Journal of Neurology, Neurosurgery, and Psychiatry 1984;47:391-398

Hypersensitivity to DNA-damaging agents incultured cells from patients with Usher's syndromeand Duchenne muscular dystrophyJAY H ROBBINS, DOMINIC A SCUDIERO,J FUJIO OTSUKA, ROBERT E TARONE,*ROGER A BRUMBACK,§ JONATHAN D WIRTSCHAFTER,II RONALD J POLINSKY,tSUSANNA F BARRETT, ALAN N MOSHELL, RONALD G SCARPINATO, MARY BGANGES, LINDA E NEE,t SHARON A MEYER,: BRIAN E CLATTERBUCKt

From the Dermatology and Biometry* Branches, National Cancer Institute, Laboratory of Clinical Science, tNational Institute of Mental Health, National Institutes ofHealth, Bethesda, Maryland, ChemicalCarcinogenesis Program, t NCI-Frederick Cancer Research Facility, Frederick, Maryland, MuscularDystrophy Association Clinic, § Fargo, North Dakota, Department of Ophthalmology, || University ofMinnesota Hospitals, Minneapolis, Minnesota, USA

SUMMARY Lymphoblastoid lines from nine Usher's syndrome (recessively inherited retinitis pig-mentosa and congenital sensorineural deafness) patients (representing eight kindreds) and fromten Duchenne muscular dystrophy patients (representing seven kindreds) showed a small butstatistically significant hypersensitivity to the lethal effects of X-rays, as measured by the cellularability to exclude the vital dye trypan blue, when compared with lines from 26 normal controlsubjects. Fibroblast lines from the Usher's syndrome patients, treated with X-rays or theradiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine, also showed astatistically significant hypersensitivity when compared to normal fibroblast lines. These findingsare consistent with the possibility that defective DNA repair mechanisms may be involved in thepathogenesis of these degenerative diseases.

The term " abiotrophy" was introduced by WRGowers in 1902 to signify the premature death ofexcitable tissues occurring in the absence of anyhistopathological evidence of the aetiology.' Gowersincluded primary neuronal degenerations and themuscular dystrophies among the abiotrophies. In1919, E Treacher Collins classified retinitis pigmen-tosa, with its premature degeneration of photo-receptors, as an abiotrophy.2 The concept of abiot-rophy has recently been reviewed.3The cause of the premature death of excitable tis-

sue in these genetic disorders has long beenunknown. We have suggested that the abiotrophicdegeneration of neural, retinal, and muscle cellsmight result from the accumulation of damagedDNA.4-'3 This DNA-damage hypothesis is based

Address for reprint requests: Jay H Robbins MD, Building 10,Room 12N238, National Institutes of Health, Bethesda, Maryland20205, USA.

Received 2 August 1983 and in revised form 23 October 1983.Accepted 5 November 1983

primarily on information obtained from the study ofthe autosomal recessive, sunlight-sensitivity diseasexeroderma pigmentosum, one form of whichinvolves a primary neuronal degeneration.4 Cul-tured cells from patients with xeroderma pigmen-tosum are hypersensitive to the lethal effects ofultraviolet radiation and ultraviolet-mimetic chemi-cals because of inherited defects in the repair ofDNA.4 '4 Fibroblasts and lymphoblastoid lines fromxeroderma pigmentosum patients with an earlyonset of neuronal degeneration are the most sensi-tive to the lethal effects of ultraviolet radiation,while cells from patients with later onsetneurodegeneration or without neuronal degenera-tion are less sensitive.56 Cells from patients withataxia telangiectasia, another inherited primaryneuronal degeneration,'5 are hypersensitive to ioniz-ing radiation9 14 16-19 and to the radiomimeticchemical N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)'820 because of a defect in either DNArepair systems'4 1820 or some other system leadingto secondary failure of DNA repair.2'23 More

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recently, cultured cells from patients with the follow-ing abiotrophic neurodegenerative diseases havealso been reported to be hypersensitive to theX-ray-type of DNA-damaging agent: Huntington'sdisease,9 16 1724 25 Friedreich's ataxia,26 familialdysautonomia,"' Alzheimer s disease," and Parkin-son's disease." We have also found hypersensitivityto MNNG in a group of nine fibroblast lines frompatients with muscular dystrophy (one limb-girdledystrophy, two Becker's muscular dystrophy, threemyotonic dystrophy, and three Duchenne musculardystrophy lines).27 We now report that cells frompatients with Usher's syndrome (recessively inher-ited retinitis pigmentosa and congenital sensori-neural deafness) are hypersensitive to X-rays and to

MNNG. We also report that lymphoblastoid lines

Robbins, Scudiero, Otsuka, Tarone et al.

from patients with Duchenne muscular dystrophyare hypersensitive to X-rays.

Methods

PatientsIn this study we evaluated lymphoblastoid lines from 26

apparently healthy normal control donors, nine patients

(representing eight kindreds) with Usher's syndrome, nine

patients (representing seven kindreds) with dominantly

inherited retinitis pigmentosa unassociated with any

neurological abnormalities, and ten patients (representing

seven kindreds) with Duchenne muscular dystrophy. Fib-roblast lines were studied from 18 normal control donors,seven patients (representing six kindreds) with Usher's

syndrome, and two patients with ataxia telangiectasia.Identification and features of the cell donors are presented

in the legend of fig 1 and in table 1. Skin biopsy and

Table 1 Post-X-ray and post-MNNG survival parameters offibroblast lines

Repository data X-ray series l* MNNG*

Designation Age, Sex Do D, Series IV Series V

yryr ~~~~~~~~DoDo, Do D

NormalCRL 1190 77 F 154 322CRL 1220 15 M 132 319CRL 1221 40 M 146 357GM 969 2 F 143 341GM 1652 11 F 104 302GM 2149 54 F 127 319 3-2 18-5GM 2987 19 M 175 409 2-5 15-0GM 3349 10 M 125 331 2-3 13-8GM 3468 5 days M 141 367 3-4 18-9GM 3651 25 F 2-6 14-6RB 4087 55 F 3-1 16-4RB 4436 57 F 2-3 13-6RB 4468 30 F 2-6 14-1RB 4470 49 M 2-7 12-5RB 4492 69 M 3-0 15-4RB 4494 71 F 2-8 15-4RB 4581 33 M 2-1 12-3RB 4582 39 F 3-0 13-8

Mean 138 342 2-8 16-1 2-7 14-2(±SE) (±6.6) (±10-7) (±0.2) (±1-0) (±0-1) (±0.5)

Usher's syndromeGM 3854 47 M 120 291 2-5 12-4GM 3889t 22 M 113 273GM 3891t 16 M 109 273 2-2 12-1RB 5061 17 F 107 247RB 5065 27 F 125 296RB 5205 6 F 133 324 1-8 11.0RB 5206 32 F 99 201 2-3 12-6

Mean 115 272 2-3 12-4(±SE) (±4-4) (±15-0) (±0-2) (±0-1)

Ataxia telangiectasiaGM 3395 13 M 45 126GM 3487 8 M 61 149

*The Do is the dose in rads of X-rays or the ,uM concentration of MNNG reducing survival from any point on the exponential portion of thesurvival curve to 37% of that point. The D.o and D0 are the doses reducing colony-forming ability to 10% and 1%, respectively. The D0values in series IV and V for the normal lines have been published previously.2"tLines GM 3889 and GM 3891 are from brothers.

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Survival of lymphoblastoid linesEpstein-Barr virus-transformed peripheral blood lympho-cytes were cultured, irradiated with X-rays, and evaluatedas described previously9' except that the lymphoblastoidlines were not centrifuged prior to treatment with X-raysbut were simply diluted with culture medium to the

* required cell concentration. The post-irradiation viabilityratio of a cell line was calculated by dividing the concentra-tion of viable (trypan-blue-excluding) cells in an irradiatedculture on the third day after irradiation by the concentra-tion of viable cells in an unirradiated culture of the same

* line on the same day.9' A limitation of this dye exclusionassay is that it is not automated and requires visual scoring

* of viable cells. Therefore, to avoid observer bias, countingof viable cells was performed using coded samples, and at

a least six replicate experiments were done on each line* * except that one normal line had only five replicate experi-* *__ ments. Even though the dye exclusion assay involves scor-.0. *0 ing single cells, results with the assay9 28 correlate well with* * colony-forming ability of the lymphoblastoid lines.'7 28 For

* the 26 normal control lines used in this study and for the 16genetic control lines reported on previously," there was nocorrelation between the post-irradiation viability ratio and

uchenne Usher's Dominant either donor age or growth rate of unirradiated lines. Forstrophy syndrorperetinitis the statistical comparison of groups of lines, analysis of

7 8 7 vanance methods were used.29 One-sided p values areOr_n07lireported.Ub.I

0-0300 28

Survival offibroblast linesWith the exception of the two ataxia telangiectasia fibro-blast lines used in the X-ray experiments, all fibroblastlines were coded so that the personnel performing andevaluating the experiments did not know the identity of thelines. There was no correlation between the post-treatmentsurvival parameters of the fibroblast lines and either donorage or plating efficiency of the untreated lines. Fibroblastlines for X-ray treatment were cultured6 and irradiated9 asdescribed previously. Fibroblast lines for the N-methyl-N'-nitro-N-nitrosoguanidine experiments werecultured and treated with MNNG after the method ofScudiero20 and exactly as in the previous MNNG seriesI-I11.'° The serum concentrations referred to therein'0 as10% and 20% were actually 8½/2% and 162/3%, respec-tively. We present here the results on all the coded normaland Usher's syndrome lines studied in MNNG series IVand V.

Fibroblast colony-forming ability after treatment wascalculated by dividing the colony-forming efficiency (thatis, "plating efficiency") of a line at a given treatment doseby the colony-forming efficiency of that line's untreatedcells in the same experiment. If the latter was less than 1 %,

the experiment was discarded. At least four colonies had tobe present at a given treatment dose for tabulation. Theaverage plating efficiency of the Usher's syndrome lineswhen untreated was not significantly different from that ofthe normal lines. Two to ten replicate experiments wereperformed with each cell line for X-rays, and at least fourwith each cell line for MNNG (except that one normal linefrom MNNG series V had only one experiment andanother had three). In each experiment a straight line wasfit to the logarithms of the colony-forming ability at allX-ray doses and at 6 ,uM and higher MNNG doses using

Fig Survival oflymphoblastoid lines treated with 100rads of X-rays. Each symbol represents the mean viabilityratio of at least six replicate experiments on a line with theexception of three Duchenne muscular dystrophy, one

Usher's syndrome, and two dominant retinitis pigmentosasymbols each ofwhich represents the average ofthe viabilityratios of lines derived from a pair of afflicted familymembers. The viability ratio ofeach afflicted member of apair from a kindred was within 2% of the average ratio ofthe pair. Each horizontal bar represents the mean ratio forits group. Thep value is for comparison with the 26 normalcontrol lines. Omission of the highest normal control line,whose viability ratio of 0-73 increases the variance of thenormal control group, would result in a mean ratio for thenormal control lines of 0.528 and in lower p values for thehypersensitive patient groups (p = 0-003 for the eightUsher's syndrome and 0-004 for the seven Duchennedystrophy kindreds). Duchenne dystrophy lines: AG 3929,GM 3780, 3782, RB 4100, 5014, 5113, 5115, 5124, 5126and NIH line C. Usher's syndrome lines: GM 3853, 3892,RB 4361, 5062, 5064, 5204, 5207, 5333 and 5360.Dominant retinitis pigmentosa lines: GM 3823, 3834,3836, 3894, 3907, 3911, RB 4562, 5058 and 5060.

venepuncture were performed after obtaining informedconsent of the donors. Lines with "GMN' and "'RB"prefixes were obtained from the Institute for MedicalResearch, Camden, NJ. Lines with "'CRL" prefixes werefrom the American Type Culture Collection, Rockville,MD.

080-

0

00

00000*

000000000

900

0

Normalcontrols

260-5360-010

0 70o207-- a

> 60c:1

2n

x

II050

040

KindredsMean ratioSEP value

DLdy

n.,U.4M0 0090 012

U40/0 0100009

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the method of least squares. From each straight line theY-axis intercept and the D0,, D,O, and Do, survival para-meters were obtained by computer as described in detailpreviously.27 The Do is the negative inverse of the slope ofthe exponential portion of the survival curve and is thetreatment dose which reduces the colony-forming abilityfrom any point on the exponential portion of the survivalcurve to 37% of that point. The D,0 and Doi values are thedoses which reduce the colony-forming ability to 10% and1 %, respectively. The methods used to estimate survivalcurves and to compare lines and groups of lines have beendescribed previously.27 One-sided p values are reported.

Robbins, Scudiero, Otsuka, Tarone et al.

from that of 0-55 (+0.030) for the seven dominantretinitis pigmentosa kindreds (last column). Themean viability ratio of 0-49 (±0-010) for the eightUshef's syndrome kindreds (third column) wassignificantly less than the mean ratio of the normalcontrol lines (p = 0.009) and of the seven dominantretinitis pigmentosa lines (p = 0.029). The meanviability ratio of 0-49 (±0.009) for the sevenDuchenne muscular dystrophy kindreds (secondcolumn) was significantly less (p = 0-012) than themean ratio of the normal control lines.

Results

SURVIVAL OF LYMPHOBLASTOID IFigure 1 shows the post-X-ray viabilymphoblastoid lines. The mean(±SE) of 0-54 (+0-010) for the 26lines (first column) was not signifi

100

Colony-formingability 101% remaining)

Ataxia telangiectasiaA GM 3395* GM 3487

0 100 200X-ray dose (

Fig 2 Post-X-ray survival ofnormal ansyndrome fibroblast lines. Each plotted pis the geometric mean of the post-X-ray co

ability obtained at the indicated dose frorrexperiments performed. The straight lineexponential portion of each survival curv

the D 0and Do estimates reported in the taxof cell lines is presented in table 1. Usher'GM 3889 and GM 3891 are from siblingnormal zone encompasses the survival cu

normal lines.

SURVIVAL OF FIBROBLAST LINESFigure 2 shows the post-X-ray survival of the nine

LINES normal fibroblast lines (shaded area), seven Usher'sility ratios of the syndrome lines (open symbols), and the two ataxiaviability ratio telangiectasia lines (solid symbols). Table 1 presentsnormal control the post-X-ray survival parameters for these lines.

icantly different The normal lines had mean Do and D0 values of 138(range 104-175) and 342 (range 302-409), respec-

Usher's syndrome tively. The two ataxia telangiectasia lines (fig 2)O RB 5205 were very sensitive to the X-rays and had mean D,,A RB 5065 and D,0 values of 53 and 138, respectively (table 1).O GM 3854 The seven Usher's syndrome lines represented sixO GM 3889 kindreds, four of which had lymphoblastoid linesR GM 3891 studied. These Usher's syndrome lines had survivalv RB 5206 curves which extended from the lowest part of the

normal zone to well below that zone (fig 2). Their.......... mean Do and D10 values (table 1) of 115 (range

99-133) and 272 (range 201-324), respectively,Normal were significantly less than the corresponding mean\2$zone2 values for the nine normal lines (p = 0 007 and

0-001, respectively). The two Usher's syndrome sib-lings, GM 3889 and GM 3891, had similar survivalcurves (fig 2; table 1). With their average Do and D,0values representing a single kindred, the mean Do(116) and D,0 (272) values for the six Usher's syn-drome kindreds were still significantly less than the

B\E corresponding values of the nine normal lines (p =\\G 0-015 and 0-002, respectively).

The post-MNNG survival parameters of the fivenormal lines in MNNG series IV (table 1) had meanDo and Do, values of 2-8 (range 2.3-3.4) and 16-1(range 13-8-18-9), respectively, and were not

300 400 significantly different from those obtained for thesemrsdl lines when they had been studied and presentedd Usher's previously in series 111.10 The Usher's syndrome lineoint for a cell line RB 5205, from one of the lymphoblastoid lineolony-forming donors, had Do and Doi values of 1-8 and 11-0,n the replicate respectively, which were significantly below those ofdepicting the the five normal lines (p = 0-02 and 0-05, respec-e corresponds to tively). The eight additional normal lines of MNNGble. Identifications syndrome lines ser0esV had mean Do and Do, values, respectively,5s. The shaded of 2-7 (range 2-1-3-1) and 14-2 (range 12.3-16-4)rves for the nine (table 1). The three new Usher's syndrome lines of

series V were from three of the Usher's syndrome

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lymphoblastoid line kindreds. Their mean Do andDoi values of 2-3 and 12-4, respectively, weresignificantly less than the corresponding mean val-ues of the normal lines (p = 0-023 and 0-006,respectively). The summary p values for the meanDo and Doi values of these three Usher's syndromelines and the Usher's syndrome line of series IVwere p = 0013 and 0-009, respectively, demonstrat-ing that these four Usher's syndrome lines werehypersensitive to MNNG.

Discussion

Ushers syndrome is the first disease with retinitispigmentosa demonstrated to have enhanced sen-sitivity to ionising radiation or to a radiomimeticchemical. Usher's syndrome cells are hypersensitivein the lymphoblastoid (fig 1) and fibroblast-X-ray(fig 2; table 1) assays and the fibroblast-MNNGassay (table 1). While there is overlap in all threeassays between the post-treatment survival of theUsher's syndrome lines and that of the normal lines,the hypersensitivity found is statistically significant.A similar degree of overlap with the lower normallimit has been reported in ionising-radiation survivalassays for the following radiosensitive disorders:Huntington's disease,'72425 Alzheimer's disease,"Parkinson's disease," Friedreich's ataxia,26 and theheterozygous state of ataxia telangiectasia.'618 Apossible reason for such overlap is that the lethaldamage responsible for the hypersensitivity repre-sents but a small portion of the total lethal lesionsinduced by the ionising radiation. A more detaileddiscussion of this possible mechanism and of itsrelationship to defective processes for repairingdamaged DNA is presented below.

Several diseases with sensorineural deafness otherthan Usher' s syndrome are also associated withhypersensitivity to DNA-damaging agents:xeroderma pigmentosum6 '4 and Cockayne' s syn-drome'4 30 3 cells are hypersensitive to theultraviolet-type of DNA-damaging agent, andFriedreich's ataxia26 cells to the ionising-radiation-type of DNA-damaging agent. Both xerodermapigmentosum and Cockayne' s syndrome are dis-eases with demonstrated inherited defects in DNArepair-fibroblasts from these diseases are unable torepair normally ultraviolet-irradiated viruses inhost-cell reactivation experiments.'2 32-34 Fried-reich's ataxia may also be a disease with defectiveDNA repair; recently, Lewis and coworkers haveshown that Friedreich' s ataxia fibroblasts havehypersensitivity to ionising radiation,26 moderatelyimpaired potentially lethal DNA damage repair,26and inhibition of post-irradiation DNA synthesis.35The Do values (mean 138; range 104-175) of our

irradiated normal fibroblast lines are similar to thevalues reported for normal lines by Chamberlainand Lewis26 (mean 150; range 125-168) and by Coxand Masson36 (mean 122; range 98-160). It is ofinterest that the mean post-irradiation Do value forthe Friedreich's ataxia fibroblasts reported byChamberlain and Lewis26 was 83% of the meanvalue for their normal lines, while the mean post-irradiation Do of our Usher's syndrome fibroblastlines also was 83% of the mean of our normal lines(table 1). If Usher's syndrome and Friedreich'sataxia are disorders with defective repair of DNA, itshould be possible to find other DNA-damagingagents which will induce a relatively larger differen-tial in post-treatment survival than that induced byionising radiation or MNNG. We have used X-raysand MNNG because of the hypersensitivity of ataxiatelangiectasia fibroblasts to these agents;'4 16 18-20however, for the reasons discussed by Chamberlainand Lewis,26 the molecular abnormality of ataxiatelangiectasia is probably different from that ofFriedreich's ataxia and, therefore, of Usher's syn-drome. In this regard, it is important to note thatneither deafness nor retinitis pigmentosa occurs inataxia telangiectasia.'3 15

Many patients with the ultraviolet-sensitivedisorder Cockayne's syndrome have a retinitis pig-mentosa,3' and Cockayne' s syndrome cells arehypersensitive to the lethal effects of ultravioletradiation'430 3' and have abnormal host-cell reacti-vation of ultraviolet-irradiated viruses.'2 33 34 In con-trast, cells from patients with Usher's syndrome,which is not clinically a sunlight sensitive disorder,can repair ultraviolet-radiation-induced DNA dam-age normally: the three Usher's syndrome lympho-blastoid lines we tested had normal post-ultravioletirradiation survival (unpublished data), and Usher'ssyndrome fibroblasts have normal host-cell reactiva-tion of ultraviolet irradiatedH simplex virus.'2 Thus,Usher's syndrome is the first retinal dystrophyshown to have hypersensitivity to the X-ray-type ofDNA-damaging agent. However, not all diseaseswith retinitis pigmentosa are associated withhypersensitivity to these DNA-damaging agents.The group of lymphoblastoid lines from patientswith dominantly inherited retinitis pigmentosa hadnormal survival after treatment with X-rays (fig 1),and the one dominant retinitis pigmentosa lympho-blastoid line tested had normal survival after treat-ment with ultraviolet radiation (unpublished data).Similarly, not all primary neuronal degenerationshave a hypersensitivity to the X-ray-type of DNA-damaging agent: groups of fibroblast lines fromeight patients classified as having motor neuron dis-ease26 and from five patients with spinal muscularatrophy38 had normal survival after treatment with

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ionising radiation and MNNG, respectively, and agroup of five lymphoblastoid lines from patientswith amyotrophic lateral sclerosis had normalpost-X-ray survival (unpublished data).

It is important to note that the absence ofhypersensitivity to the lethal effect of a specificDNA-damaging agent does not necessarily indicatethat the disease in question has no inherited defectin the repair of DNA. Thus, the lymphoblastoid linetested from one member of the xeroderma pigmen-tosum complementation group E kindred,39 as wellas the fibroblast line from the other member,40 hadpost-ultraviolet irradiation survival in the normalrange despite the fact that this kindred has well-documented defects in nucleotide excision repair4'and in host-cell reactivation of ultraviolet irradiatedvirus32 and purified viral DNA.42 Therefore, beforeconcluding that dominantly inherited retinitis pig-mentosa has no abnormal responses to DNA-damaging agents, other types of agents and tests willhave to be utilised.The presence of hypersensitivity to X-rays in the

Duchenne muscular dystrophy lymphoblastoid lines(fig 1) is consistent with our finding of low post-MNNG survival in three Duchenne muscular dys-trophy fibroblast lines.27 Thus, the association be-tween hypersensitivity of fibroblasts to MNNG"' 18 20and hypersensitivity of lymphoblastoid lines toX-rays9 1" demonstrated for neurodegenerations andfor Usher's syndrome is applicable also to theDuchenne form of muscular dystrophy.We have demonstrated in Huntington's disease,9'

Alzheimer disease," Parkinson's disease," Usher'ssyndrome, and Duchenne muscular dystrophy thatthe hypersensitivity is specific for the ionising-radiation type of DNA-damaging agent, since thereis normal survival after treatment with 254-nmultraviolet radiation in fibroblasts and/or lympho-blastoid lines from patients with these disorders.Although the molecular basis for the in vitro

Table 2 Degenerative disorders of excitable tissue withcellular hypersensitivity to DNA-damaging agents

Classification Reference

UV-radiation-type:Xeroderma pigmentosum (6, 14)Cockayne's syndrome* (14, 30, 31)

X-ray-type:Ataxia telangiectasia (9, 14, 17, 19, 20)Friedreich's ataxia (26)Huntington's disease (9, 10, 16, 17, 24, 25)Familial dysautonomia (10)Alzheimer's disease (11)Parkinson's disease (1i)Ushee's syndrome (Current study)Duchenne muscular dystrophy (Current study)

'While Cockayne's syndrome is primarily a demyelinating disease,the degeneration of photoreceptors in Cockayne's syndromeappears to be a primary retinal degeneration.'3 37

Robbins, Scudiero, Otsuka, Tarone et al.

hypersensitivity to X-rays and MNNG of these cellsis unknown, we hypothesise that X-rays and MNNGproduce their lethal effect in vitro by damagingDNA. Exposure of cells to either of these agents invitro results in a myriad of different types of lesionsin DNA.4344 Some are not lethal even if unrepaired;others are potentially lethal lesions. In normal cells acertain fraction of these potentially lethal lesions arenot repaired. The fraction of these lesions notrepaired by the patients' cells may be slightly higherdue, perhaps, to the inability of the patients' cells torepair an infrequent type of normally repairablelesion. The resulting slight increase in the numberof lethal lesions remaining in the patients' cells couldcause the small (but significant) hypersensitivitywe have demonstrated (figs 1 and 2; table 1). Pre-sumably, greater differences between disease andnormal cells would be evident in vitro if more selec-tive DNA-damaging agents were used.

In vivo, the DNA of post-mitotic excitable tissueis constantly being damaged by intracellular metabo-lites45 and spontaneous hydrolytic reactions.46 Dueto differences in the intracellular or extracellularenvironment,7 '13the types and quantities of thisDNA damage would differ among different excit-able tissues and between different neuronal popula-tions.'2 13 In normal cells the damaged DNA is read-ily repaired, but in these diseases characterised byhypersensitivity (table 2) there is defective repair ofa type of potentially lethal lesion. The excitable tis-sue which undergoes premature death would be thatin which the unrepairable type of lesion represents arelatively large proportion of the potentially lethallesions induced. The different clinical patterns ofthese various disorders would result from differentdefective repair processes or from different muta-tions in the same repair process.'2 '3 The ultraviolet-type of DNA damage accumulates in xerodermapigmentosum,6 '3 while the X-ray-type of DNAdamage accumulates in radiosensitive neurodegen-erations, Usher's syndrome, and Duchenne muscu-lar dystrophy. Examination of the DNA fromDuchenne dystrophy muscle cells in various stagesof dystrophic change should reveal increasing levelsof accumulated DNA damage. Since samples ofskeletal muscle can be safely and abundantlyobtained, it should be possible to test ourhypothesis.

References

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2 Collins ET. IX. Diseases of the retina. 1. Abiotrophy ofthe retinal neuro-epithelium or "retinitis pigmen-tosa". Trans Ophthal Soc UK 1919;39: 165-95.

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4Robbins JH, Kraemer KH, Lutzner ML, Festoff BW,Coon H. Xeroderma pigmentosum. An inheriteddisease with sun sensitivity, multiple cutaneous neo-plasms, and abnormal DNA repair. Ann Intern Med1974;80: 221-48.

5Andrews AD, Barrett SF, Robbins JH. Relation ofDNA repair processes to pathological ageing of thenervous system in xeroderma pigmentosum. Lancet1976;i: 1318-20.

6 Andrews AD, Barrett SF, Robbins JH. Xeroderma pig-mentosum neurological abnormalities correlate withcolony-forming ability after ultraviolet radiation. ProcNatl Acad Sci USA 1978;75:1984-8.

7Robbins JH. Workshop summary: xeroderma pigmen-tosum. In: Hanawalt PC, Friedberg EC and Fox CF,eds. DNA Repair Mechanisms. New York: AcademicPress 1978:603-7.

8 Robbins JH. Significance of repair of human DNA: evi-dence from studies of xeroderma pigmentosum. J NatlCancer Inst 1978;61:645-56.

9 Moshell AN, Tarone RE, Barrett SF, Robbins JH.Radiosensitivity in Huntington's disease: implicationsfor pathogenesis and presymptomatic diagnosis. Lan-cet 1980;i: 9-11.

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3 Robbins JH. Hypersensitivity to DNA-damaging agentsin primary degeneration of excitable tissue. In: Fried-berg EC, Bridges BR, eds. Cellular Responses to DNADamage. UCLA Symposia on Molecular and CellularBiology, New Series, Volume II. New York: Alan RLiss 1983, 671-700.

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