Mutation Research, DNA Repair, 273 (1992) 97-118 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00

97

MUTDNA 00182

The XPD complementation group Insights into xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy R.T. Johnson and Shoshana Squires Cancer Rt'scatch Campaign, Mammalian Cell DNA Repair Research Group, Departmou of Zoology, Unicersityof Cambridge, Cambridge CB2 3EJ (Great BritabO (Accepted 20 May 1991)

Keywords: XPD complementation group; Xeroderma pigmentosum, group D; Cellular DNA repair, defects in; Neurological abnormalities: Trichothiodystrophy; Cockayne's syndrome

Summary The xeroderma pigmentosum complementation group D is defined by more than 30 unrelated individuals of whom less than half show major abnormalities of the central nervous system, once considered to be the hallmalk of the group. Fibroblasts from the great majority of these individuals show very considerable sensitivity to UV light in vitro despite the fact that the cells carry out what appears to be substantial excision repair, as judged from repair synthesis and incision activity. This article reviews the XPD group and the defects in cellular DNA repair and examines the lack of correlation between repair and the appearance of neurological abnormalities. The article also discusses the recent awareness that at least some members of two other inherited conditions, trichothiodystrophy and Cockayne's Syndrome, carry mutations in the XPD gent.

By the mid 1970s the XPD complementation group was established on the basis of a few individuals all of whom displayed severe photosensitivity, tumours and marked neurological prcblems (Robbins et al., 1974; Cleaver and Bootsma, 1975). Since then there has been a steady increase in new XPD cases, but many of those affected individuals do not conform to the original pattern. As a group we now see that XPD individuals show great variety in the severity of their condition in terms both of tumour incidence and in abnormalities of the central nervous system. Moreover,

Correspondence: Dr. R.T. Johnson, Cancer Research Campaign, Mammalian Cell DNA Repair Research Group, Department of Zoology, University of Cambridge, Cambridge

CB2 3EJ (Great Britain).

defects or deficiencies in the immune response are becoming evident in this group. At the cellular level XPD has attracted much attention bccause great UV sensitivity is coupled to moderate and in some cases considerable repair synthesis. in addition to presenting new data on repair in some of the XPD mutants the purpose of this survey is to examine the diversity of cellular defects associated with this complementation group. Sadly, however, analysis of XPD mutants is as yet spnradic; very few have been the subject of wide investigation and the picture is therefore fragmentary. The recent demonstrations that fusions between XPD cells and cells from certain individuals with trichothiodystrophy (TTD), or XPH fail tO result in complementation of nucleotide excisiGn-repair defect suggests that mutations in the

98

XPD gene are responsible for several inherited human diseases (Wood, 1991). Each of these conditions, Cockayne's syndrome (expressed by XPH individuals), trichothiodystrophy and xeroderma pigmentosum (XP) are photosensitive, but only in XP (and in some CS in XP individuals) is this associated with the early onset and abundance of tumours in sun-exposed skin.

Abundance and distribution of XPD individuals Table 1 provides information about the abundance and geographic distribution of the three most common excision-defective XP complementation groups, A, C and D. These data, though almost certainly incomplete, comprise a reasonable guide to the occurrence of these mutations, For the XPD group the data represent unrelated individuals, and it is clear that their distribution is uneven with Europe providing more than two thirds of the known cases. Even excluding the German collection, Europe still provides more than half the individuals. This distribution contrasts sharply with that from Japan which is rich

TABLE I GEOGRAPHIC DISTRIBUTION OF THE MOST COMMON EXCISION DEFECTIVE XP COMPLEMENTA.

TION GROUPS"

North-America Europe excluding Germany

Germany lapan [~gypt

xp group A C 3 5 10 14 2 7 21 i 7 I2 43 39

Db 6~ 14 ~ 12 3~ 35

Adapted from Fischer et al. (1982), h D mutations from unrelated families; does not include the 8 independent TTD type 2 or 2 TTD type 3 mutations, " Tolal number of cases 7; 1 sib pair. d Tolal number of cases 17:3 sib pairs, including the previously designated XPH sibs who also show Coekayne's Syndrome features and the recently assigned XPD-CS individual XPgBR. c Total number of cases 4; 1 sib pair,

in XPA patients but where only three unrelated XPD families have been identified (Ichihashi et al., 1988). Nomenclature of XPD strains is provided in Appendix Table 1. The limited association between XPD mutations and severe neurological abnormalities In the early days of XP work it seemed clear that individuals falling into the XPD group were uniformly afflicted with severe neurological abnormalities (Robbins et al., 1974; Cleaver and Bootsma, 1975), sharing this characteristic with XPA individuals. Fibroblasts from XPA and D also proved to be more UV-sensitive than from other XP groups (Andrews etal., 1978). However, as Table 2 shows, the tight XPD association with abnormal neurology is now much less convincing (Pawsey et al., 1979). Taking the 35 XPD cases where data have been given more than half of the individuals show normal rather than abnormal neurology. Excluding the more recently charaeterised Mannheim group the proportion of neurologically normal individuals is 40%. The two children sharing features both of Cockayne's syndrome and XP, previously classified as belonging to a separate XP complemcntation group, XPH, display neurological symptoms associated with CS rather than with XP (Lafforet and Dupuy, 1978). It is worth pointing out that on diagnosis of XP a number of the individuals without apparent neurological symptoms were very young (Table 2) and they may therefore develop late onset CNS abnormalities (Kraemer and Slor, 1985). The XPD mutation is clearly associated with sun-induced skin tumours in many of the individuals though in a few, possibly because of effective sun screening, no tumours have been recorded (Table 2). It is also becoming clear that the quality of immune response may be an important consideration in the development of skin tumours (Bridges, 1981; Lehmann and Norris, 1989). Very few XPD individuals have been examined for the normality of their immune system (Table 2) but all that have show some unusual feature (Lafforet and Dupuy, 1978; Norris etal., 1990). Only two of these four individuals, however, are known to have developed skin tumours.

99

Verification of XPD mutations Traditionally XP cells have been placed into different complementation groups on the basis of short term studies of the recovery of normal levels of unscheduled DNA synthesis after U v challenge (Robbins et al., 1974). However, the work of Giannelli et al. (1982) indicates that caution must be exercised in the analysis of XPD complementation because this is slc,,v and may not reach normal levels of UDS. Alternative short-term assays for establishing complementation groups include recovery of growth potential of population containing fused cells after UV (Cleaver, 1982; attribution of XP1PO and XP108LO as XPD via lack of ability to complement XP2NE), or recovery of unscheduled DNA synthesis in XP x XP fused populations (Lchmann and Stevens, 1980; attribution of XPIDU via lack of ability to complement XP1BR). In many cases it is difficult to identify which XPD strains have been used to verify the status of others in UDS or other assays. Table 3, which is almost certainly incomplete, lists XPD and TTD provenance (in those TTD cases which carry the XPD mutation). A full list of XPD attribution is desirable,

Given some of the difficulties of measuring complementation by UDS in XPD (slew kinetics, sometimes very high levels of repair synthesis) an alternative route is by means of generating permanent hybrids between different XPD mutants. This can only be achieved, however, if one of the partners is a permanent cell line. There are 3 XPD lines, the SV4O transformed XP6BE and XP3NE (NIGMS Catalogue of Cell Lines 1990/1991) and the UV sensitive XP102LO - HeLa hybrid (HD2) (Johnson et al., 1985). Indeed, it was because of the unusual origin of HD2 that c r o s s e s w e r e made with other XPD fibroblasts (and w i t h X P I 0 2 L O itself) t o e s t a b l i s h its genetic identity (Johnson et al., 1985). Hybridisation with fibroblasts from each complementation group subsequently established that XPH could not be complemented by XPD and therefore should be included in the XPD group (Johnson et al., 1985, 1989). Permanent hybrids offer several advantages in the study of complementation. First they allow

the biological significance of repair to be established, and second, a wider range of repair chatacteristics can be analysed than is possible in short-term heterokaryons (Johnson et al., 1989; Johnson, 1989 and Fig. 1). Judged by complementation for UDS HD2 × XPH or x XPD heterokaryons could be considered positive, although the extent of improvement was slight compared with crosses involving fibroblasts from other complementation groups (Johnson et al., 1985, 1989). Perhaps the XPD x XPD improvement in UDS reflects a degree of intragenic complementation, though as might be expected the resulting proliferating hybrids did not show iraproved UVresistance.

20

~s

~ ~-g ~g ~~ o ,. ~

/~-

~

*..... jr.-" .. ,, ...;..-~'~':-"". . . . . . . . . =~""":-~-2-.. /~.7~..¢:':,, 4~~ ,~i/

10

I i / _ ~ ~ ' "

'. : e / ~ 0J ~ 0 2

' 4

' e

' o

uv dos, I Jm "~j Fig. 1. Accumulation of DNA breaks over 40 rain after irradiation as a funclionof UV dose in wild-typefibroblasts. [3H]Thymidine-prelabelled culturesfrom (48BR(o o): BCL-DI ( u - - t , I ; XPD heterozygotc fibroblasts. XPHI02LO ( • .... • ) : CRL 1159 (,~ .... • ) : CRL 1202 (0 .... , ) . and in hybrids produced between the permanent XPD cell line HD2TG (ra ra) and either XPD fibroblasts XPIIlLO (3/ll/e, • • ) : XPI07LO ( 3 / X F . , ,). or trichothiodystrophy fibrGblasts T T D 2 G L (3/GL2CCI. • Ill) were plated at 2 x 10"~ cells per 35-ram dish one day before irradialion. The cells were incubated for 40 rain before and after UV-irradiation with hydroxyurea and ~,:osine arabinoside. After the 30-rain postirradiation period the cells were lysed in alkali and the DNA eluted from hydroxyapatite columns to determine the frequency of repair-related DNA breaks. For other details see Squires and

Johnson(1988).

100 T a b l e 4 shows t h e c u r r e n t s t a t u s o f f u s i o n s involving XPD x XPD partners. These studies w e r e u n d e r t a k e n to verify the X P D s t a t u s o f several X P D fibroblast strains a n d one T T D strain

in p r o l i f e r a t i n g hybrids. F o r t u n a t e l y t o d a t e all crosses i n v o l v i n g D × D f u s i o n s h a v e f a i l e d to g e n e r a t e h y b r i d s t h a t have r e c o v e r e d U V resist a n c e o r e x c i s i o n r e p a i r ability.

TABLE 2 CLINICAL SYMPTOMS IN XPD INDIVIDUALS Individual I

Sex

Age

Neurological problems 2

XPIPO " ~"XP5BE b S L XP6BE h XP7BE b XPI02LO ~ XPI04LO ~ I XPI07LO c

M F F F F F M

2 26 20 !I 18 !I 5

Yes. severe Yes. severe Yes. severe Yes. severe No No 3 No

F

aborted

S

Immune systems

Turnouts 4

T b T h T b none c none c < 50~ of normal NK cytotoxicity d

L XPI08LO ~

XPII I LO c

M

7

No

< 50% of normal NK

T c.d

cytotoxicity d

XPI35LO d

M

7

XPIBR ~ XPIKC" XPKABE ,.t

F F F M F M F

6 9 22 23 28 28

M F

I0

X P I N E "'"

I XP2NE "'~ XP3NE ~`" XPI DUh XPlSPV t XPI6PV * XPl7PV S I XP-CS-2 J "Patient 7" J XP8BR k XPI0TO I XP43KO m r XP58TO m. S L XP59TO m. XPITBE h S

XP9MA a XP 12MA a XP 17MA ~ XPI8MA a xPIgMA ~ XP33MA " XP36MA ~ XP39MA p XP40MA p XP46MA p XP47MA p XP55MA q

M F M F M F M F F

M

< 50% of norm~.l NK cytotoxieity d

none d

Yes. severe

2 2 51 31 8 6

Yes. severe Yes, severe Yes. severe Yes. severe

Yes. CS-like Yes. CS-like Yes. CS-like No

No Borderline No

14

Yes. severe

11 42 I1 38 33

No No No No No No

T T

Diminished PHA response

T none T T none none T T

none none T

I01 Repair defects in XPD

(I) UV-sensitit'ity offibroblasts and limited host-cell reactivation of irradiated viruses The early work of Robbins et al. (1974), Kraemer et al. (1976) and Andrews et al. (1978) estabfished that XPD fibroblasts were extremely and uniformly UV-sensitive, second only to XPA cells and substantially more sensitive than XPC. Excepting some of the cell strains from the Mannheim collection which arc unusually resistant to ultraviolet (Appendix Table 3) (and express considerable excision repair ability), all reports (Appendix Table 2) indicate substantial killing by U V with virtually no shoulder on the survival curve. Compared with XPA cells, however, when quiescent holding is imposed after UV, X P D fibroblasts (XP7BE, XP3NE, XPKABE) show improved survival in line with their very limited repair capabilities (Simons, 1979; Kantor and Hull, 1984). Reactivation of U V damaged adenovirus 2, in X P D cells, assayed by the production of viral antigen (Rainbow, 1980) or viable virus (Day, 1976; Abrahams and van der Eb, 1976; Arase et al., 1979) has been examined in very few mutants [XPI02LO, through its derived hybrid HD2, Johnson et al., 1985; XPSBE, XP6BE (sibs) and

XP7BE (Day, 1976; Abrahams and van der Eb, 1976; Arase et al., 1979)]. In each of these strains repair of damaged virus is greatly depressed compared to wild-type cells (Appendix Table 2). Enhanced reactivation of damaged virus, an indicator of an inducible excision-repair response, has been observed in one XPD strain (XP3NE) for Herpes simplex virus type I (Abrahams et al., 1984). These studies report a similar time scale of enhanced reactivation in XPD, XPC and XPA cells as is found in normal cells, and also a similar magnitude. For XP cells, however, the amounts of UV given to the cells and to the virus were considerably less than for WT (8-fold and 4-fold respectively).

(2) UV-induced incision activity in XPD cells XP cells are deficient in one or more early steps of the excision-repair pathway. The ratelimiting UV-induced incision step is determined most readily by the use of repair-synthesis inhibitors, where incomplete repair sites accumulate as D N A breaks. These breaks are quantitared by means of alkaline unwinding followed by centrifugation in alkaline sucrose gradients or by hydroxyapatite chromatography (Collins and Johnson, 1984). The time course of UV-induced excision repair consists of two kinetics; a rapid

Notes to table 2 t S indicates affected sibs,

a blank indicates not known, •~ slightly deaf. 4 blank indicates not known, '~ NIGMS Databank. Robbins et al. (1974); Andrews et al. (1978). c Pawsey et al. (19"/9). d Norris el al. (1990), patient I, XPI35LO; patient 4, XPI07LO; patient 5, XPIIILO. e C.F. Arlett, personal commuaication. t ATCC Database. Thrush et al. (1974). h B. Johnson, personal communication. i M. Stefanini, personal communication. J Lafforet and Dupuy (1978). k D. Bootsma and A. Lehmann, personal communication. t Takebe et al. (1977); Mamada et al. (1988). m Ichihashi et al. (1988). " Fujiwara and Sato (1985). o Fischer et al. (1982). P Thielmann et aL (1985). q Thielmann et al. (1986).

102 one during the first 6 h after irradiation and a slower one that continues for at least a day. Table 5 and Fig. 2 provide comparative data

higher initial rate of arrested repair-site accumulation, and at low U V doses accumulate D N A breaks at frequencies similar to normal cells. On

for the early incision activity in a selection of XPD strains immediately after UV-irradiation. Fig. 2 shows that in the presence of DNA synthesis inhibitors the accumulation of DNA breaks during the first 40 min after UV is dose-dependent. The dose-response curves of break frequencies in the majority of the XPD strains are very similar, the number of inhibitor-arrested repair sites increasing to a maximum of around 3 breaks/10 ') dalton, which is only 10-20% of the normal level (Table 5; Appendix Table 2). There are a few exceptions which show significantly higher frequencies of DNA break accumulation. XP-CS-2 cells (previously assigned XPH) show a

the basis of its incision kinetics the XP-CS-2 cell strain was considered to be quite distinct from the 'typical' XPD cells (Squires and Johnson, 1988). The other mutant, XP1PO, can be singled out as the most unusual XPD cell strain. It shows a high level of UV-induced incision, about 60% of that of normal, as measured by DNA break accumulation and UDS (Fig. 2 and Table 5). It has been reported that XP1PO has about 2 / 3 of wild-type ability to remove angelicin monoadducts (Cleaver and Gruenert, 1984) and about 40% of normal UV induced UDS (Cleaver et al., 1984). A detailed kinetic and biochemical analysis of repair in XP1PO cells is now in progress.

TABLE 3 ATTRIBUTION OF XPD OR TTD MUTANTS BY HETEROKARYON ASSAY OF UNSCHEDULED SYNTHESIS OR REPLICATION RECOVERY AFTER UV Designated XPD mutation XPSBE (XP6BEsib)

Fusions for attribution The initial XPD strains;

XPTBE

will complement XPA, B and C but not each other

XP$BE

XPIO7LO

Author Robbins el al. (1974)

Pawsey cl al, (1979)

XPlllLO XP3NE. XP2NE XP3NE

Identifiedas X P D unpubllsh~ddata of E. de W¢~rd-Kastel¢in,W, Keijzer and D, Bootsma citedin Dc W¢¢rd-Kasteleinet al.(1976)

XP3NE

XPI02LO XPI04LO XPI08LO

XPIBR

XPIDU

Lehmann and Stevens(1980)

XP2NE

XPIPO XPI08LO

Cleaver (1982)

XPS9TO

XP43KO

lchihashi et al. (1988)

XPKABE and XPITBE

Identified as XPD, data unpublished(Andrewset al., 1978)

XP3NE

"VrDI PV TTD2PV

TTD 1PV

TTD2PV TTD4PV TTD4PV

"I~D4PV

TTD2GL

Lehmann et al. (1988)

TTD4PV

TTD1BI

Lehmann et al. (1988)

Stefanini el al. (1986)

103 TABLE 4

8

.

COMPLEMENTATION ANALYSIS USING PROLIFERATING HYBRID CELLS -

Permanent XPD line HD2TG and

Hybrids with no recovery of UV R

Hybrids with recovery of UV R

XP102LO sJ XPIBR sJ XP1NE s XPI07LO XPIIILO XP6BESV s XP-CS-2 sj "VI'D2GL s,t XPI02LO s

and XPA, B, C, E, F and G

6

/

~

SJ

XP6BESV and

I

-

~~-~

m o~

and XPI s (i.e. XPC), XPA (XPSV2OS) s

i

. , r 4

a o Q. v

HD2TG s

~

XP-CS-2 XPIPO s s XP7BE s

Fig. 2 shows the time courses of DNA break accumulation of UV-irradiatcd fibroblasts of normal, XPI PO, XP-CS-2, and two representitives of the low incision activity XPD group, XPIBR and XP135LO. In the normal and XPIPO cells, in the continuous presence of DNA-synthesis inhibitors, D N A breaks start to accumulate immediately af-

,

0

-

-

~

~

, ~

0~ 0

t

2

4

6

Fig. 2. Accumulation of DNA breaks as a function of UV dose in XPD fibroblasts. XPIPO (D C3): XP-CS-2 to--o): XPI07LO (& z~), XP7BE

(,

,): XPIDU (11 XPIBR: ( • ~

•

ii): XPI35LO (