Embed Size (px)
Citation preview
Cytogenetic Studies in Tuberous Sclerosis
Claudia U. Dietrich, Winfrid Krone, and Ralf Hochsattel
ABSTRACT: A cytogenetic study was performed with cultures derived from peripheral blood, unaffected skin, and angiofibromas of four patients suffering from the sporadic form of tuberous sclerosis (TSC). Increased frequencies of unstable chromosomal anomalies were found in lymphocytes and in fibroblasts from unaffected skin of the patients. The slight increase of the overall rate of unstable anomalies observed in angiofibroma-derived cultures above that of lymphocytes and skin fibroblasts, respectively, could almost entirely be attributed to a higher frequency of dicentric chromosomes. Of the 17 facial angiofibromas from which a total of 20 cell cultures were established, nine showed a normal karyotype, while eight exhibited stable chromosomal rearrangements, among which 19 clonal types could be identified. Unbalanced forms of various translocations caused partial trisomies of the long arms of chromosomes 1, 3, 7, 10, and 15. There was no clustering of breakpoints to a particular chromosomal region, nor was one particular chromosome preferentially involved. Frequencies and kinds of rearrangements varied between cultures derived from different angiofibromas from the same patient and between different culture charges from the same tumor. TetrQploidy was not generally more abundant in the angiofibroma-derived cultures, but there were a few culture charges with exceedingly high rates of tetraploid cells. The occurrence of premature centromere disjunction (PCD), either affecting all chromosomes or only part of them in angiofibroma-derived cultures, first described in TSC by Scappaticci et al. could be confirmed.
INTRODUCTION
Tuberous sclerosis (TSC) is an autosomal dominant phakomatos is wi th high but incomple te penetrance and variable expressivi ty (for reviews, see Koch [1] and the monography edi ted by Gomez [2]). A recent est imate of its prevalence among people below 30 years of age [3] is close to 1 : 20000, which shows that the frequency of TSC has long been underes t imated. Tuberous sclerosis is a mul t i sys tem disorder. The most important manifestat ions of the disease affect the skin, kidney, brain, eye, and heart. Visceral organs and the skeleton may also be involved. The classical complex of symptoms comprises skin lesions, seizures, and mental retardation. Like other neuro- cutaneous syndromes, TSC predisposes to benign and mal ignant tumors; TSC patients have an increased risk of developing angiomyol ipomas and renal carcinomas, cardiac rhabdomyomas and/or glial brain tumors [4]. Among the typical cutaneous manifesta- t ions of the disease [5] are red or pink nodules, p redominan t ly in the centrofacial areas. Original ly designated as "adenoma sebaceum" [6], these lesions were later identif ied as angiofibromas arising from hamartomatous growth of dermal and vascu- lar connective tissue [7]. The basic defect under ly ing TSC is unknown, but there is hope that it wil l soon be e lucidated because the TSC gene has been local ized recent ly
From the Abteilung Humangenetik (C. U, D., W. K.J and the Abteilung Dermatologie (R. H.), Universitaet Ulm, Federal Republic of Germany.
Address reprint requests to: Prof, Dr. W. Krone, Abteilung Humangenetik, Universitaet U/m, P.O. Box 4066, D-7900 U/m, FRG.
Received January 13, 1989; accepted June 1, 1989.
1 6 1
© 1990 Elsevter Science Publishing Co.. Inc. Cancer Genet Cytogenet 45:161-177 (1990) 655 Avenue of the Americas, New York, NY 10010 0165-4608/90/$03.50
162 (: U Dietrich et dl
by means of linkage studies to chromosome band 9q34 ]8, 9], although genetic hetero- geneity of the disease cannot at present be excluded [10-12].
A liability to enhanced spontaneous and/or induced chromosomal breakage is no longer an exclusive feature of the well-known recessive chromosomal instability syndromes. Albeit at a lower level, chromosomal instability has also been observed in cell cultures derived from patients with a variety ot dominant cancer-prone syn- dromes: dysplastic nevus syndrome (13-15], adenomatosis coli and recti [16-18], medullary carcinoma of the thyroid [191, and neurofibromatosis (NF-1) 120].
In earlier cytogenetic studies on TSC patients, some authors reported examples of constitutive chromosomal variants whose significance is difficult to judge 121, 22]. Others have addressed the question of chromosomal instability and obtained conflict- ing results [23, 24[. A thorough cytogenetic investigation of cell cultures derived from various skin lesions of TSC patients and of lymphocyte cultures was published recently by Scappaticci et al, [25]. Besides higher than normal frequencies of unstable chromosomal anomalies, such as breaks and dicentri(:s, these autbors observed l)re- mature centroinere division (PCD), indi(:ating an influen(:e of the TSC mutation on the mechanism of chromosome disjunction at cell division. The data to be described in this communication confirm our earlier report 1261 that chromosomal instability, especially in angiofibroma-derived cultures, apparently results in both unstable anom- alies and stable chromosomal rearraugements.
PROBANDS
The healthy parents of the TSC patients underwent thorough dermatologic and neuro- logic examination for the presence of microsymptoms, but none were detected, We therefore consider our patients as sporadic cases of TSC. Clinical data for the TSC patients are summarized in Table 1. Angiofibromas were excised from the nasolabial area of TS1, TS2, TS3, and TS4 at various occasions. Skin biopsies were taken from the retroauricular area of the same patients. In addition, patients TS1 and TS2 donated skin biopsies from the upper arm. Sixteen healthy donors (eight female, eight male[, whose ages were within the same range as those of the patients, provided skin biopsies from the upper arm. Chromosome preparations were obtained from lymphocyte cul- tures of patients TS1, TS2, TS3, TS4, TS6, and TS7. The control group comprised 20 age-matched individuals (ten female, ten male).
MATERIALS AND METHODS
Cell Culture
To ensure cytogenetic heterogeneity of the tissue samples, angiofibroma-derived cul- tures and cultures of dermal fibroblasts were initiated not only from multiple frag- ments seeded in the same culture flask, but also from very small fragments isolated in separate flasks. The cell lines were propagated in Dulbecco's modified Eagle medium (DMEM) (Biochrom) supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 IU/ml penicillin, and 10 ~tg/ml streptomycin. In parallel to each chromosome preparation, the cultures were tested for mycoplasma contamination as described by Chen [27]. Peripheral lymphocytes were cultured in supplemented MEM-JOKLIK (Biochrom) according to the method of Pfeiffer [28].
Chromosome Analysis
To arrest cells at metaphase, cultures derived from angiofibromas and from unaffected skin were exposed to colcemid (0.05 ~g/ml) for 30 minutes prior to harvest by trypsinization. Hypotonic treatment (0.4% KC1) and fixation (methanol:acetic acid
Cytogenetic Studies in Tuberous Sclerosis 163
Table 1 Clinical data on the TSC patients
Patient (year of birth/sex)
TS1 TS2 TS3 TS4 TS6 TS7 (1963/F) (1966/M) (1967/F) (1954/M) (1972/M) (1967/M)
Skin Adenoma sebaceum + + + + + + Multiple hypomelanotic + + + + + +
macula Lumbosacral connective + + - - + +
tissue nevi Koeneu tumors + + + - - - Multiple papillomatous - - + - - +
and fibrous skin tumors Car6 au lait spots . . . . +
CNS and eye Cerebral pulsy + - + # - - Para- and periventricular + + + + + +
calcifications Mental retardation - - + + - - Retinal mulberry-like + + NE - - -
tumors Kidney
Angiomyolipoma NE - NE unilateral + bilateral + +
Abbreviahon: NE, not examined.
3 : I) fol lowed standard procedures. GTG banding was carried out by a modification of the method described by Francke and Oliver [29]. Cell harvesting and GTG banding of peripheral lymphocytes was performed using standard methods. Chromosomal anomalies were scored according to the recommendat ions of the ISCN (1985) [30]. The frequency of polyploid mitoses was determined by screening the slides at low magnification and by counting the chromosomes of polyploid metaphases at high magnification.
RESULTS
An overview of the frequencies and categories of chromosomal anomalies found in cultures of lymphocytes, skin fibroblasts, and fibroblast-like cells from angiofibromas is presented in Table 2. Both lymphocytes and skin fibroblasts from the TSC patients exhibit an approximately twofold higher level of total anomalies than those from healthy donors. The increased average number of acentric fragments in TSC lympho- cytes and the higher level of unstable anomalies in TSC skin fibroblasts are notable. In the angiofibroma-derived cultures, the frequency of breaks remains wi th in the range of that of the TSC skin fibroblasts, while the frequency of both the dicentric chromosomes and the translocations is dramatically increased. As will be shown below, part of these translocations occur as clonal rearrangements.
Lymphocytes The frequency of stable chromosomal rearrangements in lymphocytes of healthy persons is low and is subject to inter individual variability. Four cells with different kinds of translocations were found in the lymphocyte cultures of one of six TSC
Ta
ble
2
Fre
qu
en
cie
s a
nd
ty
pe
s o
f c
hro
mo
som
al
ab
err
ati
on
s
No.
of
No.
of
Per
cen
t P
erce
nt
Kin
d
of l
esio
ns
Ori
gin
of
No.
of
ind
ep
en
de
nt
cell
s ab
no
rmal
to
tal
cult
ure
s p
rob
and
s cu
ltu
res
anal
yze
d
cell
s le
sio
ns
ctb
+
csb
ac
e d
ic
r ct
e t
inv
LC
O
20
21
2000
2.
2 2.
8 1.
9 0.
4 0.
3 0.
1 0.
1 0.
2 0
LT
S
6 6
435
6.0
7.6
4.8
1.6
0.2
0 0
0.9
0
FC
O
16
19
1800
3.
8 4.
0 2.
9 0
0.06
0
0.06
1.
0 0
FT
S
4 18
16
00
7.1
7 8
5.5
0.3
0.4
0.1
0.1
1 2
0.3
AT
S
4 37
39
00
28.3
30
,6
5.1
0 1.
9 0.
1 0.
1 23
.5
0
Abb
revt
at~
ons
LC
O, p
erip
hera
l ly
mph
ocyt
es f
rom
con
trol
s; L
TS
, per
iphe
ral
lym
pho{
yte
s fr
om T
SC p
atie
nts,
FC
O, s
kin
fibr
obla
sts
from
co
ntro
ls,
FT
S, s
kin
fibr
obla
sts
from
TSC
pat
ient
s; A
TS,
ang
lofi
brom
a-de
rive
d fi
brob
last
-lik
e ce
lls f
rom
TSC
pat
ient
s.
Cytogenetic Studies in Tuberous Sclerosis 165
Table 3 Frequencies and types of chromosomal aberrat ions in per iphera l lymphocytes from six TSC patients and from 20 heal thy donors
No. of No. of Total no. Kind of lesions cells abnormal of lesions
Donors/sex analyzed cells (%) (%) ctb + csb ace t dic r cte
TS1/F 60 7 (11.7) 8 (13.3) 5 3 0 0 0 0 TS3/F 59 1 (1.7) 1 (1.7) 1 0 0 0 0 0 TS2/M 90 11 (12.2) 15 (16.6) 6 4 4 1 0 0 TS4/M 66 2 (3) 2 (3) 2 0 0 0 0 0 TS6/M 60 2 (3.3) 4 (6.7) 4 0 0 0 0 0 TS7/M 100 3 (3) 3 (3) 3 0 0 0 0 0
Sum TS 435 26 (6.0) 33 (7.6) 21 7 4 1 0 0
10controls/F 1024 16 (1.6) 19 (1.9) 11 4 1 3 0 0 10controls/M 976 26 (2.7) 36 (3.7) 26 4 2 2 1 1
patients examined, and three of 20 heal thy donors exhibi ted one such cell each in their lymphocyte cultures. These figures are well wi th in the range of the normal in te r indiv idual var iabi l i ty [31]. The average number of unstable anomalies is in- creased in the lymphocytes of TSC patients, but the difference with the controls cannot be ascertained stat is t ical ly because of the high range of in ter indiv idual var iabi l i ty and the low number of probands in both groups (Table 3). There is no correlat ion between the frequencies of chromosomal anomalies in the pat ient ' s lymphocytes and the severity of the disease.
Dermal Fibroblasts
Biopsies of unaffected skin of the TSC patients were taken in part from the upper arm and in part from the retroauricular area. The latter was chosen because TSC patients were exposed repeatedly to computer tomography (CT) and some of our patients had received CT scans of their skulls a short t ime before the angiofibromas were excised. In order to account for possible regional cytogenetic heterogeneity of the angiofibromas, some of the tumor cultures were establ ished from very small isolated fragments of t issue (see below). The same procedure was fol lowed with the biopsies taken from the skin of the retroauricular area. Table 4 summarizes the results obtained with cultures from unaffected skin of TSC patients and with fibroblasts from heal thy donors, respectively. Both groups of probands exhibit in te r indiv idual var iabi l i ty wi th regard to the frequencies of the various kinds of unstable aberrations. Once again, this precludes statistical ascer ta inment of the increase by a factor of two of the overall frequency of these anomalies in the cultures from the TSC patients. Considering the kinds of unstable anomalies, however, it can be recognized that dicentrics, rings, acentric fragments, and chromat id exchanges occur very rarely in the controls (4% of unstable lesions), whi le these anomalies represent 13.7% of the unstable lesions in skin fibroblasts from the TSC patients. Some of the cultures of skin fibroblasts from both the TSC patients (three of four) and the controls (three of 16) exhibit transloca- tions, some of which occur as clonal rearrangements. If the number of i ndependen t breakage events causing stable rearrangements is counted rather than the number of cells carrying such rearrangements, there are 22 such events in the cultures from our four TSC patients and eight in those from 16 heal thy donors. On a per-donor basis there is thus a 5.5-fold higher level of breaks leading to t ranslocat ions in the cultures der ived from unaffected skin of the TSC patients than in those from heal thy donors.
Tab
le 4
F
req
uen
cies
an
d
typ
es
of
chro
mo
som
al
aber
rati
on
s in
sk
in f
ibro
bla
sts
fro
m f
ou
r T
SC
p
atie
nts
an
d
fro
m
16
hea
lth
y
do
no
rs"
No.
of
Tot
al n
o.
Ori
gin
and
no
of
ab
no
rmal
of
les
ion
s cu
ltu
res
exam
ined
ce
lls
(%)
(%)
Kin
d of
les
ion
s
ctb
+ cs
b ac
e di
c r
cte
t in
v
TS
I:
2M-A
, 3
S-R
28
(5.
6)
28 (
5,6)
27
(5.
4)
0 1
(0.2
) 0
0 0
0 T
S2
:2
M-A
, 3
S-R
26
(5.
2)
32 (
6.4)
23
{4.
6)
1 (0
.2)
5 (1
.0)
1 (0
.2)
0 2
(0.4
) 0
TS
3:3
S
-R
35 (
11 7
) 41
(13
.7)
25 (
8.3)
0
1 (0
.3)
1 (0
.3)
1 [0
.3)
9 [3
.0)
4 (1
.3J
TS
4:3
S
-R
24 (
8.0)
24
(8.
0)
13 (
4.3)
3
(1.0
1 0
0 0
8 (2
7)
4 (0
.3)
Su
m T
S1
-TS
4
113
{7.1
) 12
5 (7
8)
88 (
5.5)
4
{0.3
) 7
(0.4
) 2
(0.1
) 1
(0.0
6)
19 (
1.21
4
(0.3
)
16 c
ontr
ols
14 M
-A,
4 S
-A
69 (
3.8)
73
(4.
0)
53 (
2.9)
0
1 (0
.061
0
1 (0
.06f
18
{1)
0
Cul
ture
s w
ere
deri
ved
m p
art
from
mul
tipl
e fr
agm
ents
of s
kin
biop
sies
fro
m t
he u
pper
~rm
(M
-A],
and
m p
art
from
ver
~ sm
all
sing
le f
ragm
ents
of
ret
roau
rmul
ar s
kin
(S-R
), or
fro
m t
he u
pper
arm
[S-
A},
res
pect
wel
v O
ne h
undr
ed m
itos
es w
ere
anal
yzed
fro
m e
ach
cult
ure
Cytogenet ic S tud ies in Tube rous Sclerosis 167
Table 5 F requenc ie s and types of c h r o m o s o m a l aberra t ions in ang io f ib roma-der ived
f ibrob |as t - l ike cel ls f rom four TSC pat ients
No. of No. of Total no. Kind of lesions Origin of cells abnormal of lesions cultures analyzed cells (%) (%) ctb + csb dic r cte t
TS1 T I a 200 8(4) 10(5) 10 0 0 0 0 T II 1500 484(32.3) 514(34.3) 40 29 2 0 443 TIII F6 b 100 10{10) 11{11) 10 1 0 0 0
TS2 T I 100 47(47) 54(54) 22 0 0 0 32 T II 100 7(7) 10/10) 8 0 2 0 0 T IIl 200 22(11) 26(13) 23 3 0 0 0 T IV 100 3(3) 3(3) 2 0 0 0 1 T V 100 8(8) 8(81 2 0 0 0 6 T VI F1 300 207(69) 208(69.3) 3 4 0 0 201 T VII F1 100 2(2) 2{2) 1 1 0 0 0 T ViII F2 100 69(69/ 69(691 2 0 0 0 67
TS3 T I F2 100 5(5) 5(5) 5 0 0 0 0 T I F3 100 6(6) 8(8) 6 2 0 0 0 T II F1 100 8(8) 8(8) 8 0 0 0 0 T II F2 100 65(65) 70(70) 9 0 0 0 61 T II F3 100 100(100) 134 6 26 0 0 102
TS4 T I F6 100 1(1) 1(1) 1 0 0 0 0 T II F4 100 16(16) 17(17) 5 7 0 2 3 TIII F1 100 18(18) 19(19) 19 0 0 0 0 T IV F1 200 16(8) 16(8) 16 0 0 0 0
3900 1102(28.3) 1193(30.6) 198 73 4 2 916
° The Roman numerals indicate the various tumors studied.
b The designations F1, F2, etc., indicate that the cultures are derived from single fragments of the various tumors. When these designations are missing, the cultures are derived from mulhple fragments of the corre- sponding tumors
This figure increases to 6.8-fold if the two types of pe r icen t r i c invers ions occur r ing in skin cul tures of pat ient TS4 are inc luded .
F ib rob las t - l i ke Cel ls f rom Angiofibromas
Whi le in the ang iof ib roma-der ived cu l tures the f r equency of breaks r emains w i t h i n the range of that of the TSC skin fibroblasts, the n u m b e r of d icen t r i c c h r o m o s o m e s and espec ia l ly that of t rans loca t ions is s igni f icant ly inc reased {Tables 2 and 51. The overa l l f r equency of stable c h r o m o s o m a l anomal i e s in the ang io f ib roma-der ived cul- tures is 23.5%, w h i c h is more than 15 t imes h igher than the rate observed in cu l tures f rom the unaf fec ted skin of our TSC pat ients . The f requency of t r ans loca t ions var ies b e t w e e n pat ients and among the var ious cu l ture charges f rom each of the pat ients . This latter type of var iabi l i ty reflects the cy togene t ic he te rogene i ty of the t umor t issue. In fact, m a n y of the t rans loca t ions observed occur as c lona l rear rangements , thus r ep resen t ing example s of var iega ted t rans loca t ion m o s a i c i s m as desc r ibed by H o e h n et al. [32] in cu l tures f rom pat ients w i th Werne r ' s syndrome. Table 6 s u m m a r i z e s the number s and k inds of all c lonal r ea r rangements that occu r red in cu l tu res f rom var ious angiof ibromas of three TSC pat ients . A m o n g the cel l l ines g rown f rom 17 angiofibro- mas and e x a m i n e d at va r ious early in v i t ro passages, n ine exh ib i t ed a no rma l karyo- type. S ix of the t umor cu l tures s h o w e d m u l t i p l e c lona l rear rangements , w i t h four of these also hav ing sporadic nonc lona l anomal ies ; two tumor l ines exh ib i t ed on ly
168 l; u. Dietrich el al.
Table 6 Clonal chromosome rearrangements in cell cultures derived from angiofibromas of patients TS1, TS2, and TS3
No of No of Origin of cells abnormal cultures analyzed cells (%) Type of rearrangement
TS1 TII 1500 37(2 5)
TS2 TI 100
180[12) 19(1.3)
172(11.5) 33(2.2)
29[291
5(5)
TS2 TV 100 2(2} 3{3)
TS2 TVI F1 300 108{36) 93(31)
TS2 TVIII F2 100 44(44) 2(2)
19(19)
TS3 TII F2 100 58(58)
TS3 TII F3 100 100(100)
46,XX,-13,Tder(13)t(3;13)(q12 q13;q33 q34}+ (Gs + partial trisomy 3q12 - q13-*qter)
46,XX,t(3;13)(q 12 - ql 3;q33 - q34) 47,XX,+4,t(3;13)(q12-q13:q33 q34) 46,XX,t(1,3)(p32 - p34;q27 - q29) 46,XX,t(7:14)(p21-p22;q21 q221
46,XY,t(6:10)(10:lS) (pll p12,p14-p15p11;qll-q12)
46,XY,t(10,18}(p14 - plS;ql 1 - q12) 46,XY,t(7:l 2)(q36:q13)
46,XY,4p 46,XY,t(9;15)(q22,q25 - q26}
46,XY,t[5;6)(q35:q15) 46,XY, - 1, + der(1}t(1;101(p36;ql l] (partial trisomy
10q11-~q22) 46,XY, - 1, + der(1 )t(1 ;10)(p36;q11 ) (partial trisomy
10q11--~q24) 46,XY,- 1, + der(1)t(1:10)(p36;q11) [partial trisomv
10q11--*qter)
46,XY,t(1;7)(q21 - q23;q32 - q36) 47,XY, + 1.t(1;7)(q21 - q23;q32 - q36/ 46,XY.t(2;15)(pll;pll - p13)
46,XX,t(6:8)(q26 - q27;q 13)
46,XX,t(4:24/(p16;q22 - q24)
nonclonal rearrangements. There is, however, not only a high degree of heterogeneity among the various tumors, but also among the cultures derived from different frag- ments of the same tumor. Ten of the 19 different clones can be assigned to four groups characterized by cytogenetically related anomalies. In each of these groups there are clones apparently derived from a primordial type of clone by addit ional changes. The first and second clone of TII from patient TS1, for instance (Table 6), represent the unbalanced and balanced forms of a reciprocal translocation between chromosomes 3 and 13, except that the Gs+ occurs only in the former. The third clone is derived from the second one by nondis junct ion of chromosome 4. This kind of relationship also seems to prevail between some of the clonal and nonclonal anomalies, suggesting that the latter may represent the early stages of c]onal evolution. Another example of clonal evolution is apparently represented by the translocations characterizing two clones from TI of patient TS2. In the original type, part of 18q is translocated to 10p. Breaks at both a more proximal point of 10p and at 6p11-p 12 bring about the recipro- cal exchange of this 10p-18q element with 6p. Entirely unrelated anomalies may also occur in different areas of a tumor, as exemplified by cultures derived from different fragments of tumor TII of patient TS3.
A total of 17 chromosomes are involved in the various translocations. There is no conspicuous clustering of breakpoints on one particular chromosome or in one special region. Only three of the breakpoints occur in different translocations of different
Cytogenetic Studies in Tuberous Sclerosis 169
1 2 3 4 5
_ . . . . . . . . . . .
Q
6 7 8 9 10 11 12
13 14 15
. . . . . . . .
16 17 18
19 20 21 22 XX
Figure I G-banded karyotype of an angiofibroma-derived cell clone from patient TS1 showing 46,XX,t(3;13](q12-q13;q33-q34). Arrowheads indicate breakpoints.
patients. The clonal t(7;14) in TS1 and t(4;14) in TS3 share the breakpoint 14q22. An apparently identical t(5;6)(q35;q15) occurs in a clone of TS2 and in a single cell of TS1. Partial trisomies for segments of chromosomes lq, 3q, 7q, 10q, and 15q have arisen as a consequence of the unbalanced forms of various translocations. Figures 1-5 show examples of some of the more prominent types of clonal rearrangements.
Analysis of Ploidy The frequency of tetraploid mitoses was not generally increased in angiofibroma- derived cultures and in cultures from unaffected skin of TSC patients in comparison with that prevailing in cultures from healthy donors. There were, however, four cell lines that contained high numbers of tetraploid mitoses, three of which were derived from angiofibromas of patients TS1, TS2, and TS3, and the fourth from unaffected skin of patient TS3:TS1 T II, 27.4% (n = 700); TS2 T IV, 28.7% (n -- 3083); TS3 T II F3, 52.8% (n ~ 900); TS3 S-R 2, 31.3% (n ~ 900). In all other cultures from angiofibromas and unaffected skin of TSC patients, the rate of tetraploid mitoses varied within the same broad range as in the cultures from the skin of healthy donors: angiofibroma cultures, 1-15.5%; cultures from unaffected skin, 0.7-8.9%; skin cul- tures from healthy donors, 0-17.2%. Whether or not individual cell lines with very high levels of tetraploidy can more frequently be obtained from tissues of TSC patients than from the skin of healthy donors remains to be investigated. The occurrence of endoreduplications, with frequencies of up to more than 50% of the 4n mitoses, suggests that tetraploid cells arose de novo in our cultures. Although the rate of
170 (: u. Dietrich et al
1 2 3
• ;d ~ "
4 5
, , ,! ̧ ~ ~ ~
6 7 8 9 |0
G *
11 12
13 14 15 16 17 18
19 20 21 22 XY
Figure 2 G-banded karyotype of an anglofibroma-derwed cell clone from patient TS2 showing 46,XX,t(1;7)(q21-q23;q32-q36). Arrowheads indicate breakpoints
octaploid mitoses is about equal in skin cultures from healthy donors (0.08%, n = 18,000) and in those from unaffected skin of TSC patients (0.06%; n = 8,900), the angiofibroma-derived cultures showed about twice the frequency of 8n mitoses (0.14%; n = 37,300).
An interesting relationship exists between the frequency of dicentrics and the ploidy of the cells (Table 7). There is about a tenfold higher level of dicentrics per diploid complement in the tetra- and octoploid cells as compared with the diploid cells in the cultures derived from angiofibromas. A similar relationship prevails in the cultures from unaffected skin of the TSC patients.
Scappaticci e ta ] . [25] first described the occurrence of premature centromere disjunction (PCD) in cell cultures derived from angiofibromas and from other skin lesions of TSC patients. Up to 29% PCD of all chromosomes and up to 17% PCD of part of the chromosomes was seen by these authors at early passages of their cultures. The data shown in Table 8 confirm these results for angiofibroma-derived cultures, although with lower frequencies, which may be related to the comparatively late passages at which our chromosome slides were prepared.
DISCUSSION
We found two different kinds of chromosomal instability in cell cultures from patients with TSC: increased frequencies of unstable chromosomal anomalies in lymphocytes, skin fibroblasts, and fibroblast-like cells from angiofibromas and, in addition, a pro- nounced variegated translocation mosaicism in about half of the angiofibroma-derived
Cytogenetic Studies in Tuberous Sclerosis 171
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18
" ~ * ~ -~, ~ ~ " ! " . . . . . . . " !~a ' -"
19 20 21 22 XY
Figure 3 G-banded karyotype of an angiofibroma-derived cell clone from patient TS2 showing 46,XY,t(2;15)(p11;p11-p13). Arrowheads indicate breakpoints.
cultures. While an increase of breaks and dicentrics in lymphocytes and cultures from skin lesions of TSC patients was also observed by Scappaticci et al. [25], translocations were rarely seen by these authors. In view of the fact that stable clonal and nonclonal rearrangements occurred in only about 50% of our angiofibroma specimens and the frequency of these anomalies showed a high degree of variability even within individ- ual tumors (Table 5), this discrepancy may be explained by chance. Furthermore, a pattern of chromosomal anomalies, similar to that described here, was observed by Pandolfo and Smith (personal communication) in angiofibroma-derived cultures.
Mitelman [33] identified 83 breakpoints in the human karyotype that are preferen- tially involved in chromosomal anomalies of neoplasias characterized by a single type of aberration. Of the 24 different breakpoints found in the clonal rearrangements occurring in angiofibroma-derived cultures, 14 coincide with breakpoints defined by Mitelman [33]. This represents a 2.3-fold higher frequency of coincidence than ex- pected for a random distribution. Cytogenetic investigations of a larger sample of angiofibromas, and especially of other kinds of tumors that affect TSC patients, will help to answer the question of whether or not the coincidence of more than twice the expected number of breakpoints with "cancer breakpoints" is of any significance, e.g., for the increased frequency of gliomas and angiomyolipomas in TSC. Recent evidence from linkage studies suggests that at least one gene causing TSC is located on the most distal segment of the long arm of chromosome 9 [10-12]. It is noteworthy that chromosome 9 is only affected in one of the rearrangements observed in angiofi- bromaoderived cultures, but in a more proximal region of the long arm (TS2, TV, see Table 6). No clustering of the breakpoints of the various clonal and nonclonal
172
v
1 2 3 4 5
,
6 7 8 9 10 11 12
13 14 15 16 17 18
19 20 21 22 X X Figure 4 G-banded karyotype of an angiofibroma-derJved (:eli clone from patient TS3 shuwing 46,XX,t(6,8)(q26-q27;q13). Arrowheads indicate breakpoints.
a b
Figure 5
C
Tab le 7 R e l a t i o n s h i p b e t w e e n the n u m b e r of d i cen t r i c s a n d p l o i d y in cu l tu res de r i ved f rom ang io f ib romas and f rom unaf fec ted sk in of four TSC pa t i en t s
Dicentric chromosomes Origin of No. and percent cultures Ploidy of cells n % % per 2n complement
2 n 1050 (87.5] 14 1.3 1.3 Angiofibromas 4 n 134 (11.2) 37 27.6 13.8
8 n 16 (1.3) 9 563 14.1
2 n 582 (97J 3 0.5 0.5 Unaffected skin
4 n 18 (3) 3 16.7 8.4
Cytogenetic Studies in Tuberous Sclerosis 17 3
Table 8 Frequency of premature centromere division in cell cultures derived from retroauricular skin and from angiofibromas of four TSC patients
No. of cells with Total no. of cells
Origin No. of cells PCD of all Partial PCD with PCD of cultures analyzed chromosomes (%) (%) (%)
Retroauricular skin from 400 TS1, TS2, TS3, and TS4
Five angiofibroma-derived 500 cultures from TS1, TS2, TS3, and TS4
o o o
6 {1.2% ) 11 (2,2%) 17 (3.4°)
Abbreviation. PCD, premature centromere division.
rearrangements at one or a few chromosomal regions was observed, nor was there a single chromosome preferentially involved. This contrasts with the preferential involvement of chromosome 3 in PCD and in the formation of dicentrics reported by Scappaticci et al. [25]. In Werner's syndrome, the distribution of the breakpoints of the multiple rearrangements [32] correlates with the length of the chromosomes [34], with the exception of four hot spots that stand out above this background of an essentially random distribution.
In earlier cytogenetic studies, Suzuki [23] found a fourfold higher frequency of chromosomal anomalies in lymphocytes of four TSC patients than in those of healthy donors. The majority of these anomalies were breaks, but some rings, dicentrics, and chromatid exchanges also occurred. These findings were not confirmed in the study of lymphocyte cultures from ten TSC patients performed by Roos et al. [24]. It is difficult to judge the cause of this discrepancy as the latter authors did not present their cytogenetic results in detail. Average frequencies tend, however, to disguise interindividual variability. The accordance between the extent of increase of the frequency of chromosomal aberrations in lymphocytes of TSC patients found by Scappaticci et al. [25] and by ourselves seems to justify the conclusion that a certain degree of chromosomal instability in TSC is also expressed in lymphocytes.
The constitutional nature of the chromosomal instability in TSC is also corrobo- rated by the results obtained with cultures derived from unaffected skin of the patients (Table 4). Once again, the frequency of anomalies is increased about twofold above the control level in these cells. Besides unstable anomalies, translocations occurred in some cultures of three of the four TSC patients, some of them as clones. Clonal rearrangements were, however, also found in the cultures of three of 16 healthy donors (Table 4). On a per-donor basis there does not seem to be a difference between TSC fibroblasts and those from healthy individuals in this respect. If, however, the numbers of independent breakage events required to produce the rearrangements are compared, there is a sixfold higher frequency of such events in the TSC fibroblasts. In view of the fact that Salk found stable rearrangements (variegated translocation mosaicism) in only eight of 95 lines of fibroblast-like cells from healthy donors [34], our sample of 16 controls exhibits a higher frequency of this phenomenon. This could be due to an incidental choice of individuals with some kind of genetic predisposition to chromosomal instability or to environmental factors. Spontaneous chromosomal in- stability correlates frequently with increased sensitivity against one or the other kind of mutagenic agent [35, 36]. Two lines of fibroblasts from TSC patients were found to be hypersensitive to N-methyl-N'nitro-N-nitrosoguanidine (MNNG) [37], while the response of these and other TSC lines to x-rays was highly variable [38]. After examin-
174 ¢:. u. Dietrich et al.
ing the survival of five TSC lines after exposure to alkylating agents including MNNG, Ohno and Takeshita could not confirm hypersensitivity to this mutagen I39].
For many years, meningiomas represented the only example of a benign tumor characterized by a well-defined chromosomal aberration (for review, see [401). Mean- while, clonal chromosomal rearrangements and numerical aberrations are no longer a rarity in primary cultures of benign tumors. As discussed and summarized recently by Sandberg and coworkers [41-43] and by Helm and Mitelman [441, such anomalies have been found in mixed tumors of the salivary gland [45, 46], in lipomas [47, 48], in leiomyomas of the uterus [49, 50], in colonic adenomas I51], and in benign odontogenic tumors [52]. Consistent breakpoints and/or chromosomes preferentially involved in numerical anomalies were observed in most of these cytogenetically aberrant benign neoplasias. This contrasts with the unspecific distribution of break- points seen in the translocations of our angiofibroma-derived cell cultures. Thus, angiofibromas of TSC patients do not seem to belong to the group of benign tumors characterized by specific chromosomal anomalies. Rather, they exhibit chromosomal instability, causing increased frequencies of both stable rearrangements and unstable chromosomal anomalies. Both aspects of this chromosomal instability represent con- stitutional features of TSC. In this latter respect, TSC resembles the dysplastic nevus syndrome (DNS), where stable chromosomal anomalies have been found not only in cultures derived from dysplastic nevi, but also in those from healthy skin 115, 53]. As in TSC, there was no preferential involvement of a particular chromosome or chromosomal region in the rearrangements observed in the fibroblasts from DNS patients. And TSC shares constitutional chromosomal instability with adenomatosis coli and recti (ACR), another dominantly inherited cancer syndrome ]17, 18]. Further cytogenetic studies, including affected and unaffected cells from a larger number of TSC patients, may show that this disease is the third chromosome instability syn- drome with an autosomal dominant mode of inheritance. In contrast to ACR and DNS, where the benign tumors show a very high tendency of malignant degeneration, this does not seem to occur in angiofibromas of TSC patients. Multiple clones with stable translocations were recently described in a benign basosquamous papilloma by Mertens et al. [54].
The question of whether the frequency of polyploid cells is generally increased in angiofibroma-derived cell cultures from TSC patients remains to be clarified by more extensive studies. The observations of Scappaticci et al. [251 and our finding of individual culture changes with exceedingly high rates of tetraploid mitoses point in this direction. The most conspicuous peculiarity of the tetra- and octoploid cells was their high frequency of dicentric chromosomes (Table 7). Because the relationship between the frequency of dicentrics and polyploidy prevails in both angiofibroma- derived cultures and those from the unaffected skin of TSC patients, this phenomenon may be the consequence of a more general mechanism of polyploidization. One possibility is that dicentric chromosomes delay or inhibit the progress of karyokinesis and cytokinesis. The phenomenon is certainly worth investigating more thoroughly in an appropriate model system.
In their cell cultures grown, respectively, from two angiofibromas, three hypopig- mented macules, and one car6 au lait spot of TSC patients, Scappaticci et al. [25] found premature centromere disjunction (PCD) as a conspicuous disturbance, affect- ing either some or all chromosomes. Albeit at a lower frequency, we were able to confirm this observation (Table 8). The PCD of the X chromosome was considered as a cause of nondisjunction by Fitzgerald et al. [55]. Out-of-phase separation of centromeres, either prematurely or delayed, was proposed as a general mechanism of mitotic nondisjunction by Vig [56[, a hypothesis supported by experimental evidence. Angiofibroma-derived cells from our patient TS3 exhibited a fourfold higher fre- quency of micronuclei and lagging chromosomes than appropriate control lines
Cytogenetic Studies in Tuberous Sclerosis 175
(Krone, unpubl i shed results). German [57] and Louie and German [58] provided some cytogenetic evidence suggesting that PCD in Robert's syndrome may be due to a disturbance of the attraction of the pericentric consti tut ional heterochromatin of sister chromatids. Whether or not the PCD seen in TSC cells is caused by this kind of mechanism or by an error in the interaction between the centromeres and the spindle fibers remains to be investigated.
The relationship between one or the other type of chromosomal instabili ty found in angiofibroma-derived cultures and the increased amounts of small polydisperse circular DNA (spcDNA) in these cells [59] remains to be clarified. It is tempting to speculate, however, that the mobilization of transposable elements may be one of the factors contributing to chromosomal instability in TSC, and possibly also in other chromosomal breakage syndromes.
The authors express their sincere gratitude to the patients and their families for their exemplary and thoughtful cooperation. The help of the volunteers who gave the control biopsies is also gratefully acknowledged. Mrs. Lieselotte Mueller-Molenar and Mrs. Cornelia Staudacher pro- vided technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (Kr 362/17-2).
REFERENCES
1. Koch G {1966): Phakomatosen. In: Humangenetik, Vol. 5/1, 1st Ed., PE Becker, ed. Thieme Verlag, Stuttgart, pp. 34-111.
2. Gomez MR (1988): Criteria for diagnosis. In: Tuberous Sclerosis, 2nd Ed., MR Gomez, ed. Raven Press, New York, pp. 9-19.
3. Hunt A, Lindenbaum RH (1984): Tuberous sclerosis: A new estimate of prevalence within the Oxford region. ] Med Genet 21:272-277.
4. Bender BL, Yunis EJ {1982): The pathology of tuberous sclerosis. Pathol Ann 17:339-382. 5. Jung EG (1973): Die Hautveraenderungen der tuberoesen Hirnsklerose. Schweiz Med
Wochenschr 103:516-521.
6. Pringle JJ {1890): A case of congenital adenoma sebaceum. Br J Dermatol 2:1-14. 7. Nickel WR, Reed WB (1962): Tuberous sclerosis. Arch Dermatol 85:209-226.
8. Connor JM, Yates JRW, Mann L, Aitken DA, Stephenson JBP (1987): Tuberous sclerosis: Analysis of linkage to red cell and plasma protein markers. Cytogenet Cell Genet 44:63-64.
9. Fryer AE, Chalmers A, Connor JM, Fraser I, Povey S, Yates AD, Yates JRW, Osborne JP (1987): Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet i:659-661.
10. Northrup H, Beaudet AL, O'Brien WE, Herman GE, Lewis RA, Pollack MS (1987}: Linkage of tuberous sclerosis to ABO blood group. Lancet ii:804-805.
11. Smith M, Haines J, Trofatter J, Dumars K, Pandolfo M, Conneally M (1987}: Linkage studies in tuberous sclerosis. Human Gene Mapping 9 (Paris 1987). Cytogenet Cell Genet 46:694-695.
12. Povey S, Burley MW, Fryer AE, Osborne J, A1-Gazali LI, Mueller R (1988): Genetic recombina- tion between tuberous sclerosis and oncogene v-abl. Lancet ii:279-280.
13. Jung EG, Bohnert E, Boonen H (1987): Dysplastic nevus syndrome: Ultraviolet hypermutabil- ity confirmed in vitro by elevated sister chromatid exchanges. Dermatologica 173:297-300.
14. Caporaso N, Greene MH, Tsai S, Williams-Pickle L, Mulvihill JJ (1987): Cytogenetics in hereditary malignant melanoma and dysplastic nevus syndrome: Is dysplastic nevus syn- drome a chromosome instability disorder? Cancer Genet Cytogenet 24:299-314.
15. Jaspers NGJ, Roza-de Jongh EJM, Donselaar IG, van Velzen-Tillemans JTM, van Hemel JO, Ruemke P, van der Kamp AWM (1987): Sister chromatid exchanges, hyperdiploidy and chromosomal rearrangements studied in cells from melanoma-prone individuals belonging to families with the dysplastic nevus syndrome. Cancer Genet Cytogenet 24:33-43.
16. Gardner EJ, Rogers SW, Woodward S (1982): Numerical and structural chromosome aberra- tions in cultured lymphocytes and cutaneous fibroblasts of patients with multiple adenomas of the colo-rectum. Cancer 49:1413-1419.
176 c . U . D ie t r i ch et al.
17 Delhanty JDA, Davis MB, Wood J (1983): Chromosome instability in tymphocytes, fibro- blasts, and colon epithelial-like cells from patients with familial polyposis coil. Cancer Genet Cytogenet 8:27-50.
18. Heim S, Greff Johansen S, Kolnig A-M, Stroembeck B (1985). Increased levels of spontaneous and mutagen-induced chromosome aberrations in skin fibroblasts froin patients with adeno- matosis of the colon and rectum. Cancer Genet Cytogenet 17.333-346.
19. Krizman DB, Pathak S, Salnaan NA, Hickey RC (19871: Genetic instability in fibroblasts of patients with thyroid cam:ers (TC). Int J Cancer 39:179-181
20. Krone W, Hoegemann l (19861: Cell culture studies on neurofibr/imatosis (yon Reckhng- hausen). V. Monosomy 22 and other chromosomal anomalies in cultures from peripheral neurofibromas Hum Genet 74:453-455
21. Ridolo P, Zampollo A, De Risio C, Pasquali F (19691: In tema di sclerosl tuberosa: Rlcerca genetica e cromosomica in iina fannglia. Sist Nerv 21:11-17
22. Simon M Jr., Kelemen [, Szoerenyi A, Hammer H, Dobozy A (1979): Ein seltener Mosaizismus der Chromosom-lO-Trisomie bei emer Patientin mit Bourneville-Pringle-Syndrom. Hautarzt 30:292-294.
23. Suzuki Y {1977): Cytogenetlc study of tuberous sclerosis. Brain Nerve 29:537-542.
24. Roos RP, Kirkpatrick C, Gadoth N, Berman W, Rary I [1977): An immunological and cytoge- netic investigation of tuberous sclerosis. Ann Neurol 1:192 194.
25. Scappaticci S, Cerimele D, Tondi M, Vlvarelli R, Fols A, Fraccaro M [1988). Chromosome abnormalities in tuberous sclerosis. Hum Genet 79:151-156
26 Dietrich C. Krone W, Hesse G (1986)' Chromosomal anomalies m cell cultures derived from angiofibromas of patients with tuberous sclerosis. Seventh International Congress of Human Genetics, Berlin, 1986, pp. 569-570 (abstract).
27 Chen TR (1977): In situ detection of mycoplasma cnntaminat ion in ('ell cultures by fluores- cent HOECHST 33258 stain. Exp Cell Res 104:255-262.
28. Pfeiffer RA (19741: Cell cultures from blood and bone marrow. In: Methods in Human Cytogenetics, 1st Ed., HG Schwarzacher, U Wolf. eds. Springer Verlag, New York, pp. 1-37.
29. Francke U, Oliver N [1978): Quantitative analysis of high-resolution trypsin-Giemsa bands on human prometaphase chromosomes Hum Genet 45:137-165.
30. ISCN (1985): An International System for Human Cytogenetlc Nomenclature, Harnden DG, Klinger HP (eds,1; published in collaboration with Cytogenet Cell Genet (Karger, Basel, 19851; also in Birth Defects: Original Article Series, Vol 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985).
31. Llttlefield LG, Goh K-O (1973)' Cytogenetic studies in control men and women, l Variations in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year period. Cytogenet Cell Genet 12:17-34.
32. Hoehn H, Bryant EM, Au K, Norwood TH, Boinan H, Martin GM (1975): Variegated transloca- tion mosaicism in human skin fibroblast cultures. Cytogenet Cell Genet 15:282-298.
33. Mitelman F (1986): Clustering of breakpoints to specific chromosomal regions in human neoplasia. A survey of 5,345 cases. Hereditas 104:113-119.
34. Salk D (1982): Werner 's syndrome: A review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. Hum Genet 62:1-15.
35. Arlett CF, Lehman AR (1978): Human disorders showing increased sensitivity to the induc- tion of genetic damage. Annu Rev Genet 12'95-115.
36. Friedberg EC, Ehmann UK, Williams JI (1979): Human diseases associated with defective DNA repair. Adv Radiat Biol 8:86-174.
37. Scudiero DA, Moshell AN, Scarpinato RG, Meyer SA, Clatterbuck BE, Tarone RE, Robbins JH (1981): Lymphoblastoid lines and skin fibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizing radiation and to a radiomiinetic chemical. I Invest Dermatol 78:234-238.
38, Hayashi A, Yoshida Y, Tanaka H, Arima M, Ohno K (1985): Variable radiosensitivity m fibroblasts from patients with tuberous sclerosis. [ Invest Dermatol 84:77-78.
39. Ohno K. Takeshita K [1984): Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylatmg agents. Jpn J Human Genet 29:359-369.
Cytogenet ic S tud ies in Tube rous Sclerosis 17 7
40. Zang KD (1982}: Cytological and cytogenetical studies on human meningioma. Cancer Genet Cytogenet 6:249-274.
41. Sandberg AA, Turc-Carel C (1987}: The cytogenetics of solid tumors. Relation to diagnosis, classification and pathology. Cancer 59:387-395.
42. Sandberg AA, Turc-Carel C, Gemmill RM {1988}: Chromosomes in solid tumors and beyond. Cancer Res 48:1049-1059.
43. Sandberg AA (1988}: Editorial: Cytogeuetic route of benign tumors. Cancer Genet Cytogenet 32:11-12.
44. Helm S, Mitelman F (1987}: Cancer Cytogenetics. Alan R. Liss, New York.
45. Mark J, Dahlenfos R (1986}: Cytogenetical observations in 100 human benign pleomorphic adenomas: Specificity of the chromosomal aberrations and their relationship to sites of localized oncogenes. Anticancer Res 6:299-308.
46. Bullerdiek ], Boeschen C, Bartnitzke S (1987}: Aberrations of chromosome 8 in mixed salivary gland tumors--cytogenetic findings on seven cases. Cancer Genet Cytogenet 24:205-212.
47. Helm S, Mandahl N, Kristoffersson U, Mitelman F, Rooser B, Rydholm A, Willen H (1986): Reciprocal translocation t{3;12)(q27;q13) in lipoma. Cancer Genet Cytogenet 23:301-304,
48. Turc-Carel C, Dal Cin P, Rao U, Karakousis C, Sandberg AA (1986): Cytogenetic studies of adipose tissue tumors. I. A benign lipoma with reciprocal translocation t{3;12){q28;q14}. Cancer Genet Cytogenet 23:283-289.
49. Gibas Z, Griffin CA, Emanuel BS (1988): Clonal chromosome rearrangements in a uterine myoma. Cancer Genet Cytogenet 32:19-24.
50. Helm S, Nilbert U, Vanni R, Floderus U-M, Mandahl N, Liedgren S, Lecca U, Mitelman F (1988}: A specific translocation, t(12;14}{q14-15;q23-24), characterizes a subgroup of uter- ine leiomyomas. Cancer Genet Cytogenet 32:13-17.
51. Reichmann A, Martin P, Levin B (1985}: Chromosomal banding patterns in human large bowel adenomas. Hum Genet 70:28-31.
52. Stenman G, Sandros J, Mark J, Happonen R-P (1986}: Observations by G-banding in benign odontogenic tumors. Cancer Genet Cytogenet 19:253-259.
53. Hecht F, Kaiser-McCaw Hecht B (1988}: Chromosome rearrangements in dysplastic nevus syndrome predisposing to malignant melanoma. Cancer Genet Cyogenet 35:73-78.
54. Mertens F, Helm S, ]in Y-S, Johansson B, Mandahl N, Bioerklund A, Wennerberg J, Jonsson N, Mitelman F (1989}: Basosquamous papilloma. A benign epithelial skin tumor with multiple cytogenetic clones. Cancer Genet Cytogenet 37:235-239.
55. Fitzgerald PH, Pickering AF, Morcer JM, Meithke PM (1975): Premature centromere division: A mechanism of nondisjunction causing X chromosome aneuploidy in somatic cells of man. Ann Hum Genet 38:417-428.
56. Vig BK (1984}: Sequence of centromere separation: Another mechanism for the origin of nondisjunction. Hum Genet 66:239-243.
57. German J (1979}: Roberts' syndrome. I. Cytological evidence for a disturbance in chromatid pairing. Clin Genet 16:441-447.
58. Louie E, German J (1981}: Roberts' syndrome. I[. Aberrant Y-chromosome behavior. Clin Genet 19:71-74.
59. Neidlinger C, Assum G, Krone W, Dietrich C, Hochsattel R, Klotz G (1988}: Increased amounts of small polydisperse circular DNA (spcDNA} in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TS). Hum Genet 79:286-288.
