<Original Article> X-ray induced Sister Chromatid Exchange and Chromosomal Aberrations in the Lymphocytes from the Patients with Neurofibromatosis 利用統計を見る

Yamanashi Med. J. 5(l), l9's'23, 1990

Original

Artic}e

X-ray Induced Sister Chromatid Exchange and Chromosomal

Aberrations in the Lymphocytes from the Patients with

Neurofibromatosis

Hi￡oshi HosmNo, Tatsuya TAKEsmTA, Sumio IuiMA, Akio AsAKA,

M3saya SEGAwAi>, and Maketo HiGuRAsm2)

Dapartment ofHealth Sciences, YamanashiMedicalCollege, Yamanashi, D Segawa Neurologz'cal Clinicfor Children, 2) DePartment ofMaternaland Child Health, Facudy ofMedicine, Universitpu ofTodyo,laPan

Todyo,

Abstract: Neurofibromatosis(NF),anautosomaldominantdisease,isdiagnosedbythepresence

of more than 6 Caf6-au-lait spots and neurofibromas in late chiidhood or adolescence. We examined the frequencies of sister chromatid exchange (SCE) and chromosomal aberrations (dicentrics and rings) in cultured lymphocytes from patients with NF and normal coRtrols after

X-ray irradiation at doses of 2.5, 5.e and 7.5 Gy. No differences in the baseline SCE frequencies

were found between the NF patients and centrols. The freqttency of SCE increased with the irradiation dose in both patients and controls with no apparent clifferences between the two groups. IR additlon, the frequencies ofspontaneous and X-ray induced chromosomal aberratlons

in the patieRts were not significant}y different from those in the controls. The presence of genetic

heterogeneity causing NF was suggested.

Key words: Neurofibromatosis, sister chromatid exchange, chromosomal aberrations

IN rRoDucTION

Neurofibromatosis (NF) is an autosomal, dominantly inherited disease with a high rate of new mutations and a wide range of man-ifestatiens, including somatic, neurelogical, and psychiatric symptoms. Manifestations on

the skiR are involved in the majority of

patients. Cafe'-au-lait spot, irregularly shaped

areas of increased skin pigmentation, are a hallmark of the disease. The presence of more than 6 spets, which are greater than 1.5 cm in diameter is pathognomonic of NF. Cutaneous

and subcutaneous neurofibromas commonly

appear in late childhood or adolescence. The

formation of neurofibromas on cranial or

spinal nerve roots may lead ￡o a variety of

neurolegical symptoms. Mild impairment of intellectual function is common, but severe mental defect rarely occurs. Sarcomatous de-generation of neurofibromas occurs in about 10% of patients. There is also an increased

incidence of other types of neura} tumors, such

as glioma of the optic nerve and optic chiasm,

meningioma, and pheochromocytorna.

Re-cently Hafez et al.i) reported that the

frequen-cies ef sister chromatid exchange (SCE) in

lyrr}phocytes from pa￡ieRts with NF

significaRt-ly iRcreased with the dose of gamma ray

irradiation. The present study examined the effects of X-ray irradiation oR chromesomal

aberration and SCE in lymphocytes from

patients with NF.

Received, October 27, Accepted, December 2,

1989

l989

M[ATERIALS AND METHODS

unre-Iated patieRts with NF, S ma}es and 1 female, and 4 unrelated Rormal controls, 3 males and I

female. Ages of ￡he patieRts ranged from 2 ￡o 12 years. Among the patients, a 7-year-old boy

with NF showed generalized tonic convulsion,

and a 12-year-old boy with NF combined

mental retardation and autism, the other two patients had ne symp￡oms except Caf6-au-lait spots and neurofibromas.

ANALYSIS OF SISTER CHROMATID

EXCHANGE. Heparinized peripheral blood

samples were obtained from the four patien￡s aitd the four controls. Who}e blood from each

sllbject (O.3 ml) was exposed to X-rays (O, 2.5,

5.0, 7.5 Gy) emitted from aR MBR-1505R

(HITACHI) operatiRg at I45 V, 4.5 mA.

Immediately af￡er irradiation, the blood sam-ples were added to 5 ml RPMI 1640 medium (GIBCO) containing 15% fetal bovine serum

(FBS, GIBCO), 3% phytohemagglutinin

(PHA, DIFCO) and 1%

peRicillin--streptomycin (GIBCO). The medium also con-tained 40 paM Bromodeoxyuridine (BrdUrd, Sigrna) during the en￡ire culture period. The

cultures were incubated at 370C for 72 hrs in a

C02 incubator. Colcemid (2×10rw7 M final concentration, Wako) was added to each

cul-ture during the last 6 hrs. The ce}ls were then

collected by centrifugation, exposed to O.075 M KCI hypotonic solution for 8 min., and fixed three timesin a mixture ofethanol: acetic acid

(3:l). Air dried chromosome preparations

were made, aRd a modification ofthe fiuoresc-ence-plus-Giemsa (FPG) method of Goto et al.2) was applied to obtain differential staining of

the sis￡er chrornatids. Fifty metaphase cells of

the second-division phase were scored for SCE per dose level per person.

CHROMOSOMAL ABERRATION

ANALYSIS, In respect to chromosomal

aberration analysis, the culture medium was the same except addition of BrdUrd, as in the SCE assay. The cultures were incubated for 50 hrs. Colcemid was added to each culture at 2e

hrs to arrest the cells at their first division.

Chroraesome preparatioRs were stained with Giemsa. Dicentrics and rings were scored for chromosomal aberrations. Fifty first division metaphase cells were analyzed per dose point per person. The other methods ttsed were the same as those mentioned above for {he analysis of SCE.

R￡suLTs

The frequencies of baseline and X-ray

in-'rable 1. ×-ray induced SCE frequencies ilt the tients with NF and the controls

cultured lymphocytes from the

X-ray dose (Gy)

O 2.5

5.0 7.5

NF

1 2 3 4 4.6 5.8 5.0 4.9 7.2 7.1 8.0

85

7.4 7.4 8.4 9.0 l1.5 9.1 ll.4 1l.4

mean±S.E. 4.9±O.25 7.7±e.32 8.0 ! O.39 _IO.8±G6

o 2.5 5.0 7.5 controls l 2 3 4 5.6 5.I 4.5 4.6 6.9 7.6 6.4 5.2 8.I 8.4 8.6 8.9 IO.8 11.9 IG2 1l.O

X-ray Induced Chromosomal Changes ln Neurofibromatosis 21

=

￠ o

x

ur

o

co 1 O.O 5.0 ee NF O Control

=

e. 2.o 2 ..o ."

g

8 i.o

es

@

o

NF

Con{rol Fig.

O 2.5 5.e 7.5

×--ray dose (Gy)

1. X-ray induced sister chromatid exchange frequencies in the cultured lymphocytes

from the patients with NF and the

trols,

Fig. 2.

O 5.0Z5

X-ray dose(Gy)

X-ray induced chromosomal aberration

(dicentrics and rings) frequencies in the cultured lymphocytes from the patients with NF and the controls.

Table 2. X-ray induced chromosomal aberration

cultured lymphocytes from the patients

controls

X-ray dose (Gy)

O 5.0

frequencies in with NF and 7.5 the the

NF

1 2 3 4 o.oo o.oo o.oo o.oo O.68 O.76 O.64 O.62 2.52 l.98 1.68 1.l2 mean±S.E. o.oo±o.oo

o

o.6s±o.es 5.0 1.82±O.29

75

controls I 2 3 4 o.oo o.oo o.oo o.oo O.84 O.68 O.84 O.92 2.00 1.24 l.82 O.76

mean±S.E. o.oo±o.oo O.82±OD5 l.46±O.28

dt}ced SCE in NF and centrol cells are summa-rized in Table 1 and Fig. I. The baseline SCE

frequencies in NF and contrel cells were

4.9±O.25 and 4.9±O.23, respectively. The

fre-quencies of SCE increased proportionate to increases in the doses of irradiation iR both groups. However, tkere were Ro significaRt differences iR SCE frequencies between the

patients and the controls. The number of

dicentrics and rings per cell, after exposure to various doses of X-rays, in NF and coRtrol cells

are summarized in Table 2 and Fig. 2. The baseline frequencies of dicentrics and ring chromosomes per cel} in NF and control cells were equally O.O±e.O. The X-ray treatment induced a clearly dose-yelated increase in the

frequeRcies of dicentrics and rings in both groups. At doses of 5.0 Gy, the NF cells showed rather lower frequencies of aberra-tions but these frequencies were not

signi-ficantly different from those in the ceRtrol cells (t-test, p>e.05). At 7.5 Gy, the NF cells showed higher frequencies but not significantly

diffe-rent from the controls (t-{est, p>O.05).

DIscussloN

The results obtained from the present ex-periments indicated ￡hat there were definite

X-ray dose-related responses in SCE and

aberratien frequencies in the lymphocytes of both NF patients aRd normal controls.

Howev-er, no differences were found between NF

patients and normal controls in respect to the frequency of SCE and chromosomal aberra-tions at the baseline ner under X-ray induced conditiolts. On the con￡rary, Hafez et al.3) reported highly significaRt differences in SCE and chromosomal aberration frequencies in lymphocytes of NF patients following gamrr}a-ray treatment. According to the findings of Hafez, the baseline frequencies of SCE and

aberration in NF pa￡ients were not significantly

different from those in normal coRtrols, which

is in accordance with the results of the present

study. However, their study showed much

higher frequencies of SCE induced by gamma-ray (e.75, 1.5, 3.0 Gy) irradiation in NF patients, compared to normal controls. One of the differeRces between the present study and that of Hafez is to be found in ￡he type of

ionizing radiation, that is, X-ray in the former

vs. gamma-ray iR the latter. However, X-ray and gamma-ray are almost similar in nature and therefore, will produce almost similar effects in breaking DNA straBd. Therefore, it is uniikely that the difference in the type of

ionizing radiation used significantly influenced the results. Secondly, ￡he age distribu￡ions of the subjects are slightly different. The age of

our patients ranged from 2 to I2 years,

whereas the ages in their study ranged from 8

to 22 years. In general, aging does not strongiy affect the frequencies of SCE4), so that a slight

difference in the age distribution cannot

ex-plain the discrepancy in the results. In regard

to the metheds, in both studies whole blood samples, after irradiation, were irr}mediately added to the rnedium with PHA used as the

mitogen. RPMH640 medium with 15% FBS

was used in the present study, while the same

medium with 20% autologous medium was

used in their s￡udy. The concentration of BrdUrd in the present study was 40 @M, in centrast to 10 paM in their study. The incuba-tion time in the present study was 72 hrs, in coRtrast to 64 or 68 hrs in their study.

Although we need a re-examination which

carries out under the same culture condition as

those ef Hafez et al., it is difficult te attribute the observed discrepancy to these differences in the culture conditions.

AIthough all patients met the same criteria of diagnosis, there may exist some sub-groups of NF which differ in the respoRsible genetic defects. A genetic defect which results in a

greater sensitivity to ionizing radiation could

have been responsible for the findings of the earlier study, whereas some other different genes could be involved in our cases. At least we can conclude that, not all NF patients have a higher sensitivity to ionizing radiation be-cause of the genetic heterogeneity mentioned above.

Recently Seizinger et al.5) suggested that the

gene for peripheral neurofibromatosis was eR chromosome l7 by a linkage with the nerve growth factor receptor gene, but one family

did llot show the linkage. Barker et al.6> also

fbund the liRkage of the NF gene with the probe located at the pericentromeric regioR of

chromosome 17, and they found no

heter-ogenei￡y. Further studies are required to de-termine whether or Rot gene￡ic heterogeneity

exists arnong NF patien￡s.

This work was supported, in part, by a grant from the Ministry of Heal￡h and Welfare of Japan and by Grants-in-Aid for Scientific

X-ray Induced Chromosomal Changes in Neurofibromatosis 23

Research -from the Science and Culture

･Minis￡ry of Education, of Japan.

REFERENCES

1) Hafez M, Abd El-Nabi SM, El-Wehedi G,

Toltbary Y. Enhanced response to the induction of sister chromatid exchanges by gamma

tion in neurofibromatosis. CaRcer 1986' 57: , l937-1940,

2) Goto K, Maeda S, Kano Y,Sugiyama T. Factors involved in differential Giemsa-staining of sister chromatids. Chromosoma 1978; 66: 351-S59. 3) Hafez M, Abd El-Nabi SM, El-Wehedi G,

Tonbary Y. Evidence ofchromosomal instability

in neurofibromatosis. Cancer l985; 55: 2434-2436.

4) Wu-nan W, Tai-lin Liew. The effect of age and cell proliferation on the frequency of sister

chromatid exchange ln human lymphocytes cultured in vitro. Mech. Ageing Dev. 1983; 21:377-384.

5) Seizinger BR, Rouleau GA, Ozelius lj et al.

Genetic linkage of von Recklinghausen

neurofibromatosis to the nerve growth factor receptor geRe. Cell 1987; 49: 589-594. 6) Barker D, Wright E, Nguyen K et al. Gene for von Recklinghausen Reurofibromatosis is in the pericentromeric region of chromosome 17.