A third complementation group in xeroderma pigmentosum

5lutation Research, 22 (I974) 87 9~

((:'.Elsevier Scientific l'ublishing Company, Amsterdam

-Printed

in The Netherlands

87

A third complementation group in xeroderma pigmentosum As previously described complementation is observed in binuclear cells originating from fusions between cells of two types of XP, the classic and the DSC form of the disease s. In both types of X P severe skin lesions are produced following exposure to sunlight. DSC patients have severe neurological abnormalities in addition to the skin lesions. Both forms of the disease are inherited in an autosomal recessive way. I n I968 CLEAVER demonstrated that cultivated skin fibroblasts of these patients were not able to perform UDS after UV light exposure 2. A D N A repair mechanisn~ operating in h u m a n cells after UV-irradiation, which can be demonstrated by UDS, is impaired in cells of X P patients. The ability to perform repair was restored after fusion of cells of classic X P patients with cells of DSC patients, indicating t h a t different mutations are involved in these two forms of the X P syndromeS,L In the present paper we report a third eomplementation group represented by two patients ( X P 2 R O and X P 3 R O ) with moderately severe and relatively mild s y m p t o m s of the classic form of the disease ~,~. In both cases the first skin lesions were observed relatively late in childhood: Patient X P 2 R O was z4 years old when the first skin tumors developed, I n contrast to the cells of patients belonging to the earlier reported complementation groups (e.g. Fig. I ; X P 4 R O ) having low or negligible levels of UDS after UV light exposure, cultivated cells of these patients have a 5o°/'o U1)S activity in response to Ioo e r g / m m ~ UV light and slightly more after higher doses when compared with normal h u m a n fibroblasts~, ~ (Fig. ~). To study complementation, fusions were performed between X P z R O cells and cells of a classic X P patient ( X P I 6 R O ) and between X P 2 R O cells and cells of a DSC patient (XPz5RO). Hybridizations were performed as previously described*. Recognition of the hybrid binuclear cell was carried out b y means of atebrin staining of the sex chromoSOlnes in the interphase nuclei in a female/male fusion s. The labelling procedure, UV . ~ - [ m

I

{

3O

DB 20 C

~:io 0

100

'r~

500 erg/ram2

_

XP4RO

w

1000

Fig. I. Repair I ) N A s y n t h e s i s after U V exposure of control h u m a n fibroblasts (DB), cells from p a t i e n t X P z R O (moderately, severe case of X P ) and p a t i e n t X P 4 R ( ) (classic form of XP). Exposure t i m e : 3 days.

Abbreviations: DSC, De Sanctis-Cacchione; UI)S, unscheduled DNA synthesis; X I>, xerodeI'ma pigmentosum.

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exposure conditions and preparation of the autoradiographs were the same as described earlier 8. Table I presents the results of the fusion between the female X P 2 R O cells an(1 the male X P I 6 R O cells of the classic X P type. Unfused X P 2 R O cells exhibited 5 oc}/o of the U D S of normal h u m m l fibroblasts, while X P z 6 R O cells had nmch lower U D S activity after a dose of IOO erg/inm 2 UV light. In the fused cell population XP2RO/XPz6RO, binuclear cells having two female ( X X / X X ) or two male ( X Y / X Y ) nuclei were labelled at a level as seen in the t)arental X P 2 R O and X P I 6 R O cells, respectively. In the hybrid binuclear cells with a female and a male nucleus ( X X / X Y ) , the mean grain n u m b e r per nucleus ( 2 0 and 28 grains over female and male nuclei respectively) was comparable with the level of UI)S seen in control cells (2() grains per nucleus). The same result was obtained after fusion nf X P 2 R O cells with male X P 2 5 R O I)SC cells (Table II). In this experiment the parental X P e R O cells have about b5(~.() UDS compared with control hulnan fit)roblasts after a dose of IOO e r g / m m a UV light. UDS in the DSC cells did not exceed the background level. In the fused cell t)opulation "l',,\ I:;l~I~

1

UNSCHEI)ULI~;I) I)NA POPULATIONS, USING

SYNTHFSIS I , ' O L L O \ V I N G l ~ \ " L I G H T I~:XPOSURI~2 O F PARt-~NTAL A N D I~'tISF;I) C E L l . T\VO I3IFFERENT TYPES OF CLASSIC XERODERMA PIGMENTOSUM

T h e cells w e r e e x p o s e d t o i o o e r g / m n l 2 [TV l i g h t . T h e e x p o s u r e t i m e of t h e a u t o r a d i o g r a p h s w a s t w e e k . C6 is a c o n t r o l h u m a i l f i b r o b l a s t s t r a i n , w h e r e a s X P 2 R ( ) cells o r i g i n a t e f r o m a m o d e r a t e l y s e v e r e c l a s s i c c a s e of X P a n d X I q 6 R ( ) cells a r e f r o l n a s e v e r e c l a s s i c c a s e of X I ' . T h e d e s i g n a t i o n s u s e d f o r t h e X P cell s t r a i n s f o l l o w a p r o p o s a l l)y CLEAVER ANI) BOOTSMA f o r t h e s t a n d a r d i z a t i o n of t h e n o m e n c l a t u r e f o r X P cell s t r a i n s . T h e s t r a i n s a r e c h a r a c t e r i z e d b y a s e r i a l n u m / ) e r o r t)y t w o l e t t e r s (e.g. t h e i n i t i a l s of t h e p a t i e n t ) g i v e n in t h e i n s t i t u t e w h e r e t h e cells h a v e 1)een b r o u g h t i n t o c u l t u r e . T h e l a s t t w o l e t t e r s d e n o t e t h e c i t y w h e r e t h i s i n s t i t u t e is s i t u a t e d (e.g. R ( ) for R o t t e r d a m ) . T h e a v e r a g e n u m l ) e r of g r a i n s p e r n u c l e u s mad t h e s t a n d a r d e r r o r of t h e m e a n (SF.M) in p a r e n t a l m o n o n u c l e a r cells w a s e s t i m a t e d l)y i n c l u d i n g o n l y cells w i t h 5 o o r less g r a i n s o v e r t h e i r n u c l e i . ] n t h e fuse(l b i n u c l e a r cells t h i s e s t i m a t i o n w a s p e r f o r m e d b y c o u n t i n ~ o n l y cells w i t h o 5 ° g r a i n s o v e r e a c h of b o t h n u c l e i . F o r e a c h e s t i m a t i o n o n e sli(le w a s u s e d . H o w e v e r , f o r t h o s e e s t i m a t i o n s c o n c e r n i n g o n e t y p e of n u c l e u s in a. b i n u c l e a r cell, d i a g n o s e d l)y m e a n s of a t e b r i n s t a i n i n g , 2 t o 4 s l i d e s of t h e s a m e c u l t u r e w e r e u s e d . T h e s l i d e t o s l i d e v a r i a t i o n as e v a l u a t e d f r o m t h e s e e s t i m a t i o n s w a s v e r y s m a l l , c o n t r i b u t i n g less t h a n 2 ° , in t h e SI£M a s q u o t e d hel-e. T h e h a c k g r o u n d l e v e l of [TI)S in t h i s e x p e r i m e n t , m e a s u r e d as t h e m e a n l m m b e r of g r a i n s o v e r u n i r r a d i a t e d n u c l e i , w a s 2.o t7 o.e t o - . - ! o. 3 (5 o n u c l e i c o u n t e d in e a c h s t r a i n ) .

(a) Unschcd;tlcd 1)5:,4 s3mlh,'sis ;;; par~';;lal cells (:ell strai;;

Grain cott~ls per ;~ucl.eus ( m e a n ~: £'E31)

Number (~/ ;zltclei cou;ztcd

c()

2().r ! 0. 7

lOO

XP21{() (XN) XP[6R() (XV)

~3.7 ) o.0 3.9 ::: o.2

leo Joe

(b) UJ;sched*ded D N A sy;#hesis jbllowi;~g cell/lts~o~ lgltsioJz

Type of ;~,clcus i;z diagnosed binuclear cells

XPzlU)/XIq6t~()

XX XY XX XY

in in in in

XX/NX XY/XY XX/XY XX/XY

Grain cot~tnts /~er nitclc'~ls (mea~7 ~ SE.,II) I5.O

4

Nttmber (?/ izltc[ei colt~zl~d

(-).7

g6

4.2 ± 0.3 26. 4 ~_ 0 . 8 2 8 . o ~ o.S

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89

TABLE II UNSCHEDULED

DNA

SYNTHESIS

POPULATIONS,

USING

A MODERATELY

XERODERM

FOLLOWING SEVERE

1Jr

LIGHT

CLASSIC

EXPOSURE AND

A

DE

OF PARENTAL

AND FUSED

SANCTIS-CACCHIONE

CELL

FORM

OF

A PIGMENTOSUM

XPz5RO cells are from a DSC patient. The background level of UDS in this experiment, nleasured as the mean number of grains over unirradiated nuclei, was 2. 3 2 o.3 to 2.7 ~ o.2 (5o nuclei counted in each strain). For other details see legend to Table I. (a) U~zscheduh'd DNA synthesis in parental cells Cell strab~

Grain counts per nucleus (mean ± S E M )

C6 XP2RO (XX) XP25RO(XY)

21. 9 ~

I.I

~5.4 ± o.7 2.4 _- (>.3

Number o/ nuch'i counted 5°

5° 5°

(b) U~zscheduled D N A synthesis following cell fusiotz Fusion

Type of nucleus in diagnosed binuclear cells

Grain counts per nucleus (mean ± S E M )

Number of nuclei cou~ted

NP2

X X in XX/XX XY in XY/XY XX in XX/XY XY in XX/XY

~3.5 ~ 0.5 5.3 ~: 0.9 2o.8 ± o.8

96 86 95

22.2

95

RO/X1)25R()

~

0. 7

X P 2 R O / X P 2 5 R O , binuclear cells, in which b o t h nuclei were either of tile female ( X X / X X ) or the male ( X Y / X Y ) p a r e n t a l origin, exhibited UDS comparable with the p a r e n t a l cells (i 4 a n d 5 grains per nucleus, respectively). UDS found in h y b r i d binuclear cells, with a female a n d a male nucleus ( X X / X Y ) was at the level found in control h u m a n fibroblasts (about 2I grains per nucleus). A second cousin of p a t i e n t X P 2 R O also suffered from the disease, showing relatively mild s y m p t o m s a n d i n t e r m e d i a t e levels of UDS (patient X P 3 in ref. r; X P 3 R O in Table III). Cells of this p a t i e n t were hybridized with X P 2 R O cells a n d the control fusion X P 2 R O / X P 2 R O as well as X P 3 R O / X P 3 R O were performed. Identification b y a t e b r i n staining of the three types of binuclear cells in the fused cell population X P 2 R O / X P 3 R O was n o t possible because b o t h p a r e n t a l strains were of female origin. In the three fused populations IOO binuclear cells were r a n d o m l y selected and the n u m b e r of grains per nucleus following a UV light dose of IOO e r g / m m '2 was counted (Table III). The n u m b e r of grains c o u n t e d above the nuclei of the p a r e n t a l cells X P 2 R O a n d X P 3 R O were n o t significantly different. The grain n u m b e r s found over the nuclei of binuclear cells in the fused populations X P 2 R O / X P 2 R O a n d X P 3 R O / X P 3 R O were comparable with the n u m b e r s found over the nuclei of their respective p a r e n t a l cells, lqnally, no difference was observed between the n u m b e r of grains c o u n t e d above the nuclei of binuclear cells in the fused p o p u l a t i o n X P 2 R O / X P 3 R O a n d the n u m b e r found over b o t h p a r e n t a l cells. These d a t a suggest t h a t the same m u t a t i o n is present in these two X P patients, which is expected because of the family relationship. However, the recognition of small changes in the n u m b e r of grains over tim h y b r i d b i n u c l e a r cells, might have been h a m p e r e d b y the presence of binuclear cells

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T,-\ 1~1.I~ 111 UNSCHEDULED I ) N A SX/NTHESIS FOLLO\VING [ ; \ LIGHT EXPOSURE O1; I'ARENTAL AND FUSED CELL POI)L'LATIONS, USING CELLS OF T\VO RELATED PATIENTS, BOTH CLASSIC MOI)ERATELY SEVERE CASES ()P" XEROI)ERMA PIGMP2NTOSUM ( 3 is a c o n t r o l h u l n a n l i b r o b l a s t s t r a i n , w h e r e a s X P 3 R ( ) cells o r i g i n a t e f r o m a s e c o n d c o u s i n o l X P e R ( ) p a t i e n t , h a v i n g r e l a t i v e l y m i l d s y m p t o m s of t h e c l a s s i c f o r m of t h e d i s e a s e . T h e b a c k , ~ r o u n d level of I ; ] ) S in t h i s e x l ) e r i m e n t , m e a s u r e d a s t h e I]]OEHI IIIIlHBeF of g r a i n s o v e r u n i r r a d i a t e d n u c l e i , ~ a s 3.2 : o. 3 t o 3 o : o.3 5o n u c l e i c o u n t e d in e a c h s t r a i n ) . F o r o t h e r d e t a i l s see l e g e n d t o T a b l e I.

(a) 1.'~ls~kcdldcd D N d

sy~dkcsis lJl /)arenla/ cells

Cell strait1

Grail1 coztJlls per ~ucltuts (mca~l 5"1£3l)

.\'umber of nuclei coltuh'd

C3 XI}2R() (XX)

19.o 1.30

Joo lo{}

XP3R{)(XX)

~4-7 i (}.{}

: o. 5 ! {I. 5

~o{}

(b) U~sctu'dlth'd DNd syutkcsis./o/lowing cdl /usw~7 Fusion

Grain counts per nucleus i[tl blltltclcar c~lls (mca~l Nl£~]I)

\ l i m b e r of nltclci coltnlcd

X P 2 R( )/XI'21~( ) XP3RO/XI}31({} XP2R()/XP31(()

I-'-7 ().4 ~5.-' J o.5 14.8 I o. 5

2oo 2oo 2oo

originating frolll one parent, which have been included in our calculations. Since the s t a n d a r d error of the mean of the grain n u m b e r per nucleus above binuclear cells is

similar in all three fused populations, and the frequency distribution of these grain countings in the fused population XP2RO/XP3RO showed one peak (not presented here) there is no evidence of a c o m p l e m e n t a t i o n effect. F r o m these experiments we conclude t h a t a third c o m p l e m e n t a t i o n group in X P is represented b y the X P 2 R O a n d X P 3 R O cell strains, which show i n t e r m e d i a t e

levels of UDS. Biochemical analyses of these cell strains sofar 5 have indicated that an early step of DNA repair is defective in each of the three complementation groups. These d a t a tire in agreement with prior work on the classical X W a n d the DS(" svndronle:/,Q

R e c e n t l y KRAEMER et al." presented evidence for at least three different complementation groups in XP. Experiments are in progress to compare the three groups t)resented in this paper and those reported by KRAI~;.m~Ret al.

Detbartme~zl of Cell Biology am/GcPvtics, Erasmus University, Rotterdam (Tkc Nctkerlal~ds)

T ) E. A. DE \,~'EERI-KAsTI£LEIN

\V. KEUZER D. BOOTSMA

I I{OOTSMA, l)., M. P. ~IULDIgR, l;. POT AND J . A. ('OttEN, 1 ) i f f e r e n t i n h e r i t e d l e v e l s of D N A r e p a i r r e p l i c a t i o n in x e r o d e r m a t ) i g m e n t o s u m cell s t r a i n s a f t e r e x p o s u r e t o u l t r a v i o l e t i r r a d i a t i o n , M u t a t i o n /fes., 9 (197 o) 507 .516. 2 CL]tAVER, J . E., D e f e c t i v e r e p a i r r e p l i c a t i o n of D N A in x e r o d e r m a p i g m e n t o s u m , Nature, -,iS (]968) 652-650. 3 CLEAVI0;R, J. E., X e r o d e r m a p i g m e n t o s u m : a h u m a n d i s e a s e in w h i c h a n i n i t i a l s t a g e of I ) N . \ r e p a i r is d e f e c t i v e , Proc. Natl. Acad. ~qci. ( U . S . ) , 63 (1969) 4 2 8 4.3.54 CLEAVER, J . l~., AND J . l(. TROSEO, A b s e n c e of e x c i s i o n of u l t r a v i o l e t - i n d u c e d c y c l o b u t a n e d i m e r s in x e r o d e r m a p i g m e n t o s m n , Pkotockem. Pkotobiol., i T ( i 9 7 o ) .547 .5.5°.

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91

5 ]?~LEIJER, W . J., l';. A. DE WEERD-KASTELEIN, .~'[. L. SLUYTER, W . I~EIJZER, J. DE W I I AYD D. t~JOOTSMA, U V - i n d u c e d D N A repair s y n t h e s i s in cells of p a t i e n t s w i t h different forms of

x e r o d e r m a p i g m e n t o s m n a n d of h e t e r o z y g o t e s , laJutation Res., 2o (1973) 417 428. 6 NRAEMER, ~x:. H., J. t-I. I~OBBINS AND H. G. CooN, T h r e e ~'enetic f o r m s of x e r o d e r m a p i g m e n t o -

s u m , Clin. Res., 21 (1973) 48o. 7 SETLOW, R. B., J. D. REG.¢N, J. GERMAN AYD \V. L. CARRn:.R, E v i d e n c e t h a t x e r o d e r m a t l i g m e n t o s u m cells do n o t p e r f o r m t h e first step ill t h e repair of ultraviolet d a m a g e to their DNA, Proc. Natl. Acad. Sci. ( U . S . ) , 64 (:969) Io35-IO4I. g WEERD-I'~ASTELEIN, E. A. DE, ~V. tXEIJZER AND D. BOOTSMA, Genetic h e t e r o g e n e i t y of xeroderm a p i g m e n t o s u m d e m o n s t r a t e d b y s o m a t i c cell h y b r i d i z a t i o n , Nature N e w t~iology, 238 (i972) 80 83 . 9 WEERI) KASTELEIN, ]L. A. DE, \~7. j . KLEIJER, ~'I. L. ~LUYTER AND \ g . [¢EIJZER, Repair replication in h e t e r o k a r y o n s derived from different repair-deficient x e r o d e r m a pignlentosun~ strains, Mutation Res., 19 (r973) 237-243. Received

August

z6th, i973