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Chromosomal instability in ataxia telangiectasia
Chromosomal Instability in Ataxia Telangiectasia P. H. Kohn, J. Whang-Peng, and W. R. Levis
ABSTRACT: We have examined various aspects of lymphocyte chromosomal instability in three families comprised of five individuals affected with ataxiatelangiectasia (AT), their obligate heterozygous parents, and their unaffected sibs. We found that neither baseline sister chromatid exchanges (SCEs) nor mitomycin-C-induced increments in SCEs showed any significant differences among family members or between AT heterozygotes or homozygotes. Chromosome breakage in first-division metaphases was found to be moderately elevated in three of the five AT homozygotes (range 5-12%); breakage in the six AT obligate heterozygotes was within normal limits (0-4%). Analysis of Giemsa-banded metaphases indicated the presence of a clone bearing a paracentric inversion of chromosome #14 in addition to other chromosome #14 abnormalities in one AT homozygote. The same inversion was also found in this individual's affected sister and his obligate heterozygaus father. A discussion regarding the relationship of the specificity of breakage and reunion of bands q12 and q32 on chromosome #14 and the high incidence of malignancy in AT is included. INTRODUCTION Ataxia telangiectasia (AT) is an autosomal recessive disease characterized c l i n i c a l l y by progressive neurological degeneration, ocnlocutaneous telangiectases, i m m u nodeficiency, and a p r e d i s p o s i t i o n to neoplasia, particularly of the l y m p h o r e t i c u lar system [1-3]. Chromosomal instability has also been s h o w n to be an inherent characteristic of AT [4]. Elevated levels of spontaneous c h r o m o s o m e breakage have been reported in fibroblasts and p e r i p h e r a l l y m p h o c y t e s from some, but not all, affected i n d i v i d u a l s [5-8]. Another cytogenetically significant feature of AT has been the finding of cell clones w i t h a long D-group c h r o m o s o m e [9-13]. In studies where b a n d i n g techniques have been e m p l o y e d this c h r o m o s o m e has been identified as a t a n d e m rearrangement of two #14 chromosomes, with breakpoints a p p a r e n t l y at q12 and q32 [t(14; 14) (q32; q12)] [14-19]. The association between these types of c h r o m o s o m a l instability and the high p r e d i s p o s i t i o n to n e o p l a s i a in AT has been suggested to be more than just fortuitous by several authors [4,7,11]. We present here cytogenetic data from three families with five i n d i v i d u a l s affected w i t h AT. In one of these families a clonal rearrangement of c h r o m o s o m e #14, not p r e v i o u s l y described in association w i t h AT, was f o u n d in three members. From the Department of Pediatrics, University of Florida, Gainesville, Florida; the Cytogenetic Oncology Section, MB, COP, DCT, NCI, Bethesda, Maryland; and the Department of Dermatology, USPHS Hospital, Staten Island, New York. Address requests for reprints to Dr. Peter H. Kahn, Division of Genetics, Department of Pediatrics, Box J-296, J. Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL 32610. Received October 2, 1981; accepted December 7, 1981. 289 © Elsevier Science Publishing Co., Inc., 1982 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics6, 289-302 (1982) 0165-4608/82/080289-1452.75
290
Kohn et al.
CASE REPORTS Family G Detailed clinical findings regarding this family have been previously reported [20,21]. At the time of the present study the 26-year-old affected male (S.G.) (Fig. 1A) was confined to a wheelchair and required constant care. His 20-year-old affected sister (D.G.) (Fig. 1A), although not entirely wheelchair-bound, was not able to eat or walk unassisted. Both affected individuals showed the typical progressive central nervous system (CNS) findings characteristic of AT and had telangiectases of the bulbar conjunctivae, ears, and malar areas. Pertinent laboratory findings included elevated ~-fetoprotein (AFP) levels and borderline detectable IgA. Both had long histories of sinopulmonary infections. S.G. died 3 months after initiation of the study as a result of primary hepatocellular carcinoma and generalized disseminated candidiasis. Postmortem examination revealed that the hepatocellular carcinoma was nondisseminated, and no evidence of neoplasia involving the lymphoreticular system was found (complete autopsy findings will be published elsewhere). Family H C.H., an 8-year-old affected male (Fig. 1B), was found to have clinical and laboratory findings similar to those of the two affected individuals from Family G described above. Briefly, these involved typical CNS findings, with perhaps a greater involvement of the basal ganglia in this patient. Progression of motor loss, although not as severe as in D.G. from Family G, nevertheless necessitated some wheelchair confinement. Telangiectases involving the bulbar conjunctivae and malar areas Figure 1 Pedigree of Family G (A), Family H (B), and Family R (C). Solid symbols indicate individuals affected with AT, and a slash through a symbol indicates a deceased individual. Individuals included in the present study are indicated by a set of initials below the symbols.
A. FamilyG
s.+ Family H L(~)~.n. . N ~ ~<~, ,
6~ B .
D.H. C. FamilyR
L.H.
~j'-]
C.H. K.H.
P.R.~M.R.
+, + ,,+, A.R.
i
6 ,,+,
T.R. D.R. K.R. B.R.
Chromosomal Instability in Ataxia Telangiectasia
291
were noted. Serum AFP levels were found to be elevated, IgA found to be barely detectable, and an acetylcholine receptor autoantibody shown to be present. Disease progression in C.H. has followed essentially the same course with approximately the same degree of severity as in S.G. and D.G. from Family G. Family R Several features serve to distinguish affected members in this family (Fig. 1C) from the affected individuals in Family G and Family H. First, cerebellar CNS involvement predominated, and loss of motor function was decreased. A.R. at age 20, although occasionally confined to a wheelchair, was able at the time of this study to perform other functions such as eating with a minimal amount of aid. T.R., her 18-year-old affected brother, was able to ambulate with the aid of a walker, and B.R., her 10-year-old affected brother, was able for the most part to walk unassisted. Second, although serum AFP values in these three affected individuals were elevated, IgA values were found to be normal. Histories of infection in these individuals were found to be generally unremarkable, although T.R. was not able to participate in the present study because of an episode of pneumonitis. MATERIAL AND METHODS Peripheral blood lymphocytes were grown by a microculture technique (0.7 ml whole blood/lO ml culture) for 72 hr in the dark in Ham's F-12 medium (without thymidine) supplemented with 20% fetal calf serum (Gibco) and 5-bromo2'deoxyuridine (BrdU, final concentration 25 ~M, Sigma). Mitomycin C (MMC, 0.1 ~g/ml, Sigma) was added to a duplicate set of cultures from all individuals from Family H. Colchicine (0.2 ~g/ml) was added for the final hour of culture. Harvesting was accomplished by a lO-min incubation with warm (37°C) hypotonic (0.075 M) KC1 followed by two changes of ice-cold fixative (absolute methanolglacial acetic acid, 3:1). Cell suspensions were dropped on ice-cold, clean, wet slides, steamed, and then dried on a slide warmer set at 60°C. A bone marrow aspirate obtained from S.G. (Family G) was processed directly by a modification of the method of Tijo and Whang [22]. Staining for sister chromatid differentiation (SCD) was accomplished by the method of Goto et al. [23], and Giesma banding was performed by a modification of the technique of Seabright [24]. Slides were coded, and all scoring and examination performed by a single observer. The Kruskal-Wallis test was used to analyze sister chromatid exchange (SCE) data [25]. RESULTS Sister Chromatid Exchange Staining for SCD allows the clear distinction of metaphase chromosome spreads obtained from cells that have gone through one, two, and three or more S phases [26]. Thirty second-division metaphase chromosome spreads from each member of the three families were scored for SCEs. In Family H, an additional 30 spreads from duplicate cultures containing MMC were scored for SCEs for each family member. Although SCE frequencies in the obligate heterozygous parents in the three families were found to be generally higher than those of their affected children, these differences were found not to be statistically significant. In addition, no significant differences were found between affected and nonaffected sibs (Table 1). Baseline-
292
Kohn et al. corrected increments in SCE frequencies due to MMC induction also showed no significant differences among any of the members of Family H (Table 1).
Chromosome Breakage One hundred first-division metaphases from each obligate heterozygous parent and each affected offspring from the three families were scored for chromosome breakage according to conventional standards [27]. Chromosome and chromatid gaps were not scored. Control values for chromosome breakage in this laboratory vary from 0% to 4% using the same techniques (Kohn et al., unpublished data). Using these criteria, three individuals were found to have moderately increased levels of chromosome breakage (range 5-12%). This group consisted of the two affected individuals from Family G and the single affected individual from Family H (Table 2). Both affected individuals from Family R and all the obligate heterozygous parents were found to have chromosome breakage within normal limits (Table 2). No clear preference of chromatid- over chromosome-type breakage could be discerned from any of the data (Table 2).
Chromosome Abnormalities A minimum of 30, and generally at least 50, Giemsa-banded metaphase chromosome spreads from each of the affected AT individuals and their parents were analyzed for chromosome abnormalities. Only the affected male from Family H (C.H.) Fig. 1B), the two affected individuals from Family G (S.G. and D.G.) (Fig. 1A), and their father (E.G.) (Fig. 1A) showed any evidence of karyotypic abnormalities. In 1 cell out of 57 examined from the affected male from Family H (C.H.), t(7q14q) was found (Fig. 2A); no other karyotypic abnormalities were observed. The affected male from Family G (S.G.) was found to have a single metaphase spread with t(7p14q) (Fig. 2B). Although the arms involved, and therefore the break points, in chromosome #7 in the two cells from these two individuals differed, the break point in chromosome #14 in both cases was determined to be the same (namely, q12). An unusual abnormality in chromosome #14 was detected in 32 out of 100 metaphases analyzed from cultures obtained from S.G. (Figs. 3A and 4, S.G.). Detailed analysis indicated that the abnormal chromosome #14 had a paracentric inversion with break points at q12 and q32 (Fig. 3B). In addition to this chromosome #14 abnormality, eight other spreads showed the tandem rearrangement of two #14 chromosomes (Fig. 4, S.G.) frequently seen in association with AT. One cell was detected having an extremely unusual chromosome #14 abnormality involving a tandem triplication of chromosome #14, with what we interpreted as a terminal deletion (Fig. 4, S.G.). In addition to S.G.'s chromosome #14 abnormalities, two cells were found to be monosomic for chromosome #14 and trisomic for chromosome #13 (Fig. 5A). An additional metaphase spread was detected that contained, in addition to one normal chromosome #13, two chromosomes each of which composed end-to-end (qto-q) translocations of two #13 chromosomes. One of these resulted in a dicentric, and the other contained a deletion of the centromere and proximal q arm of one of the #13 chromosomes (Fig. 5B). This metaphase also contained an inverted chromosome #14 of the type described above. The 33 analyzable metaphases obtained from the direct bone marrow preparation from S.G. showed no evidence of chromosome breakage or rearrangement. Other members of Family G also had chromosome #14 abnormalities. D.G., the
293
Chromosomal Instability in Ataxia Telangiectasia
Table 1 Patient Family G E.G.° M.G.° S.G? D.G? Family H E.H.° L.H.a D.H. Lo.H. K.H. C.H? Family R P.R.a M.R.° D.R. K.R. B.R.b A.R?
SCE in AT patients and first-degree relatives Baseline SCEs per metaphase c
12.60 11.72 8.98 9.62
_+ 4.82 _+ 5.41 -+ 3.24 -+ 4.11
11.43 11.83 11.30 9.77 11.50 10.90
_ 3.60 _ 4.07 _+ 3.20 -+ 3.95 _ 3.95 _+ 3.63
13.27 14.00 10.33 9.73 9.97 9.37
_+ 4.07 _+ 4.25 _+ 3.41 _+ 3.47 _+ 2.86 +_ 3.39
MMC SCEs per metaphase c'~
NT NT NT NT 24.83 25.67 25.07 24.00 25.30 23.57
-+ 5.52 -+ 5.98 _+ 5.61 _+ 5.53 --- 5.31 _+ 5.17
SCEse
m
m
w
13.40 13.83 13.77 14.23 13.80 12.67
NT NT NT NT NT NT
°AT obligate heterozygote. ~AT homozygote. CMean -+ standard deviation. dNot tested. eSCEs equals MMC SCEs minus baseline SCEs.
affected younger sister of S.G. (Fig. 1A), was found to have 1 metaphase out of 52 analyzable spreads possessing the same paracentric inversion of c h r o m o s o m e # 1 4 as observed for her brother S.G. (Fig. 4, D.G.). The father of these two affected sibs, E.G., was found to have 1 cell out of 100 with an extremely u n u su al t a n d e m translocation of two # 1 4 chromosomes. The proximal c h r o m o s o m e # 1 4 in this translocation contained the same paracentric inversion as that found w i t h varying frequency in his two affected offspring, whereas the distally translocated piece was that of a normal c h r o m o s o m e # 1 4 (Fig. 4, E.G.).
DISCUSSION Chromosomal instability in AT does not appear to be manifested by differences in SCE frequencies, in contrast to Bloom's syndrome w h e r e SCE frequencies have been found to be highly elevated [28]. Although the present data [Table 1) and that of others [29] suggest a decreased frequency of SCEs in AT homozygotes in comparison with normals and AT heterozygotes, these differences were not statistically significant. Similarly, although M M C - in d u ced increments in SCE frequencies appear to differentiate i n d i v i d u a l s homozygous for the Fanconi anemia gene [30], data from the present study [Table 1) and that of others [31] have not s h o w n any differential SCE response for either AT homozygotes or AT heterozygotes. One of the c o m p o n e n t s of AT c h r o m o s o m a l instability is the finding of increased levels of c h r o m o s o m e breakage in both l y m p h o c y t e s [32] and fibroblasts [7] ob-
294 Table 2
Kohn et al. Chromosome breakage in AT obligate heterozygotes and affected offspring Types of breakage
Patient Family G E.G.° M.G? S.G.b D.GP Family H E.H.a L.H.° C.H? Family R P.R.° M.R.° B.RP A.RP
Breakage (%)c
Chromosome breaks and acentric fragments
2 4 12 7
-1 3 6
2 1 5
1 -2
2 0 2.2d 3.6d
-----
Dicentrics
Chromatid breaks
Triradials and quadriradials
m
1
-
3
-
6
D
-
3
m
m
1 1 2
1 -
m
m
1
2 1 2
aAT obligate heterozygote. bAT homozygote. CNumberof cells with ~'1 break per cell from a total of 100 cells scored, expressed as a percentage. dFor B.R. and A.R., 45 and 55 metaphases were scored, respectively.
tained from some AT homozygotes. In the present study, only three of the five AT patients showed moderately increased levels of chromosome breakage (Table 2). Our and other investigators' inability to show increased levels of breakage i n cells from all AT i n d i v i d u a l s may be attributable to disease progression and related evolution and changes i n manifestation of chromosomal instability, as suggested previously [16]. Alternatively, it is conceivable that elicitation of chromosome breakage in AT cells may be related to a genetically heterogeneous i n vitro response to the a m o u n t and kind of stress placed u p o n the cells in culture, w h i c h could vary d e p e n d i n g u p o n the types and components of culture media employed, as has been s h o w n with the marker X chromosome in c o n n e c t i o n with X-linked mental retardation [33] and in patients with other forms of chromosomal instability [34]. This possibility remains to be tested. The finding of increased chromosome breakage and formation of clones i n AT lymphocytes and fibroblasts does not appear to extend to results obtained from bone marrow samples or to E p s t e i n - B a r r virus (EBV)-transformed lymphocytes [35,36]. In the present study and in seven of n i n e other reported cases of AT where bone marrow chromosomes have been examined [7,11,15,18,37,38], no chromosome abnormalities have been found. In one of the two r e m a i n i n g cases, a deleted G-group chromosome was seen in bone marrow metaphases from an AT patient with acute lymphocytic leukemia (ALL) [39]. Neither any increase i n chromosome breakage nor any clonal i n v o l v e m e n t of D-group chromosomes was apparent, making it u n l i k e l y that the observed chromosome abnormality was related to those c o m m o n l y seen i n association with AT. In the second of these two cases [40], breaks were found in 4 of 12 bone marrow metaphases examined; the significance of this is not clear, as few cells were observed and the nature of the abnormalities was not well described. It appears that i n general the reported in vitro
295
A
q
t(7;14)(q36;q12)
14
B
i
7
t(7;14}(p15;q12)
14
t(7;14)(p15;q12)
Figure 2 Partial karyotypes showing t(7q14q) from C,H. of Family H (A) and t(7p14q) from S.G. of Family G (B). Representative diagrams are shown below their respective chromosomes.
296
Kohn et al.
A
¢
g
¢
1\
¢
/
¢
¢
14
inv(14)(q12q32)
Figure 3 Partial karyotypes showing the paracentric inversion of chromosome #14 found in lymphocytes from S.G. of Family G. (A) Two pairs of chromosome #14 homologs; the normal chromosome #14 is presented as the left-hand member of each pair and the inverted chromosome #14 as the right-hand member. (B) chromosomes identical to those found in (A); in this instance, however, the right-hand member of each pair has been rotated 180 deg to obtain correct alignment of the banding patterns to better show the nature of this inversion. [Misalignment of bands of q23 and q24.2 occurs in (A). Correct alignment of these bands is achieved in (B).] Representative diagrams are found at the bottom of the figure, c, Centromere.
c h r o m o s o m a l instability is not n o r m a l l y a p p a r e n t in vivo. It has been suggested [7] that the lack of positive in vivo findings m a y be due to e x a m i n a t i o n of o n l y the d i v i d i n g m y e l o i d elements in the bone marrow; however, l y m p h o i d and other actively d i v i d i n g cell types s h o u l d also be present in bone marrow aspirates, making it likely that cytogenetic analysis w o u l d have i n c l u d e d a s a m p l i n g of these elements also. Whether c h r o m o s o m a l instability in AT is an in vitro p h e n o m e n o n related to culture conditions or w h e t h e r AT gene expression of c h r o m o s o m e abnormalities is restricted only to certain cell types, perhaps at different levels of maturation, cannot be d e t e r m i n e d at present. Thus far in AT, in only one instance has a l e u k e m i c bone m a r r o w s p e c i m e n been e x a m i n e d for c h r o m o s o m e abnormalities; unfortunately, a p e r i p h e r a l blood s p e c i m e n was not also i n c l u d e d [39]. There have been no reports of c h r o m o s o m e analyses of solid t u m o r specimens from AT patients with
14
i |
inv(14)(q12q32)
-
14
q
t(14;14)(q3~;q12)
inv(14)(q12q32),
-'-%-
14
--
t(14;14)(q32;q12)
3~ 1 23
'!
14
i
4
__
___
(q32;q12q32;q12) del(14)(q22)
t114;14;141
Z
-T
~.
P-~
i i
Figure 4 Partial karyotypes showing chromosome #14 abnormalities in the father (E.G.), affected son (S.G.), and affected daughter (D.G.) from Family G. Representative diagrams are shown below their respective chromosomes.
D.G.
3.G.
-.G.
13
~
-
dic(13)(q34)
1
q -
q
P ~
ii P
t(13; 1 3 ) ( q 3 4 ; q 3 4 ) , del(13)(q14)
1
~ 2 2
3~
2;
1--
z 13 4
14
inv(14)(q12q32)
a
F i g u r e 5 Partial karyotypes showing additional chromosome abnormalities from S.C. of Family G. (A) Trisomy 13 ~nd monosomy 14. /B) Complex abnormalities of chromosomes #13 and #14 {see text for further explanation]. Representative diagrams are shown below their respective chromosomes.
q
,~
i
,
P
jl ~,'i¸
A
Chromosomal Instability in Ataxia Telangiectasia
299
other types of cancer. Cytogenetic analysis of both malignant and nonmalignant tissue types such as these could certainly help resolve the question of whether chromosomal instability in AT is an in vivo process or whether it is strictly an in vitro phenomenon. The other component of chromosomal instability in AT is the formation of cytoganetically abnormal clones involving rearrangements of chromosome #14 as the common feature. Although tandem rearrangements of two chromosome #14 have been shown to occur with the greatest frequency, translocation of chromosome #14 to other nonhomologous chromosomes has also been reported in AT [10,16,17,41]. Among the latter types, the most common finding has been the reciprocal translocation of chromosome #14 to either the long or short arm of chromosome #7 [7,14,16,19], as found in one cell from each of two of our patients (C.H. and S.G., respectively). Longitudinal studies on patients with translocations of chromosome #14 to nonhomologous chromosomes indicate that these clones either do not show any significantly consistent increase in frequency or that they actually decrease in frequency, and neoplasia has not been reported to have resulted [7,10,16]. In all cases where banding studies have been performed, the break point on chromosome #14 has been at band q12. In contrast, chromosome #14 tandem rearrangements were formed following breakage at bands q12 and q32. They have been shown to increase in frequency over time and to be associated with the development of malignancy in several instances [7,16,18,38]. In one such case where ALL developed in an AT patient, a preexisting tandem chromosome #14 rearrangement was the progenitor of the subsequent leukemic clone [16]. These tandem chromosome #14 rearrangements occur as apparently reciprocal translocations, both with [7,18,38] and without [7,15,16,17,40] loss of the small centric translocated chromosome #14 and also in combination with a normal chromosome #14 [7,8,9,11,12,16] (Fig. 4, S.G.), resulting in triplication of most of the genetic material on the long arm of chromosome #14. It has been suggested that loss of the centric translocated chromosome #14 leads to an increase in the potential for malignancy [7], presumably because of functional monosomy for the region 14pter--~14q12. Indeed, this has been the finding in four cases of malignancy in AT patients [7,16,18,38]; however, our patient, S.G., seems to contradict this thesis, as none of his 41% of cells with various chromosome #14 abnormalities were found to be monosomic for this portion and yet he died as a result of primary hepatocellular carcinoma. Increased potential for malignancy, at least in our patient, can therefore not be correlated with partial monosomy 14. All tandem rearrangements of chromosome #14, regardless of whether they are balanced translocations, partial monosomies, or partial trisomies, share the common feature of having been formed following breakage of one chromosome #14 at band q12 and another chromosome #14 at band q32. The physical result in all cases has been rearrangment of the genetic material on chromosome #14, juxtaposing gene sequences at band q12 with gene sequences at band q32. We submit that it is this repositioning of genetic material on chromosome #14 that confers a selective growth advantage on cells possessing it, concurrently increasing the potential for the development of malignancy. At the present time all available data from the literature are consistent with this hypothesis of genetic position effect. The data from our patient (E.G.), whose major clonal abnormality before his death from liver cancer was a paracentric inversion of chromosome #14 with break points at q12 and q32, lend further support for this hypothesis. Presumably this inversion involved no loss or gain of genetic material. Genetic position effect has been shown to operate in a similar manner in mice [42] and other organisms [43]. Although hard evidence is lacking in humans, the operation of genetic position effect follow-
300
Kohn et al. ing c h r o m o s o m a l rearrangement w i t h subsequent onset of n e o p l a s i a has been suggested by data obtained from patients w i t h hereditary renal cell c a r c i n o m a [44], chronic myelogenous l e u k e m i a [45], and other leukemias and l y m p h o m a s [46]. Although c h r o m o s o m a l instability has been s h o w n in cells obtained from some i n d i v i d u a l s heterozygous for the AT gene [7,11,19], this has been a relatively unc o m m o n finding [6,9,17,18,39,41]. The risk of m a l i g n a n c y for relatives of AT patients has been shown to be elevated, although not to the extent found in affected AT i n d i v i d u a l s [47]. If c h r o m o s o m a l instability is intrinsically related to neoplastic processes in AT, one w o u l d expect to find increased breakage and clonal formation in some AT heterozygotes, albeit at a r e d u c e d level in c o m p a r i s o n with AT homozygotes. In the present study we were able to show the presence of c h r o m o s o m a l instability as e v i d e n c e d by c h r o m o s o m e # 1 4 rearrangements in only one of six heterozygotes (E.G.). Although c h r o m o s o m e breakage levels were w i t h i n n o r m a l limits, a c h r o m o s o m e # 1 4 a b n o r m a l i t y was detected having break points at q12 and q32. Longitudinal studies on AT heterozygotes with c h r o m o s o m e abnormalities such as this are necessary to establish the general relationship of genetic p o s i t i o n effect and m a l i g n a n c y in AT. Chromosomal instability in AT, as e v i d e n c e d by increased levels of chromosome breakage and the formation of clones with c h r o m o s o m e # 1 4 abnormalities, appears to be related to a high p r e d i s p o s i t i o n for neoplasia. Breakage at bands q12 and q32 on c h r o m o s o m e #14, with subsequent repositioning of the genetic material, is a finding peculiar to i n d i v i d u a l s heterozygous and homozygous for AT genes. Whether the p h e n o m e n o n of genetic position effect can be invoked either as an explanation for an increase in the potential for m a l i g n a n c y or as a direct cause of malignancy remains to be proven. At this time more cytogenetic data are n e e d e d from malignant tissues and from longitudinal studies on m u l t i p l e tissue types obtained from heterozygous and homozygous AT individuals.
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C h r o m o s o m a l Instability in A t a x i a T e l a n g i e c t a s i a
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12. Hayashi K, Schmidt W (1975): Tandem duplication q14 and dicentric formation by endto-end chromosome fusions in ataxia telangiectasia (AT). Humangenetik 30, 135-141. 13. Goodman WN, Cooper WC, Kessler GB, Fischer MS, Gardner MD (1969): Ataxia telangiectasia--A report of two cases in siblings presenting a picture of progressive spinal muscular atrophy. Bull Los Angeles Neurol Soc 34, 1-22. 14. Hecht F, McCaw BK (1973): Evidence for a consistent chromosomal abnormality in ataxia-telangiectasia (A-T) lymphocytes. Am J Hum Genet 25, 32A. 15. Hecht F, McCaw BK, Koler RD (1973): Ataxia-telangiectasia--Clonal growth of translocation lymphocytes. N Engl J Med 289, 286-291. 16. McCaw BK, Hecht F, Harnden DG, Teplitz RL (1975): Somatic rearrangement of chromosome 14 in human lymphocytes. Proc Natl Acad Sci USA 72, 2071-2075. 17. Oxford JM, Harnden DG, Parrington JM, Delhanty JD (1975): Specific chromosomes aberrations in ataxia telangiectasia. J Med Genet 12,251-262. 18. Levitt R, Pierre RV, White WL, Siekert RG (1978): Atypical lymphoid leukemia in ataxia telangiectasia. Blood 52, 1003-1011. 19. Aurias A, Dutrillaux B, Buriot D, Lejeune J (1980): High frequencies of inversions and translocations of chromosomes 7 and 14 in ataxia telangiectasia. Mutat Res 69, 369-374. 20. Fireman P, Boesman M, Gitlin D (1964): Ataxia telangiectasias: A dysgammaglobinemia with deficient ~,A(~2A)globulin. Lancet 1, 1193-1195. 21. Bar RS, Levis WR, Rechler MM, Harrison LC, Siebert S, Podskalny J, Roth J, Muggeo M (1978): Extreme insulin resistance in ataxia telangiectasia: Defect in affinity of insulin receptors. N Engl J Med 298, 1164-1171. 22. Tijo JH, Whang J (1962): Chromosome preparations of bone marrow cells without prior in vitro culture or in vivo colchicine administration. Stain Technol 37, 17-20. 23. Goto K, Maeda S, Kano Y, Sugiyama T (1978): Factors involved in differential Giemsastaining of sister chromatids. Chromosoma 66, 351-359. 24. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2, 9 7 1 972. 25. Sokal RR, Rohlf FJ (1969): Biometry. Freeman, San Francisco, pp. 387-391. 26. Craig-Holmes AP, Shaw MW (1976): Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique. Exp Cell Res 99, 79-87. 27. Buckton KE, Evans HJ (1973): Methods for the Analysis of Human Chromosome Aberrations. WHO, Geneva, pp. 18-53. 28. Chaganti RS, Schonberg S, German J (1974): A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71, 4508-4512. 29. Hatcher NH, Brinson PS, Hook EB (1976): Sister chromatid exchanges in ataxia telangiectasia. Murat Res. 35, 333-336. 30. Latt SA, Stetten G, Juergens LA, Buchanan GR, Gerald PS (1975): Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia. Proc Natl Acad Sci USA 72, 4066-4070. 31. Galloway SM (1977): Ataxia telangiectasia. The effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes. Mutat Res 45, 343-349. 32. Hecht F, Koler RD, Rigas DA, Dahnke GS, Case MP, Tisdale V (1968): Leukemia and lymphocytes in ataxia-telangiectasia. Lancet 2, 1193. 33. Sutherland GR (1979): Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture. Am J Hum Genet 31, 125-135. 34. Keck M, Emerit I (1979): The influence of culture medium composition on the incidence of chromosomal breakage. Hum Genet 50, 277-283. 35. Cohen MM, Sagi M, Ben-Zur Z, Schaap T, Voss R, Kohn G, Ben-Bassat H (1979): Ataxia telangiectasia--Chromosomal stability in continuous lymphoblastoid cell lines. Cytogenet Cell Genet 23, 44-52. 36. Kohn PH, Kraemer KH, Buchanan JK (1982): Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and replication inhibition in human lymphoblastoid lines. Exp Cell Res 137, 387-395. 37. Hecht F, Case M (1969): Emergence of a clone of lymphocytes in ataxia-telangiectasia. American Society of Human Genetics Annual Meeting, San Francisco, October 1-4.
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Kohn et al. 38. Sparkes RS, Como R, Golde DW (1980): Cytogenetic abnormalities in ataxia telangiectasia with T-cell chronic lymphocytic leukemia. Cancer Genet Cytogenet 1,329-336. 39. Lampert F (1969): Akute lymphoblasticke leuk~mie bei Geschwistern mit progressiver Kleinhirnataxie (Louis-Bar-Syndrom). Dtsch Med Wochenschr 94, 217-220. 40. Lisker R, Cobo A (1970): Chromosome breakage in ataxia-telangiectasia. Lancet 1,618. 41. Harnden DG (1974): Ataxia telangiectasia syndrome: Cytogenetic and cancer aspects. In: Chromosomes and Cancer, J German, ed. John Wiley, New York, pp. 619-636. 42. Russell LB, Bangham JW (1961): Variegated-type position effects in the mouse. Genetics 46, 509-525. 43. Demeree M, Slyzinska H (1937): Mottled white 258-18 of Drosophila melanogaster. Genetics 22, 641-649. 44. Cohen AJ, Li FP, Berg S, Marchetto DJ, Tsai S, Jacobs SC, Brown RS (1979): Hereditary renal-cell carcinoma associated with a chromosomal translocation N. Engl J Med 301, 592-595. 45. Rowley JD (1973): A new consistent chromosomal abnormality in chronic myelogeneous leukemia identified by quinacrine fluorescence and giemsa staining. Nature 243, 290293. 46. Rowley JD (1980): Chromosome abnormalities in cancer. Cancer Genet Cytogenet 2, 175198. 47. Swift M, Sholman L, Perry M, Chase C (1976): Malignant neoplasms in the families of patients with ataxia telangiectasia. Cancer Res 36, 209-215.
ABSTRACT: We have examined various aspects of lymphocyte chromosomal instability in three families comprised of five individuals affected with ataxiatelangiectasia (AT), their obligate heterozygous parents, and their unaffected sibs. We found that neither baseline sister chromatid exchanges (SCEs) nor mitomycin-C-induced increments in SCEs showed any significant differences among family members or between AT heterozygotes or homozygotes. Chromosome breakage in first-division metaphases was found to be moderately elevated in three of the five AT homozygotes (range 5-12%); breakage in the six AT obligate heterozygotes was within normal limits (0-4%). Analysis of Giemsa-banded metaphases indicated the presence of a clone bearing a paracentric inversion of chromosome #14 in addition to other chromosome #14 abnormalities in one AT homozygote. The same inversion was also found in this individual's affected sister and his obligate heterozygaus father. A discussion regarding the relationship of the specificity of breakage and reunion of bands q12 and q32 on chromosome #14 and the high incidence of malignancy in AT is included. INTRODUCTION Ataxia telangiectasia (AT) is an autosomal recessive disease characterized c l i n i c a l l y by progressive neurological degeneration, ocnlocutaneous telangiectases, i m m u nodeficiency, and a p r e d i s p o s i t i o n to neoplasia, particularly of the l y m p h o r e t i c u lar system [1-3]. Chromosomal instability has also been s h o w n to be an inherent characteristic of AT [4]. Elevated levels of spontaneous c h r o m o s o m e breakage have been reported in fibroblasts and p e r i p h e r a l l y m p h o c y t e s from some, but not all, affected i n d i v i d u a l s [5-8]. Another cytogenetically significant feature of AT has been the finding of cell clones w i t h a long D-group c h r o m o s o m e [9-13]. In studies where b a n d i n g techniques have been e m p l o y e d this c h r o m o s o m e has been identified as a t a n d e m rearrangement of two #14 chromosomes, with breakpoints a p p a r e n t l y at q12 and q32 [t(14; 14) (q32; q12)] [14-19]. The association between these types of c h r o m o s o m a l instability and the high p r e d i s p o s i t i o n to n e o p l a s i a in AT has been suggested to be more than just fortuitous by several authors [4,7,11]. We present here cytogenetic data from three families with five i n d i v i d u a l s affected w i t h AT. In one of these families a clonal rearrangement of c h r o m o s o m e #14, not p r e v i o u s l y described in association w i t h AT, was f o u n d in three members. From the Department of Pediatrics, University of Florida, Gainesville, Florida; the Cytogenetic Oncology Section, MB, COP, DCT, NCI, Bethesda, Maryland; and the Department of Dermatology, USPHS Hospital, Staten Island, New York. Address requests for reprints to Dr. Peter H. Kahn, Division of Genetics, Department of Pediatrics, Box J-296, J. Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL 32610. Received October 2, 1981; accepted December 7, 1981. 289 © Elsevier Science Publishing Co., Inc., 1982 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics6, 289-302 (1982) 0165-4608/82/080289-1452.75
290
Kohn et al.
CASE REPORTS Family G Detailed clinical findings regarding this family have been previously reported [20,21]. At the time of the present study the 26-year-old affected male (S.G.) (Fig. 1A) was confined to a wheelchair and required constant care. His 20-year-old affected sister (D.G.) (Fig. 1A), although not entirely wheelchair-bound, was not able to eat or walk unassisted. Both affected individuals showed the typical progressive central nervous system (CNS) findings characteristic of AT and had telangiectases of the bulbar conjunctivae, ears, and malar areas. Pertinent laboratory findings included elevated ~-fetoprotein (AFP) levels and borderline detectable IgA. Both had long histories of sinopulmonary infections. S.G. died 3 months after initiation of the study as a result of primary hepatocellular carcinoma and generalized disseminated candidiasis. Postmortem examination revealed that the hepatocellular carcinoma was nondisseminated, and no evidence of neoplasia involving the lymphoreticular system was found (complete autopsy findings will be published elsewhere). Family H C.H., an 8-year-old affected male (Fig. 1B), was found to have clinical and laboratory findings similar to those of the two affected individuals from Family G described above. Briefly, these involved typical CNS findings, with perhaps a greater involvement of the basal ganglia in this patient. Progression of motor loss, although not as severe as in D.G. from Family G, nevertheless necessitated some wheelchair confinement. Telangiectases involving the bulbar conjunctivae and malar areas Figure 1 Pedigree of Family G (A), Family H (B), and Family R (C). Solid symbols indicate individuals affected with AT, and a slash through a symbol indicates a deceased individual. Individuals included in the present study are indicated by a set of initials below the symbols.
A. FamilyG
s.+ Family H L(~)~.n. . N ~ ~<~, ,
6~ B .
D.H. C. FamilyR
L.H.
~j'-]
C.H. K.H.
P.R.~M.R.
+, + ,,+, A.R.
i
6 ,,+,
T.R. D.R. K.R. B.R.
Chromosomal Instability in Ataxia Telangiectasia
291
were noted. Serum AFP levels were found to be elevated, IgA found to be barely detectable, and an acetylcholine receptor autoantibody shown to be present. Disease progression in C.H. has followed essentially the same course with approximately the same degree of severity as in S.G. and D.G. from Family G. Family R Several features serve to distinguish affected members in this family (Fig. 1C) from the affected individuals in Family G and Family H. First, cerebellar CNS involvement predominated, and loss of motor function was decreased. A.R. at age 20, although occasionally confined to a wheelchair, was able at the time of this study to perform other functions such as eating with a minimal amount of aid. T.R., her 18-year-old affected brother, was able to ambulate with the aid of a walker, and B.R., her 10-year-old affected brother, was able for the most part to walk unassisted. Second, although serum AFP values in these three affected individuals were elevated, IgA values were found to be normal. Histories of infection in these individuals were found to be generally unremarkable, although T.R. was not able to participate in the present study because of an episode of pneumonitis. MATERIAL AND METHODS Peripheral blood lymphocytes were grown by a microculture technique (0.7 ml whole blood/lO ml culture) for 72 hr in the dark in Ham's F-12 medium (without thymidine) supplemented with 20% fetal calf serum (Gibco) and 5-bromo2'deoxyuridine (BrdU, final concentration 25 ~M, Sigma). Mitomycin C (MMC, 0.1 ~g/ml, Sigma) was added to a duplicate set of cultures from all individuals from Family H. Colchicine (0.2 ~g/ml) was added for the final hour of culture. Harvesting was accomplished by a lO-min incubation with warm (37°C) hypotonic (0.075 M) KC1 followed by two changes of ice-cold fixative (absolute methanolglacial acetic acid, 3:1). Cell suspensions were dropped on ice-cold, clean, wet slides, steamed, and then dried on a slide warmer set at 60°C. A bone marrow aspirate obtained from S.G. (Family G) was processed directly by a modification of the method of Tijo and Whang [22]. Staining for sister chromatid differentiation (SCD) was accomplished by the method of Goto et al. [23], and Giesma banding was performed by a modification of the technique of Seabright [24]. Slides were coded, and all scoring and examination performed by a single observer. The Kruskal-Wallis test was used to analyze sister chromatid exchange (SCE) data [25]. RESULTS Sister Chromatid Exchange Staining for SCD allows the clear distinction of metaphase chromosome spreads obtained from cells that have gone through one, two, and three or more S phases [26]. Thirty second-division metaphase chromosome spreads from each member of the three families were scored for SCEs. In Family H, an additional 30 spreads from duplicate cultures containing MMC were scored for SCEs for each family member. Although SCE frequencies in the obligate heterozygous parents in the three families were found to be generally higher than those of their affected children, these differences were found not to be statistically significant. In addition, no significant differences were found between affected and nonaffected sibs (Table 1). Baseline-
292
Kohn et al. corrected increments in SCE frequencies due to MMC induction also showed no significant differences among any of the members of Family H (Table 1).
Chromosome Breakage One hundred first-division metaphases from each obligate heterozygous parent and each affected offspring from the three families were scored for chromosome breakage according to conventional standards [27]. Chromosome and chromatid gaps were not scored. Control values for chromosome breakage in this laboratory vary from 0% to 4% using the same techniques (Kohn et al., unpublished data). Using these criteria, three individuals were found to have moderately increased levels of chromosome breakage (range 5-12%). This group consisted of the two affected individuals from Family G and the single affected individual from Family H (Table 2). Both affected individuals from Family R and all the obligate heterozygous parents were found to have chromosome breakage within normal limits (Table 2). No clear preference of chromatid- over chromosome-type breakage could be discerned from any of the data (Table 2).
Chromosome Abnormalities A minimum of 30, and generally at least 50, Giemsa-banded metaphase chromosome spreads from each of the affected AT individuals and their parents were analyzed for chromosome abnormalities. Only the affected male from Family H (C.H.) Fig. 1B), the two affected individuals from Family G (S.G. and D.G.) (Fig. 1A), and their father (E.G.) (Fig. 1A) showed any evidence of karyotypic abnormalities. In 1 cell out of 57 examined from the affected male from Family H (C.H.), t(7q14q) was found (Fig. 2A); no other karyotypic abnormalities were observed. The affected male from Family G (S.G.) was found to have a single metaphase spread with t(7p14q) (Fig. 2B). Although the arms involved, and therefore the break points, in chromosome #7 in the two cells from these two individuals differed, the break point in chromosome #14 in both cases was determined to be the same (namely, q12). An unusual abnormality in chromosome #14 was detected in 32 out of 100 metaphases analyzed from cultures obtained from S.G. (Figs. 3A and 4, S.G.). Detailed analysis indicated that the abnormal chromosome #14 had a paracentric inversion with break points at q12 and q32 (Fig. 3B). In addition to this chromosome #14 abnormality, eight other spreads showed the tandem rearrangement of two #14 chromosomes (Fig. 4, S.G.) frequently seen in association with AT. One cell was detected having an extremely unusual chromosome #14 abnormality involving a tandem triplication of chromosome #14, with what we interpreted as a terminal deletion (Fig. 4, S.G.). In addition to S.G.'s chromosome #14 abnormalities, two cells were found to be monosomic for chromosome #14 and trisomic for chromosome #13 (Fig. 5A). An additional metaphase spread was detected that contained, in addition to one normal chromosome #13, two chromosomes each of which composed end-to-end (qto-q) translocations of two #13 chromosomes. One of these resulted in a dicentric, and the other contained a deletion of the centromere and proximal q arm of one of the #13 chromosomes (Fig. 5B). This metaphase also contained an inverted chromosome #14 of the type described above. The 33 analyzable metaphases obtained from the direct bone marrow preparation from S.G. showed no evidence of chromosome breakage or rearrangement. Other members of Family G also had chromosome #14 abnormalities. D.G., the
293
Chromosomal Instability in Ataxia Telangiectasia
Table 1 Patient Family G E.G.° M.G.° S.G? D.G? Family H E.H.° L.H.a D.H. Lo.H. K.H. C.H? Family R P.R.a M.R.° D.R. K.R. B.R.b A.R?
SCE in AT patients and first-degree relatives Baseline SCEs per metaphase c
12.60 11.72 8.98 9.62
_+ 4.82 _+ 5.41 -+ 3.24 -+ 4.11
11.43 11.83 11.30 9.77 11.50 10.90
_ 3.60 _ 4.07 _+ 3.20 -+ 3.95 _ 3.95 _+ 3.63
13.27 14.00 10.33 9.73 9.97 9.37
_+ 4.07 _+ 4.25 _+ 3.41 _+ 3.47 _+ 2.86 +_ 3.39
MMC SCEs per metaphase c'~
NT NT NT NT 24.83 25.67 25.07 24.00 25.30 23.57
-+ 5.52 -+ 5.98 _+ 5.61 _+ 5.53 --- 5.31 _+ 5.17
SCEse
m
m
w
13.40 13.83 13.77 14.23 13.80 12.67
NT NT NT NT NT NT
°AT obligate heterozygote. ~AT homozygote. CMean -+ standard deviation. dNot tested. eSCEs equals MMC SCEs minus baseline SCEs.
affected younger sister of S.G. (Fig. 1A), was found to have 1 metaphase out of 52 analyzable spreads possessing the same paracentric inversion of c h r o m o s o m e # 1 4 as observed for her brother S.G. (Fig. 4, D.G.). The father of these two affected sibs, E.G., was found to have 1 cell out of 100 with an extremely u n u su al t a n d e m translocation of two # 1 4 chromosomes. The proximal c h r o m o s o m e # 1 4 in this translocation contained the same paracentric inversion as that found w i t h varying frequency in his two affected offspring, whereas the distally translocated piece was that of a normal c h r o m o s o m e # 1 4 (Fig. 4, E.G.).
DISCUSSION Chromosomal instability in AT does not appear to be manifested by differences in SCE frequencies, in contrast to Bloom's syndrome w h e r e SCE frequencies have been found to be highly elevated [28]. Although the present data [Table 1) and that of others [29] suggest a decreased frequency of SCEs in AT homozygotes in comparison with normals and AT heterozygotes, these differences were not statistically significant. Similarly, although M M C - in d u ced increments in SCE frequencies appear to differentiate i n d i v i d u a l s homozygous for the Fanconi anemia gene [30], data from the present study [Table 1) and that of others [31] have not s h o w n any differential SCE response for either AT homozygotes or AT heterozygotes. One of the c o m p o n e n t s of AT c h r o m o s o m a l instability is the finding of increased levels of c h r o m o s o m e breakage in both l y m p h o c y t e s [32] and fibroblasts [7] ob-
294 Table 2
Kohn et al. Chromosome breakage in AT obligate heterozygotes and affected offspring Types of breakage
Patient Family G E.G.° M.G? S.G.b D.GP Family H E.H.a L.H.° C.H? Family R P.R.° M.R.° B.RP A.RP
Breakage (%)c
Chromosome breaks and acentric fragments
2 4 12 7
-1 3 6
2 1 5
1 -2
2 0 2.2d 3.6d
-----
Dicentrics
Chromatid breaks
Triradials and quadriradials
m
1
-
3
-
6
D
-
3
m
m
1 1 2
1 -
m
m
1
2 1 2
aAT obligate heterozygote. bAT homozygote. CNumberof cells with ~'1 break per cell from a total of 100 cells scored, expressed as a percentage. dFor B.R. and A.R., 45 and 55 metaphases were scored, respectively.
tained from some AT homozygotes. In the present study, only three of the five AT patients showed moderately increased levels of chromosome breakage (Table 2). Our and other investigators' inability to show increased levels of breakage i n cells from all AT i n d i v i d u a l s may be attributable to disease progression and related evolution and changes i n manifestation of chromosomal instability, as suggested previously [16]. Alternatively, it is conceivable that elicitation of chromosome breakage in AT cells may be related to a genetically heterogeneous i n vitro response to the a m o u n t and kind of stress placed u p o n the cells in culture, w h i c h could vary d e p e n d i n g u p o n the types and components of culture media employed, as has been s h o w n with the marker X chromosome in c o n n e c t i o n with X-linked mental retardation [33] and in patients with other forms of chromosomal instability [34]. This possibility remains to be tested. The finding of increased chromosome breakage and formation of clones i n AT lymphocytes and fibroblasts does not appear to extend to results obtained from bone marrow samples or to E p s t e i n - B a r r virus (EBV)-transformed lymphocytes [35,36]. In the present study and in seven of n i n e other reported cases of AT where bone marrow chromosomes have been examined [7,11,15,18,37,38], no chromosome abnormalities have been found. In one of the two r e m a i n i n g cases, a deleted G-group chromosome was seen in bone marrow metaphases from an AT patient with acute lymphocytic leukemia (ALL) [39]. Neither any increase i n chromosome breakage nor any clonal i n v o l v e m e n t of D-group chromosomes was apparent, making it u n l i k e l y that the observed chromosome abnormality was related to those c o m m o n l y seen i n association with AT. In the second of these two cases [40], breaks were found in 4 of 12 bone marrow metaphases examined; the significance of this is not clear, as few cells were observed and the nature of the abnormalities was not well described. It appears that i n general the reported in vitro
295
A
q
t(7;14)(q36;q12)
14
B
i
7
t(7;14}(p15;q12)
14
t(7;14)(p15;q12)
Figure 2 Partial karyotypes showing t(7q14q) from C,H. of Family H (A) and t(7p14q) from S.G. of Family G (B). Representative diagrams are shown below their respective chromosomes.
296
Kohn et al.
A
¢
g
¢
1\
¢
/
¢
¢
14
inv(14)(q12q32)
Figure 3 Partial karyotypes showing the paracentric inversion of chromosome #14 found in lymphocytes from S.G. of Family G. (A) Two pairs of chromosome #14 homologs; the normal chromosome #14 is presented as the left-hand member of each pair and the inverted chromosome #14 as the right-hand member. (B) chromosomes identical to those found in (A); in this instance, however, the right-hand member of each pair has been rotated 180 deg to obtain correct alignment of the banding patterns to better show the nature of this inversion. [Misalignment of bands of q23 and q24.2 occurs in (A). Correct alignment of these bands is achieved in (B).] Representative diagrams are found at the bottom of the figure, c, Centromere.
c h r o m o s o m a l instability is not n o r m a l l y a p p a r e n t in vivo. It has been suggested [7] that the lack of positive in vivo findings m a y be due to e x a m i n a t i o n of o n l y the d i v i d i n g m y e l o i d elements in the bone marrow; however, l y m p h o i d and other actively d i v i d i n g cell types s h o u l d also be present in bone marrow aspirates, making it likely that cytogenetic analysis w o u l d have i n c l u d e d a s a m p l i n g of these elements also. Whether c h r o m o s o m a l instability in AT is an in vitro p h e n o m e n o n related to culture conditions or w h e t h e r AT gene expression of c h r o m o s o m e abnormalities is restricted only to certain cell types, perhaps at different levels of maturation, cannot be d e t e r m i n e d at present. Thus far in AT, in only one instance has a l e u k e m i c bone m a r r o w s p e c i m e n been e x a m i n e d for c h r o m o s o m e abnormalities; unfortunately, a p e r i p h e r a l blood s p e c i m e n was not also i n c l u d e d [39]. There have been no reports of c h r o m o s o m e analyses of solid t u m o r specimens from AT patients with
14
i |
inv(14)(q12q32)
-
14
q
t(14;14)(q3~;q12)
inv(14)(q12q32),
-'-%-
14
--
t(14;14)(q32;q12)
3~ 1 23
'!
14
i
4
__
___
(q32;q12q32;q12) del(14)(q22)
t114;14;141
Z
-T
~.
P-~
i i
Figure 4 Partial karyotypes showing chromosome #14 abnormalities in the father (E.G.), affected son (S.G.), and affected daughter (D.G.) from Family G. Representative diagrams are shown below their respective chromosomes.
D.G.
3.G.
-.G.
13
~
-
dic(13)(q34)
1
q -
q
P ~
ii P
t(13; 1 3 ) ( q 3 4 ; q 3 4 ) , del(13)(q14)
1
~ 2 2
3~
2;
1--
z 13 4
14
inv(14)(q12q32)
a
F i g u r e 5 Partial karyotypes showing additional chromosome abnormalities from S.C. of Family G. (A) Trisomy 13 ~nd monosomy 14. /B) Complex abnormalities of chromosomes #13 and #14 {see text for further explanation]. Representative diagrams are shown below their respective chromosomes.
q
,~
i
,
P
jl ~,'i¸
A
Chromosomal Instability in Ataxia Telangiectasia
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other types of cancer. Cytogenetic analysis of both malignant and nonmalignant tissue types such as these could certainly help resolve the question of whether chromosomal instability in AT is an in vivo process or whether it is strictly an in vitro phenomenon. The other component of chromosomal instability in AT is the formation of cytoganetically abnormal clones involving rearrangements of chromosome #14 as the common feature. Although tandem rearrangements of two chromosome #14 have been shown to occur with the greatest frequency, translocation of chromosome #14 to other nonhomologous chromosomes has also been reported in AT [10,16,17,41]. Among the latter types, the most common finding has been the reciprocal translocation of chromosome #14 to either the long or short arm of chromosome #7 [7,14,16,19], as found in one cell from each of two of our patients (C.H. and S.G., respectively). Longitudinal studies on patients with translocations of chromosome #14 to nonhomologous chromosomes indicate that these clones either do not show any significantly consistent increase in frequency or that they actually decrease in frequency, and neoplasia has not been reported to have resulted [7,10,16]. In all cases where banding studies have been performed, the break point on chromosome #14 has been at band q12. In contrast, chromosome #14 tandem rearrangements were formed following breakage at bands q12 and q32. They have been shown to increase in frequency over time and to be associated with the development of malignancy in several instances [7,16,18,38]. In one such case where ALL developed in an AT patient, a preexisting tandem chromosome #14 rearrangement was the progenitor of the subsequent leukemic clone [16]. These tandem chromosome #14 rearrangements occur as apparently reciprocal translocations, both with [7,18,38] and without [7,15,16,17,40] loss of the small centric translocated chromosome #14 and also in combination with a normal chromosome #14 [7,8,9,11,12,16] (Fig. 4, S.G.), resulting in triplication of most of the genetic material on the long arm of chromosome #14. It has been suggested that loss of the centric translocated chromosome #14 leads to an increase in the potential for malignancy [7], presumably because of functional monosomy for the region 14pter--~14q12. Indeed, this has been the finding in four cases of malignancy in AT patients [7,16,18,38]; however, our patient, S.G., seems to contradict this thesis, as none of his 41% of cells with various chromosome #14 abnormalities were found to be monosomic for this portion and yet he died as a result of primary hepatocellular carcinoma. Increased potential for malignancy, at least in our patient, can therefore not be correlated with partial monosomy 14. All tandem rearrangements of chromosome #14, regardless of whether they are balanced translocations, partial monosomies, or partial trisomies, share the common feature of having been formed following breakage of one chromosome #14 at band q12 and another chromosome #14 at band q32. The physical result in all cases has been rearrangment of the genetic material on chromosome #14, juxtaposing gene sequences at band q12 with gene sequences at band q32. We submit that it is this repositioning of genetic material on chromosome #14 that confers a selective growth advantage on cells possessing it, concurrently increasing the potential for the development of malignancy. At the present time all available data from the literature are consistent with this hypothesis of genetic position effect. The data from our patient (E.G.), whose major clonal abnormality before his death from liver cancer was a paracentric inversion of chromosome #14 with break points at q12 and q32, lend further support for this hypothesis. Presumably this inversion involved no loss or gain of genetic material. Genetic position effect has been shown to operate in a similar manner in mice [42] and other organisms [43]. Although hard evidence is lacking in humans, the operation of genetic position effect follow-
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Kohn et al. ing c h r o m o s o m a l rearrangement w i t h subsequent onset of n e o p l a s i a has been suggested by data obtained from patients w i t h hereditary renal cell c a r c i n o m a [44], chronic myelogenous l e u k e m i a [45], and other leukemias and l y m p h o m a s [46]. Although c h r o m o s o m a l instability has been s h o w n in cells obtained from some i n d i v i d u a l s heterozygous for the AT gene [7,11,19], this has been a relatively unc o m m o n finding [6,9,17,18,39,41]. The risk of m a l i g n a n c y for relatives of AT patients has been shown to be elevated, although not to the extent found in affected AT i n d i v i d u a l s [47]. If c h r o m o s o m a l instability is intrinsically related to neoplastic processes in AT, one w o u l d expect to find increased breakage and clonal formation in some AT heterozygotes, albeit at a r e d u c e d level in c o m p a r i s o n with AT homozygotes. In the present study we were able to show the presence of c h r o m o s o m a l instability as e v i d e n c e d by c h r o m o s o m e # 1 4 rearrangements in only one of six heterozygotes (E.G.). Although c h r o m o s o m e breakage levels were w i t h i n n o r m a l limits, a c h r o m o s o m e # 1 4 a b n o r m a l i t y was detected having break points at q12 and q32. Longitudinal studies on AT heterozygotes with c h r o m o s o m e abnormalities such as this are necessary to establish the general relationship of genetic p o s i t i o n effect and m a l i g n a n c y in AT. Chromosomal instability in AT, as e v i d e n c e d by increased levels of chromosome breakage and the formation of clones with c h r o m o s o m e # 1 4 abnormalities, appears to be related to a high p r e d i s p o s i t i o n for neoplasia. Breakage at bands q12 and q32 on c h r o m o s o m e #14, with subsequent repositioning of the genetic material, is a finding peculiar to i n d i v i d u a l s heterozygous and homozygous for AT genes. Whether the p h e n o m e n o n of genetic position effect can be invoked either as an explanation for an increase in the potential for m a l i g n a n c y or as a direct cause of malignancy remains to be proven. At this time more cytogenetic data are n e e d e d from malignant tissues and from longitudinal studies on m u l t i p l e tissue types obtained from heterozygous and homozygous AT individuals.
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