybrids, therefore ADA+ only was scored.

-

** -

2 2

7

I2 I2

18

PEPA GPI ADA SODA ACON, Gd

3

19 20 21

7

I7

GALK

I I

I I

-

I

-

2

I I

-

I

-

I

-

I

I I

-

-+

3

2 2

2

2 I

-

+-

h

--

Total I

h

>

Subclones

...............................................................................................................................................................................................................

MPI APRT

NP

PGMJENOJPEPC MDH,/ICD, HEXfi M.E,/SODB BGUS AKlIAK, GOT, LDH, PEPB ESD

Enzyme marker

r

Table 2. Segregation of aGLU and 25 marker enzymes i n the MOG hybrids (primary clones and subclones)

F

s

b

ELLENSOLOMON AND

276 ( a ) Human fibroblasts

OTHERS

(b) Humanhouse hybrids and parental cells

Fig. 1. Photographs of two starch gels stained for ~LGLU;( a )showing the three aGLU phenotypes in human fibroblasts and ( b ) showing the separation of mouse and human aGLU activity in the MOG hybrids, and in the human and mouse parental cells. + ,Human enzymo present; - , human enzyme absent.

The enzymes used as chromosome markers are listed in Table 2. In some cases, more than one marker was used for a particular chromosome, but in many cases only one of these markers was tested.

Quantitative assay of a-glucosidase This was done as described previously (Swallow et al. 1975), except that the buffer used was 0-2 M citrate at pH 3-5. Thermostability comparisons Method 1. Samples were subjected to electrophoresis in the usual way. One half of the sliced gel was incubated between heating plates (McAlpine, Hopkinson & Harris, 1970) at 60 "C for 20 min and then both halves were stained for a-glucosidase. Method 2. Aliquots of cell extract supernatant obtained by centrifugation at 20000g for 30 min were heated for 0-30 min at 55 "C and then assayed for a-glucosidase. Residual activity was expressed as a percentage of the original activity.

RESULTS AND DISCUSSION

The electrophoretic pattern of aGLU in human fibroblasts of the three phenotypes aGLU 1, aGLU 2-1 and aGLU 2, in mouse cells and in the MOG hybrids is shown in Fig. 1. Fifteen independent primary MOG hybrids and 32 subclones were analysed. The human aGLU 2 isozyme segregated in the hybrid clones (Fig. 1). The segregation analysis of aGLU and the marker enzymes is shown in Table 2. It can be seen from Table 2 that aGLU is probably not on chromosomes 1, 2, 5, 7, 9, 10, 11, 12, 13, 15, 16, 21, 22 (or X) as there are more than two clones discordant for aGLU and the enzyme markers for each of these chromosomes. There were no discordances with galactokinase, the marker for chromosome 17. Since these hybrids all contained a large number of human chromosomes one of the MOG subclones was subcloned

... I

-

PGM,/ENO,/PEPC

+

f -

++ + + ++

f

5 HEXfl

+ + + + + + + n.t. + 4+ ++ + + n.t. + + ++ n.t.

+ + n.t. + + +

6 I0 Ws GOT, I1

++

n.t. -

+ + + + + + + ++

LDH,

I2

-

+

2i

n.t. n.t.

+ + n.t. + + + +-

PEPB

G d was present in MOD 3.4A but was not tested in the MOG 3.4A subclones. +, Enzyme present; - , enzyme absent; f , enzyme present but weak; n.t., not tested.

I3

I2

I1

10

9

7 8

6

5

3 4

2

I

MOG 3.4 A MOG 3.4 A subclones

Chromosome no. Enzyme marker

+ n.t. + + n.t. + ++ n.t. + ++

n.t.

+

+ + + + + + + + ++ n.t. + ++

13 14 ESD N P

+ + + + + + ++ + + ++

+ + + + + + + + ++ + + ++

15 16 MPI APRT

+ +-

-

+ ++ + +-

+

n.t,.

+

IS PEPA

Table 3. Analysis of aGLU and 16 marker enzymes in the subclones of MOG 3.4A

f

+ + + + + +

+ + -

n.t. -

+

I9 GPI

21

I7

f

n.t. n.t. n.t. n.t. n.t. n.t. n.t.

+ + + + +

-

++ +

+ n.t.

+ + + +

+ + +

SOD, GALK

-

F

+ %

+';

278

ELLENSOLOMON AND

OTHERS

Fig. 2. ( a ) Photographs of three pieces of starch gel which were heated for 0, 15 and 20 mins at 60 "C after electrophoresis of human and mouse cell extracts, showing the difference in thermostability of the human and mouse enzymes. fb) Photographs of two halves of a starch gel, one heated and one unheated showing the presence of heat-stable aGLU in some human-mmse hybrids. + , Human enzyme present; - , human enzyme absent.

again. Enzymes present in the original clone (MOG 3.4A) were tested in the subclones, and the detailed analysis is shown in Table 3. aGLU again showed complete concordance with galactokinase (GALK), but segregated from nucleoside phosphorylase (14), peptidase A (18) and glucose phosphate isomerase (19), so that these three chromosomes could also be eliminated as candidates. Enzyme analyses were not carried out for markers for chromosomes 3, 4 or 8, but two MOG clones were karyotyped. Both were negative for aGLU. MOG 7 contains chromosomes 1, 3 , 4 , 7, 8, 10, 11, 12, 13, 15, 16, 18, 21, X, Y and another unidentified marker chromosome, and MOG 13 contains 1, 3, 4, 20, 21, 22, X and Y. Thus chromosomes 3, 4 and probably also 8 and 20 can be excluded. The combined data suggest strongly that aGLU is located on chromosome 17. A wide range of other hybrids, in which the human aGLU phenotype was aGLU 1, was available to us. We therefore investigated various properties of a-glucosidase which might be exploited to distinguish the human and mouse enzymes in these hybrids. Using both a qualitative method (method 1) and a quantitative method (method 2) we were able to demonstrate a difference in thermostability.

Assignment of the human acid aGLU to chromosome 17

279

-

Table 4. Segregation of aGLU (by thermostability after electrophoresis) and 21 marker enzymes in 23 independent hybrids Concordant Marker enzymes

7h-----7

Chromosome

PGM, MDH, PGM, HEX, ME, BGUS AKJAK, GOT, LDH, PEPB ESD

I0 I1 I2 13

NP

I4

MPI

I5

++

I 2

I0 I1

4 5 6 7

6

9

3 I4

3 I3 2

5

-3 2

I

I

3

5

2 2 I

15 I1

I I

9

Discordant + 7

+-

-+

7 7

-

I I1 I

5 5 13 I1

7 I0

3 6 4

I

-

I I

I I

Total

Concordant

Discordant

13 I3

7 7

7 3 14

I I2 I

3

5

I4

5

5 7

I3 I2

I1

8

6

I0

16 I2

4 7 4

APRT 16 I4 I 15 .......................................................................................................................................................................................................... 22 GALK 17 I9 3 .................................................................................................................................................................................... PEPA 18 I3 3 3 16 I 7 GPI I9 5 2 I5 ADA 20 I7 2 4 I9 SODA 21 6 2 I1 8 Gd x 16 3 3 16

-

....-

................... 3

16 4 I1

6

See footnotes to Table 2.

Comparison of the thermostability of mouse and human aGLU by the first method involved heating strips of sliced gel after electrophoresis, as described by McAlpine et al. (1970). After 20 min heating a t 60 "Cboth the human aGLU 1 and aGLU 2 isozymes were still active, whereas the mouse aGLU was completely inactivated (Fig. 2). Twenty-three human/mouse hybrids were analysed using this method. Heat stable aGLU was detected in some hybrids but not in others. A typical gel is shown in Fig. 2. The segregation analysis of aGLU and the marker enzymes is shown in Table 4. aGLU and GALK showed complete concordance. Several of these hybrids have been karyotyped. Two contain chromosome 17 and very little other human material. Both of these contained thermostable (human) aGLU. They were : DFP 12.1 with 17 and 20 and part of 6 and 9, and P7A/2 containing 3q and 17q (M. Bobrow, personal communication). The hybrid P7A/2R was selected from P7A/2 by growth in BUdR to select against thymidine kinase and thus chromosome 17. This hybrid contained no thermostable aGLU and had lost GALK. The second, quantitative method of comparing the thermoatability of the human and mouse enzyme is laborious and consumes large amounts of material and thus was used only for the two hybrids P7A/2 and P7A/2R, to confirm the results obtained by the qualitative method. The thermal decay of human and mouse (C1 1D) aGLU and the aGLU present in the two hybrids is shown in Fig. 3. P7A/2 clearly contains some thermostable aGLU, whereas the P7A/2R aGLU shows a decay comparable to the aGLU of the mouse parent C1 1D.

ELLEN SOLOMON AND

280 100

A

OTHERS

A A

&

\

10

8

-

>.

.-c .-c>

-mCu 2

0 W

n

human libroblast a mouse Ci ID

A

1

0

P7A/2 P7AIZR

\b 5

10 15 Time heating a t 55 "C (rnin)

20

Fig. 3. Plot of the thermal decay of aGLU present in mouse and human cells and two hybrids. After 10 min at 55 "C the mouse cells and the hybrid P7A/2R had insufficient residual activity to measure accurately (C1 lD, less than 0.3%; P7A/2R less than 0.13%). A,Human fibroblast; 0 , mouse C11D; m, P7A/2; 0, P7A/2R. CONCLUSIONS

Using two methods and different sets of hybrids we have demonstrated that the gene determining human a-glucosidase is located on chromosome 17 in man. Evidence from the hybrid P7A/2 suggests that the gene is on 17q. SUMMARY

Hybrid clones (MOGs) were made between the mouse line RAG and a primary fibroblast line from an individual of the rare aGLU 2 phenotype. Fifteen independent primary clones and 32 subclones were tested for the presence of human aGLU after separation of the human and rodent enzymes by starch gel electrophoresis. Twenty-three other human-mouse hybrids from six different crosses were analysed for the presence of human aGLU by exploiting a difference in the thermostability of the human and mouse enzymes. The hybrids were also analysed for up to 25 other enzymes which were used as markers for different human chromosomes. Two of the MOG hybrids were karyotyped and karyotype data were already available for a number of the other hybrids. The combined results demonstrate that aGLU is located on chromosome 17, and probably on 17q.

Assignment of the humaN acid aGLU to chromosome 17

281

We would like to thank Dr Sue Povey and Dr Martin Bobrow for helpful advice and Alison Tanyar and Steve Jeremiah for running many of the gels.

REFERENCES

BOBROW, M. & CROSS,J. (1974). Differential staining of human and mouse chromosomes in interspecific cell hybrids. Nature, Lond. 251, 77. FELLOUS, M., COUILLIN,P., NEWPORT-FAUTES, C., FREZAL, J., BILLARDON, C. & DAUSSET, 5. (1973). Studies of human alloantigens on man-mouse hybrids: possible synteny between HLA and P system. Eur. J . Immun. 3, 543. HARRIS,H. & HOPKINSON, D. A. (1976). Handbook of Enzyme Electrophoresis in H u m a n Genetics (and Appendix 1977). Amsterdam, Oxford : North-Holland Publishing Company. VAN HEYNINGEN, V., CRAIG, I. W. & BODMER, W. F. (1973). Genetic control of mitochondria1 enzymes in human-mouse somatic cell hybrids. Nature, Lond. 242, 509. JONES, E. A., GOODFELLOW, P. N., KENNETT,R. H. & BODMER, W. F. (1976). The independent expression of HLA and p2 microglobulin on human-mouse hybrids. Somatic Cell Genet& 2, 483. MCALPINE, P. J., HOPKINSON, D. A. & HARRIS, H. (1970). Thermostability studies of the isozymes of human phosphoglucomutase. Ann. H u m . Genet., Lond. 34, 61. NABROLZ, M., MIGGIANO, V. & BODMER, W. (1969). Genetic analysis with human-mouse somatic cell hybrids. Nature, Lond. 223, 358. SOLOMON, E., BOBROW, M., GOODFELLOW, P. N., BODMER, W. F., SWALLOW, D. M., POVEY,S. & N ~ E LB. , (1976). Human gene mapping using an X/autosome translocation. Somatic Cell Genetics 2, 125. SWALLOW, D. M., CORNEY,G., HARRIS, H. & HIRSCHRORN, R. (1975). Acid a-gluoosidase: A new polymorphism in man demonstrable by ‘affinity’ electrophoresis. Ann. Hum. Genet., Lond. 38, 391. WINNIPEQCONFERENCE1977 (1978). Fourth International Work8hop on Human Gene Mapping. Birth Defects: Original Article Series (in the Press). New York: The National Foundation.