Hum. Genet. 55, 375-381 (1980)

© by Springer-Verlag 1980

Assignment of the Genes for Human Lysosomal Acid Lipases A and B to Chromosomes 10 and 16

Nguyen Van Cong, D. Weil, M. C. Hors-Cayla, M. S. Gross, S. Heuertz, C. Foubert, and J. Frezal

Clinique et Unit6 de Recherches de G~n6tique M6dicale (INSERM U 12) H6pital des Enfants Malades, 149, rue de Savres F-75730 Paris, Cedex 15, France

Summary. Twenty-three independent man-hamster (CH/HGPRT-) hybrids were analysed for human acid lipases and for some other human enzyme markers (PP, GOT1, PGP) and chromosomes. Eighteen in- dependent man-mouse ( L A / H G P R T - T K ) hybrids were analysed for human acid lipases, human enzyme marker PGP and human chromosomes.

Three fibroblasts from unrelated patients with WD (Wolman's disease), one fibroblast from a hetero- zygote for WD, and 15 normal fibroblasts were analysed for acid lipases.

The following results were obtained: 1) A positive correlation was observed between acid

lipase A and Chr. 10, and between acid lipase B and Chr. 16. In fact, among 23 independent man-hamster hybrids, 6 were Chr. 10+ PP+ GOT 1+ LIPA+ and 17 were Chr. 10- P P - GOT 1- L I P A - , and among 41 independent man-rodent hybrids 23 were Chr. 16+ P G P + L I P B + and 18 were Chr. 1 6 - P G P - L I P B - . Except for Chr. 10, the other autosomes were observed in hybrids LIPA-, and except for Chr. 16, the other autosomes were observed in hybrids LIP B- .

These results indicate that the gene for lipase A is on Chr. 10 and the gene for lipase B is on Chr. 16.

2) The acid lipase A is deficient in WD fibroblasts. Therefore the mutation responsible for WD is on Chr. 10. The B, C and at least three additional lipases were observed in WD fibroblasts and in WD hetero- zygote fibroblasts at pH 4.0 and with 4-methylumbelli- feryl oleate as substrate. The relationship between these different acid lipases are obscure. In the normal fibro- blasts from healthy control subjects a considerable variation in acid lipase A activity was observed. In some normal fibroblasts from healthy control subjects,

Offprint requests to: Nguyen Van Cong (address see above)

in which the lipase A is reduced, we observed the same acid lipase zymogram pattern as in WD heterozygote fibroblasts.

Introduction

In human fibroblasts, lysosomal acid lipase activity can be separated by gel electrophoresis into three isozymes, from the cathode to the anode, namely A, B, and C (Cortner et al. 1976; Coates et al. 1978). The acid lipase A is absent in fibroblasts and other tissues from patients with Wolman's disease (WD) (which is usually fatal within the first 6 months of life) and from patients with the related disorder, cholesteryl ester storage disease (CESD) (Cortner et al. 1976; Coates et al. 1978). Reduced lipase A is observed in heterozygotes for WD and CESD (Cortner et al. 1976; Coates et al. 1978). According to Cortner et al. (1976), Kyriakides et al. (1972), and Beaudet et al. (1974), the heterozygotes for WD or for CESD have about 50% of control lysosomal acid lipases activity but these results are not consistent with the data of Coates et al. (1978). The residual (>_ 10%) activity measured in WD and CESD is probably attributable to the B isozyme (Cortner et al. 1976). The relationships among bands A, B, C are obscure, although some preliminary results suggest that B differs from A in molecular weight and substrate specificity.

The lipase A (LIPA) locus was assigned to Chr. 10 by analysis of the karyotype and the Chr. 10 marker GOT1 in human hamster hybrids (Koch et al. 1979).

In this work we present a study of man-rodent hybrids, the analysis of different sorts of electro- phoretic bands of acid lipases observed in different

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376 Nguyen Van Conget al.: Assignment of the Genes for Lysosomal Acid Lipases

Fig.1. Acid lipases (LIP). l, human heterozygote for Wolman's disease; 2, mouse LA; 3, normal human fibroblast; 4, Wolman (fibroblast); 5, hamster CH; 6-9, man-hamster hybrids; 10-12, man-mouse hybrids. 6, L I P A - LIPB+; 7, LIPA + LIPB+; 8, LIPA +LIPB- ; 9, L I P A - L I P B - ; 10 and 11, LIPB+; 12, LIPB-

h u m a n f ib rob las t s (normal , W D , he te rozygote for WD) , and the results o f loca l iza t ion o f different loci respons ib le for acid lipases.

F o r this s tudy we ana lysed the Chr. 10 markers , soluble g lu tamic -oxa loace t i c t r ansaminase ( G O T 1 ) (Creagan et al. 1973; M c A l p i n e et al. 1975; Chern et al. 1975; Nguyen Van Cong et al. 1975), inorganic p y r o - phospha t a se (PP) (Nguyen Van Cong et al. 1975; F i sher et al. 1976; McAlp ine et al. 1975), and the Chr. 16 marke r p h o s p h o g l y c o l a t e phospha t a se (PGP) ( D o n a l d et al. 1979; Wei l et al. 1979; Povey et al. 1980).

Materials and Methods

Parental Cells

The parental rodent lines with selected markers were mouse CL1D, LA/TK-, HGPRT- and Chinese hamster CH/HGPRT-. The parental human cells were cultured fibroblasts possessing a balanced reciprocal translocation: Strain 34: 46,XY,t(X;2) (p22;q32); strain BL: 46,XX,t(X;2)(p22;q32); strain 56: 46,XY, t(X;5)(q21;ql 1).

Hybrid Cells

Ten 34-CH-independent man-hamster hybrids were obtained with Sendai virus, selected and maintained in HAT medium (hypoxanthine, amethopterine, thymidine) (Littlefield 1964; Davidson and Ephrussi 1965). Twelve 56-CH-independent man- hamster hybrids, one BL-CH man-hamster hybrid, and eighteen 56-LA-independent man-mouse hybrids were obtained with PEG (polyethylene glycol) (Pontecorvo 1975; Davidson and Gerald 1976), selected in HAT+ ouabaine medium and main- tained in HAT medium.

Other Human Cells

Fifteen normal fibroblasts, three unrelated WD fibroblasts and one fibroblast heterozygote for WD were used for acid lipase analysis.

Electrophoresis

Preparation of Cell Extracts. Human fibroblasts, mouse cells, hamster cells, man-mouse and man-hamster cells were washed in PBS three times and finally centrifuged at 2500 r/min for 5 rain. The final pellet was homogenized in an equal volume of distilled water. The suspension was then frozen and thawed three times.

Enzyme Studies

The enzyme markers used for the mapping are listed as follows: lipase A and B (LIPA and LIPB) (Cortner et al. 1976), in- organic pyrophosphatase (PP) (Fisher et al. 1974), soluble glutamic-oxaloacetic transaminase (GOT1) (Van Someren et al. 1974), and phosphoglycolate phosphatase (PGP) (Barker and Hopkinson 1978).

Staining

Fluorescent bands of acid lipase activity were observed after staining with 4-methylumbelliferyl oleate pH 4.0.

Chromosome Studies

The chromosomes were stained according to the Dutrillaux and Lejeune technique (1971). About 12 mitoses were analysed for different hybrids.

Results

Human Cells

With the e lec t rophore t ic technique used three p r inc ipa l bands o f acid l ipases were observed in var ious h u m a n f ibroblas ts , f rom the ca thode to the anode , namely A, B, and C (Figs. 1 and 2). The A band is very intense in compar i son with the B and C bands . The C band is the least intense. A cons iderab le var ia t ion in acid l ipase A act ivi ty was observed a m o n g 15 f ibroblas ts f rom heal thy con t ro l subjects. In two samples of these f ibro-

Nguyen Van Cong et al.: Assignment of the Genes for Lysosomal Acid Lipases 377

Fig. 2. Acid lipases. 1, normal human fibroblasts; 2-4, Wolman fibroblasts; 5, human fibroblasts from heterozygote for Wolman's disease

blasts, because of the prominence of A, the B and C bands were not observed easily. In the other fibroblasts from healthy control subjects, the B and C bands were observed more or less easily according to the promin- ence of the A band. In the fibroblast from a hereto- zygote subject for WD, because of the decrease of the A band, the B and C bands were observed clearly. For some fibroblasts from healthy control subjects in which the lipase A activity is reduced the B and C bands were also observed clearly; these normal fibroblasts present exactly the same acid lipase zymogram pattern as the fibroblast from a heterozygote for WD. In the three unrelated WD fibroblasts the acid lipase A was absent; we observed, in addition to acid lipase B and C, a series of additional acid lipases with low intentity. Compared with B and C lipases, one of these additional lipases is more cathodal and the others are more anodal. In the fibroblast f rom a heterozygote for WD and in a normal fibroblast, where lipase A was not prominent, the cathodal additional band was also observed.

Rodent Cells

In the mouse cell extracts, in the zone of human lipase A migration we observed a significant band of lipase, slightly more cathodal than human lipase A and a series of bands of low intensity, and more anodal than human lipase B. In the hamster cell extracts we observed only a series of bands of low intensity and more anodal than human lipase B (Fig. 1.).

Hybrids Cells

Human lipase A was identified easily in man-hamster hybrids. In man-mouse hybrids, because of the import-

ance of mouse acid lipase A, human lipase A is not observed easily (Fig. 1).

'Consequently only the man-hamster hybrids were used for the mapping of LIPA. Human LIPB was observed without difficulty in different man-rodent hybrids. Human lipase C, because of its identical migration with the mouse and hamster isozymes, was not identified in the hybrid cells. The additional human acid lipases, because of their weakness in activity, were not analysed in hybrid cells.

Different man-rodent hybrid series were analysed for human acid lipases and for some other human markers. The following results were obtained:

Man-Hamster Hybrids

1) An independent segregation between lipases A and B was observed. Among 23 independent hybrids three were EIPA+ LIPB+, eight were L I P A - L I P B - , three were L IPA+ L1PB- , and nine were L I P A - L IPB+ (Table 3).

2) A positive correlation was observed between Chr. 10, PP, GOT 1, and LIPA. In fact six hybrids were Chr. 1 0 + P P + G O T I + and 17 hybrids were Chr. 10- P P - G O T 1- (Tables 1, 3, and 4).

3) The percentage of dissociation between L I P A and the Chr. 10 is zero, whereas the percentage of dis- sociation between LIPA and the other autosomes (except Chr. 22) is significant (between 20 and 50) (Table 4).

4) Except for Chr. 10, the other autosomes were observed in hybrids L I P A - (Table 4).

5) Another positive correlation was observed between Chr. 16, PGP and LIP B. Among 23 in-

378 N g u y e n V a n C o n g et al.: A s s i g n m e n t o f the G e n e s fo r L y s o s o m a l A c i d L ipa se s

Table 1. A n a l y s i s o f h u m a n c h r o m o s o m e s a n d e n z y m e m a r k e r s

in 23 i n d e p e n d e n t m a n - h a m s t e r h y b r i d s

H y b r i d s E n z y m e s a C h r o m o - b

PP G O T 1 L I P A L I P B P G P s o m e s

10 16

3 4 - C H C . . . . . . .

E - - - + + - +

O . . . . . . .

T - - - + + - +

u + + + + + + +

V - - - + + - +

Z + + + + + + +

BY . . . . . . .

F U - - - + + - +

G T + + + - - + -

5 6 - C H C + + + - - + -

D . . . . . . .

F - - - + + - +

G . . . . . . .

K - - - + + - +

L - - - + + - +

N . . . . . . .

S - - - + + - +

V + + + + + + +

W - - - + + - +

X . . . . . . .

BY . . . . . . .

Tab le 2. A n a l y s i s o f h u m a n c h r o m o s o m e s a n d e n z y m e m a r k e r s in 18 i n d e p e n d e n t m a n - m o u s e h y b r i d s

H y b r i d s E n z y m e s a C h r o m o s o m e 16 b

L I P B P G P

5 6 - L A A + + +

B + + +

D - - -

F - - -

G + + +

H + + +

I + + +

J + + +

K + + +

L + + +

N + + +

O - - - - - -

p - _ _

R - - -

S - - -

T + + +

U + + +

W - - -

a E n z y m e s : + , the h u m a n e n z y m e is p r e s e n t in the h y b r i d cells;

- , the h u m a n e n z y m e is a b s e n t in the h y b r i d cells

b C h r o m o s o m e : +, the p e r c e n t a g e o f h y b r i d cells wi th the

h u m a n c h r o m o s o m e is _>30%; - , the p e r c e n t a g e o f h y b r i d

cells wi th the h u m a n c h r o m o s o m e is = 0%

B L - C H C + + + - - + -

a E n z y m e s : + , the h u m a n e n z y m e is p r e s e n t in the h y b r i d cells;

- , the h u m a n e n z y m e is a b s e n t in the h y b r i d cells

b C h r o m o s o m e s : + , the p e r c e n t a g e o f h y b r i d cells w i th the

h u m a n c h r o m o s o m e is _>30%; - , the p e r c e n t a g e o f h y b r i d

cells wi th the h u m a n c h r o m o s o m e is = 0 %

d e p e n d e n t h y b r i d s , 12 w e r e C h r . 1 6 + P G P + L I P B +

a n d 11 w e r e C h r . 1 6 - P G P - L I P B - ( T a b l e s 1 a n d 3) .

Man-Mouse Hybrids

A positive correlat ion was observed between Chr. 16, P G P and LIPB. In fact, a m o n g 18 independent man- mouse hybrids 11 were Chr. 16+ P G P + L I P B + and 7 were Chr. 1 6 - P G P - L I P B - (Tables 2 and 3).

Man-Rodent Hybrids

1) The correlat ion between Chr. 16, PGP, and acid lipase B was very positive. In fact among 41 in- dependent man- rodent hybrids 23 were Chr. 16+ P G P +

Table 3. S e g r e g a t i o n a n d c h r o m o s o m e a s s i g n m e n t o f ac id l ipases A a n d B in m a n - h a m s t e r (a) , m a n - m o u s e (b), a n d m a n - r o d e n t (c)

cell h y b r i d s

a L I P A L I P A L I P B

+ - - + - - + - -

L 1 P B + 3 9 C h r . 1 0 , P P , G O T 1 + 6 0 C h r . 1 6 , P G P + 12 0

- 3 8 - 0 1 7 - 0 1 1

b L I P B c L I P B

+ - + -

C h r . 1 6 , P G P + 11 0 C h r . 1 6 , P G P + 23 0

- 0 7 - 0 18

Nguyen Van Conget al.: Assignment of the Genes for Lysosomal Acid Lipases

Table 4. Segregation of human acid lipases A, B and chromosomes in man-rodent hybrids a

379

Chromosome LIP A/Chromosomes LIP B/Chromosomes

+/+ - / - + / - - / + +/+ - / - + j - - / +

1 1 15 4 2 7 16 14 1

2 0 16 6 1 5 13 17 4

3 2 9 4 8 17 9 5 9

4 4 7 0 9 14 7 5 8

5 2 8 4 6 11 12 7 3

6 6 9 0 8 13 6 10 12

7 1 13 4 1 11 13 8 4

8 5 8 1 9 18 8 4 7

9 3 12 3 5 5 15 18 3

[ ] 16 17 0 01 5 12 16 5 11 3 '10 3 7 14 10 7 7

12 5 9 1 7 15 7 6 10

13 6 13 0 4 7 13 16 5

14 1 12 5 4 5 16 17 1

15 1 12 4 2 5 14 16 1

[ ] 3 8 3 9 123 18 0 0 I 17 0 15 5 2 11 12 10 6

18 1 14 4 1 7 14 12 2

19 2 9 3 8 16 10 5 5

20 2 10 2 4 15 9 6 7

21 4 10 0 5 13 5 5 9

22 5 15 0 2 8 15 12 3

a Twenty-three independent man hamster were analysed for the segregation of human acid lipase A and chromosomes. Forty-one independent man-rodent (23 man-hamster and 18 man-mouse) hybrids were analysed for the segregation of human lipase B and chromosomes. In some cases less than 23 man-hamster or less than 41 man-rodent hybrids were used because we did not take into account the hybrids where one given human chromosome is present in less than 30% of hybrid cells

L IPB+ and 18 were Chr. 1 6 - P G P - L I P B - (Tables 3 and 4).

2) The percentage of dissociation between LIPB and the Chr. 16 was zero, whereas the percentage of dissociation between LIP B and the other h u m a n autosomes was significant (between 24 and 53).

3) Except for Chr. 16, the other autosomes were observed in hybrids LIP B - (Table 4).

The different results obta ined indicate that at least two independent genes control led the acid lipase synthesis: the gene for acid lipase A is on Chr. 10 and the gene for acid lipase B is on Chr. 16.

D i s c u s s i o n

In a pre l iminary study (unpubl ished data) we found a positive correlat ion between lipase B and P G P (a marker of Chr. 16), with a low percentage of dis- sociation between these two markers. The gene for h u m a n lipase A was recently assigned to Chr. 10 (Koch et al. 1979). To increase efficiency of the mapp ing

of the gene for h u m a n lipases A and B we selected a sample of 41 independent man- roden t hybrids accord- ing to the following criteria:

For the man-hamste r hybrids, we retained the hybrids + + + or - - - for the markers Chr. 10, PP, GOT1 and the hybrids + + or - - for the markers Chr. 16, PGP. The hybrids with the dissociated results - + + , - + - , - - + for the markers Chr. 10, PP, GOT1 and - + for the markers Chr. 16, P G P were el iminated.

For the man-mouse hybrids we selected the hybrids + + or - - for the markers Chr. 16, PGP.

Concerning h u m a n chromosome detection, we recorded C h r . + when the h u m a n chromosome was present in at least 30% of hybr id cells and Chr. - when the h u m a n chromosome was absent in all hybrid cells analysed. The results (+) (weakly positive) correspond- ing to a presence of the h u m a n chromosome in less than 30% of hybrid cells were eliminated.

In the enzyme analysis, only the results + or - (presence or absence of the h u m a n enzyme in the hybrid cells) were recorded. The results (+) (weakly positive)

380 Nguyen Van Cong et al.: Assignment of the Genes for Lysosomal Acid Lipases

or + (weakly ambiguous ) were e l iminated . Wi th our sample o f 41 independen t m a n - r o d e n t hybr ids selected accord ing to these cr i ter ia no d issoc ia ted results between acid l ipase A and Chr. 10 or between ac id lipase B and Chr. 16 were observed. The posi t ive cor- re la t ion de tec ted between l ipase A and Chr. 10 con- firms the ass ignment o f L I P A on this c h r o m o s o m e by Koch et al. (1979). The posi t ive cor re la t ion de tec ted between l ipase B and Chr. 16 indicates that at least one gene on Chr. 16 is necessary for the f o r m a t i o n of l ipase B. In fact the re la t ionship between the B, C and add i t iona l bands are unclear . As b a n d B is an inter- media te band , the B isozyme m a y be a he te ropo ly - meric isozyme, and it depends in this case on two different genes. In this hypothes is (as no d issoc ia ted result was found be tween Chr. 16 and LIPB) , one of these two genes is on Chr. 16, the o ther on a h u m a n c h r o m o s o m e a lways present in different m a n - r o d e n t hybr ids used. This second gene m a y be on Chr. X, as a segment of X is a lways present in the different hybr ids used. In fact our hybr ids issued f rom the roden t lines H G P R T - and selected in H A T med ium had to re ta in a segment o f h u m a n Chr. X, bear ing the h u m a n H G P R T gene. To test this hypothes is of a second gene of X for the f o r m a t i o n of l ipase B, a series of m a n - r o d e n t ( T K - ) selected for the h u m a n T K gene (Chr. 17) was used. We re ta ined only the hybr ids negat ive for the c h r o m o s o m e X m a r k e r G 6 P D . In these hybr ids , in the absence of X, the l ipase B was observed. This result indicates tha t Chr. X was not necessary for the f o r m a t i o n o f l ipase B. The l ipase B was observed only in the hybr ids P G P + . Consequen t ly it appea r s tha t only the h u m a n Chr. 16 is necessary for the f o r m a t i o n o f l ipase B. However the re la t ionships between B, C, and different add i t iona l l ipases remain unclear . Accord ing to our results the ca thoda l add i t i ona l l ipase, l ipase A, was the rmolab i le , while the B, C and anoda l add i t iona l l ipases were the rmos tab le . Af te r i ncuba t ion at 60°C for 20 min the l ipase A was m a r k e d l y reduced and the ca thoda l add i t i ona l l ipase was comple te ly inactive, whereas the B, C and a n o d a l add i t i ona l l ipases r ema ined clear ly active. NO3Ag (2mM) comple te ly inh ib i ted l ipase A, while the o the r different l ipases remained active. In W D f ibroblas t s only l ipase A was deficient, the B, C and different add i t i ona l l ipases being active. These different obse rva t ions indicate that l ipase A is in- dependen t o f o ther different l ipases. The gene for h u m a n l ipase A is on Chr. 10. The W D and C E S D muta t ions are on this ch romosome . The gene for l ipase B is on Chr. 16. The poss ib i l i ty tha t the B, C and a n o d a l add i t iona l l ipases depend on the same gene on Chr. 16 and tha t the ca thoda l add i t i ona l l ipase depends on a different gene has xmt been excluded. Our conclus ions is that at least two independen t genes (Chr. 10 and Chr. 16) con t ro l the acid l ipases synthesis.

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Received April 16, 1980