Hereditas 86: 121-128
(1977)
Cytogenetic studies in seven individuals with an i(Xq) karyotype E. NIEBUHR AND F. SKOVBY’ University Institute of Medical Genetics, Copenhagen,Denmark
NIEBUHR, E. and SKOVBY, F. 1977. Cytogenetic studies in seven individuals with an i(Xq) karyotype. 86: 121-128. Lund, Sweden. ISSN 0018-0661. Received March 19, 1977
- Herediras
Seven individuals with stigmata of the Turner syndrome, large Barr bodies and a late replicating, structurally abnormal X-chromosome, have been reinvestigated with different banding techniques to elucidate the mechanisms leading to formation of isochromosomes. Dicentric chromosomes With separated C-blocks were found in five of these individuals. The two remaining patients had a slightly larger and a nearly double sized block of centromeric heterochromatin. Using the centromeric model proposed by Lima-de-Faria in 1956, the size of these blocks can be related to different locations of break points within the centromere or in the short arm. The presence of a 45,X cell line in all cases is probably due to anaphase lag secondary to instability of an abnormal isochromosome centromere. The mechanisms behind centromere inactivation in dicentrics are still obscure and not likely to be disclosed before the ultrastructure of these abnormal centromeres is known in more detail, The necessity of a more specific centromere nomenclature is emphasized. Additional rare or unexpected findings were normal menstruation in one individual, bipartite Barr bodies in two cases and one pericentric inversion of chromosome 9. Erik Niehuhr, University Institute of Medical Genetics, Tagensvej 14, DK-2200 Copenhagen, Denmark
Misdivision of a centromere may lead to an unstable, telocentric chromosome and subsequently to formation of an isochromosome (DARLINGTON 1939). Such a mechanism has been favoured in most reports of subjects with an abnormal X chromosome resembling an autosome No. 3. DE LA CHAPELLE et al. (1966) suggested an alternative mechanism of misdivision in the centromeric region near the short arm or in the short arm itself, leading to formation of a dicentric X isochromosome by fusion of the sister chromatids. This possibility was predicted by DARLINGTON (1939) and the terms “dicentric isochromosome” or “isodicentric” (DARLINGTON and WYLIE 1953) were proposed. In order to test these two mechanisms we have reexamined the chromosomes in ten females with a missing C-group chromosome and an extra No. 3-like chromosome. A brief clinical description and the results of different banding techniques, autoradiography and sex chromatin studies are presented and discussed in view of the morphology of the centromere as proposed by LIMA-DE-FARIA (1 956). Case reports
The patients reported on were referred for chromosome analysis because of a Turner-like phenotype
and/or short stature. We were able to repeat the cytogenetic study on ten of eleven patients with presumptive i(Xq) chromosomes. Three of these cases will not be considered further because the abnormal chromosomes were identified as two Xautosome translocations and one 6p + . Case 1. - BGH 24/73 was referred at age 25 for infertility and short stature. She had never menstruated. Her height was 142 cm. Pubic and axillary hair was absent. The uterus was small and the adnexae were not palpated at pelvic examination. She was of normal intelligence. X-rays showed a slightly android pelvis and open iliac epiphyses. Case 2. - HBA 119/73 was referred at the age of 11 for short stature and obesity. Birth weight was 2400 gm. She had had generalized seizures since age 1. Her height at referral was 119 cm. The extremities were short with cubitus valgus. The intelligence was within normal limits. X-rays showed a horseshoeshaped kidney. Case 3. - DAS 242173 was referred at age 16 for obesity, short stature and amenorrhea. Her height
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Present address: Department of Obstetrics and Gynecology, Johns Hopkins Hospital, Baltimore, Maryland 21205, U.S.A.
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was 134 cm. Webbing of the neck was present. The breast development was poor with widely spaced nipples. The extremities were short with cubitus valgus. Pubic and axillary hair was scant. The uterus and the adnexae were not felt at pelvic examination. The intelligence was normal. Case 4 . - LES 29/74 was referred at age 45 for short stature. She had entered puberty normally and had menstruated until the age of 32. Laparotomy at age 36 showed a small uterus. Microscopy of ovarian tissue revealed normal stroma with corpora albicantia but without primordial follicles and without corpora lutea. Her height at referral was 135 cm and the lower extremities were disproportionally short. Breast development and pubic hair were both within normal limits. She was of normal intelligence. X-rays showed a small sella turcica and brachymetacarpalism. Case 5. - HRM 30/74 was referred at age 10 because of obesity, impaired hearing and short stature. Actual height was 117 cm. Her nipples were widely spaced, and the labia majora and the clitoris were absent. She had cubitus valgus. The intelligence was normal. Case 6. - KIM 91/74 was referred at the age of 12 for short stature and obesity. When seen at age 16, she had not menstruated. Her height was then 135 cm. There was no webbing of the neck but the hair line was low. A shield chest and a large number of pigmented nevi were present. Breast development was absent and the nipples were widely spaced. Pubic and axillary hair was scant. The uterus and the adnexae were not palpable at pelvic examination. Her intelligence was normal. X-rays showed a small sella turcica but no brachymetacarpalism. Case 7. - M K P 332/75 was referred at the age of 8 for precocious puberty on the basis of incipient pubic hair. Family history was negative. Her height was 123 cm. The neck was short. X-rays showed a normal sella turcica; skeletal age was two years advanced.
Material and methods Buccal smears were stained with Feulgen. Five hundred cells from each proband were examined for the presence of X-chromatin. Peripheral blood leucocytes were cultured for three days and prepared for chromosomal analysis. Conventional staining was performed with lactoacetic Orcein. Cultures for autoradiography were labelled with
3H-thymidine (Amersham, specific activity 1,9 pCi/mM) at a final concentration of 0.5 pCi/ml of culture medium for the last four hours of incubation. The slides were stained with Orcein, coated with photographic emulsion (Ilford K 2) and exposed for 11-13 days. Fifty metaphase plates were photographed in each case. Q-, R- and C-banding patterns were obtained according to the techniques described by CASPERSSON et al. (1970), DUTRILLAUX and LEIEUNE(1972) and (1972), respectively. SUMNER
Results All patients were X-chromatin positive with lower mean values than our routine counts from normal females (Table I). The Barr bodies were larger than normal. Cells with two large Barr bodies were observed in case 5, 6 and 7. Bipartite Barr bodies were observed on buccal smears in cases 6 and 7. Two cell lines were found in all seven cases: a cell line with a modal count of 45 in which a C-group chromosome was missing, and a cell line with 46 chromosomes in which a C-group chromosome was replaced by a metacentric or nearly metacentric chromosome resembling a No. 3. A third cell line with a chromosome number of 47 and two such chromosomes was observed in cases 5 , 6 and 7. The frequencies of the different cell lines are shown in Table 1. A wide centromeric constriction or a dicentric morphology was often observed in cases 3-7. A monocentric appearance was always observed in case 1 and 2 and occasionally in cases 3-7 (e.g. Fig. 1, cases 5 and 7). Autoradiography of all seven probands showed a late replicating chromosome resembling a No. 3. Its pattern of DNA synthesis appeared symmetrical around the centromere. Q- and R-banding patterns in all patients showed one normal X chromosome only. In most cells with a chromosome count of 46, the banding pattern of both arms of the abnormal C-group chromosome was identical to that of the long arm of a normal X chromosome (Fig. 1). However, in some cells (cases 3-7) the banding pattern was asymmetric around the centromere (e.g. Fig. I , case 7). C-banding of cases 1 and 2 showed only one block of heterochromatin in the isochromosome. The size of the block in case 1 was slightly larger than that of a normal X-chromosome and it was nearly of double size in case 2 (Fig. 2). One C-group chromosome in case 1 was abnormal
INDIVIDUALS WITH AN I(XQ) KARYOTYPE
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Table I . Chromosome number and X-chromatin counts Case No.
Blood cultures
X-chromatin' Percentage frequencies of the different cell lines (n = 100) Percentage frequencies of Barr bodies (n = 500)
45,XO
46,XX
46,X,i(Xq)
47,X,i(Xq),i(Xq) 0
1
2
Bipartite
0
92 94 90 74 78 25 46
0 0 0 0 4 3 7
22 24 19 13 18 11 8
0
3 4 5 6 1
8 6 10 26 18 12 47
0 0 0 0 0 3 4
'
normal range 20-32%
1
7
0 0 0
0 0 0
with a large heterochromatic block located on the short arm. Q- and C-bands revealed a pericentric inversion of chromosome No. 9: inv(9)(p12;q21) (Fig. 3). Twice as much centromeric heterochromatin as in a normal X was observed in the isochromosomes of cases 3-7. The heterochromatin was observed as two average sized and clearly separated blocks (Fig. 2). In some metaphase plates from case 7, one of the centromeric blocks appeared smaller and more weakly stained than the other (Fig. 4b). The distance between the blocks varied from case to case; the largest distance was observed in case 7. Intrapersonal variance was not observed. The identity of the large metacentric chromosomes containing the additional C-band material was in all cases verified by successive application of Q- and C-band staining to the same metaphase plates. (Fig. 3, 4) - Isochromosomes with only one average sized heterochromatic block were never observed in cases 3-7.
Discussion 1. General considerations
The first description of the Turner syndrome associated with an isochromosome for the long arm of et al. the human X was published by FRACCARO (1960). According to SCHMIDet al. (1974), this chromosomal abnormality accounts for about 13% of patients with the Turner syndrome. Patients with i(Xq) are reported to be clinically indistinguishable from those in whom an entire X is missing. This held true in the present material (because of the
78 76 81 87 80 84 85
0
0 0 2 2 3
mode of ascertainment) with exception of case 4 who menstruated regularly for 18 years. A few cases of the Turner syndrome with regular menstruation and ROSENBERG (e.g. ZARATEet al. 1969; GILBOA 1975) or even pregnancy (e.g. BAHNERet al. 1960; PHILIPand SELE1976) have been reported. In 1966 DE LA CHAPELLE et al. suggested that some i(Xq) were dicentric, but the cytological methods available at that time did not allow any confirmatory investigations. More recently, DE LA CHAPELLE and STENSTRAND (1974) were able to demonstrate twice the normal amount of centromeric heterochromatin in two out of four patients with i(Xq), consistent with the previous interpretation as idic(Xq). Similar cases have been reported by YANACISAWA (1973), COHENet al. (1975), PRIESTet al. (1979, and HOWELL et al. (1976). At least five of the seven individuals (cases 3-7) in the present study had dicentric X-chromosomes, suggesting that this type is more frequent than estimated by DE LA CHAPELLE and STENSTRAND (1974). Case 2 might also be a dicentric. The true incidence of idic(Xq) can only be determined by applying a C-band technique to each case of i(Xq), particularly since dicentric chromosomes sometimes appear monocentric by conventional staining methods (NIEBUHR 1972a, e.g. case 4). A monocentric appearance in 6 out of 11 i(Xq) cases studied without heterochromatin staining (SCHMIDet al. 1974) does not give any additional information concerning the frequency of dicentric i(Xq) chromosomes. The presence of a 45,X cell line in all patients is most likely due to anaphase lag of an isochromosome present in the zygote. If the formation of the isochromosome was a postzygotic event, a 46,XX cell line should also be present which was not the case.
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3
3
i.(Xq) X
Case 1
Case 2
Case 3
Case 3
Case 4
Case 5
P
Case 6
Case 7 I
2
Hereditas 86 (1977)
INDIVIDUALSWITH AN I(XQ) KARYOTYPE
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k
*
"I
0-
4b Fig. 3 and 4. Details of two mitoses successively stained with quinacrine mustard (a) and C-band technique (b). x 2700. - Fig. 3. Case 1; pair No. 9 with a pericentric inversion, the normal X and the i(Xq) with a somewhat larger heterochromatic block. - Fig. 4. Case 7; arrows indicating two abnormal X-chromosomes (47,X,i(Xq), i(Xq)); note the smaller size and less intense staining of one centromere.
It is difficult to assess the significance of a pericentric inversion of chromosome 9 in case 1. According to BouE et al. (1979, the estimated incidence of this inversion is about 1%. COHENet al. (1975) reported a pericentric inversion involving chromo-
some 2 in a patient with another dicentric X-isochromosome. Preferential inactivation of the abnormal X and large Barr bodies are common features of cases with i(Xq) chromosomes. The relatively low frequency of
Fig. 1 and 2. - Fig. I . Partial karyotypes showing pair No. 3, abnormal and normal X stained with quinacrine mustard (case 3 also with acridine orange). Note asymmetry around the centromere (case 3, acridine orange and case 7). the dicentric appearance in cases 3 , 4 and 6, and the monocentric appearance in cases 1,2,5 and 7. x 2500. - Fig. 2. C-band stained partial karyotypes showing pair No. 3, abnormal X and in case 1 and 2 also the normal X chromosome. Note the larger appearance of the heterochromatic block on the isochromosome than that of the normal X (case 1 and 2) and the two separated blocks in cases 3 7. ~ 2 5 0 0 .
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A
Hereditas 86 (1977)
u B
C
D
E
Fig. 5. A: the centromere model as proposed by LIMA-DE-FARIA (1956) with possible break points and centromeric heterochromatin. B, C, D, E: centromere structure after breakage at points 1-4, sister strand fusion and replication.
Barr bodies in our study may be due to a 45,X cell line in all our patients. The 47,i(Xq), i(Xq) cell line in cases 5, 6 and 7 was probably responsible for the occasional presence of cells with two large Barr bodies. Bipartite Barr bodies as seen in cases 6 and 7 are a rare, but not unusual observation in patients with dicentric i(Xq) chromosomes (THERMAN et al. 1974). 2. Formation of isochromosomes Monocentrics. In the centromere model, as proposed by LIMA-DE-FARIA (l956), breakage may occur at different points (Fig. 5). A break at point 1 (inner zone) at the G , or G2 stage will produce a chromosome which is unstable during meiosis or mitosis because of insufficient centromeric material to which the spindle apparatus may attach. However, the sister parts of the centromere may heal, creating a monocentric isochromosome which behaves like a normal chromosome during meiosis and mitosis. In such isochromosomes the amount of centromeric heterochromatin would be similar to that of a normal X. Two of the patients studied by DE LA CHAPELLE and STENSTRAND (1 974) showed a single, normal sized heterochromatic block. Our case 1 might be another example of a true isochromosome, but the somewhat larger heterochromatic block seems to indicate a more distal break in the centromere. ~
Dicentrics. Breaks at point 2, 3 or 4 (Fig. 5) will produce a telocentric chromosome or an acrocentric. ~
If fusion between sister chromatids occur, a breakagefusion cycle will be established during mitosis or the second meiotic division due to the presence of two functioning centromeres. The outcome will usually be elimination of the cell or loss of the chromosome. Little attention has been paid to the sequence of events which makes isochromosome formation possible when a chromosome break has occurred at point (1939, p. 357) and HAMERTON 2 , 3 or 4. DARLINGTON (1971) named this “second misdivision” but it does not at all imply a division. The essentiel events are a centromere non-disjunction, allowing the chromatids to pass to the same cell, preceded or followed by a fusion of the chromatids at the point of breakage. As discussed below, the inactivation of one of the two centromeres in the spindle apparatus is necessary in order to secure the transmission of the isochromosome in future cell divisions. If an isochromosome is due to a break at point 2 or 3, the centromeric heterochromatin should maximally be twice as large as that of a normal X. It would appear as one larger block, as demonstrated in case 2. However, the cases of DE LA CHAPELLE and STENSTRAND and our cases 3-7 showed a clear separation of the two heterochromatic blocks in most cells. This indicates that the breaks leading to the dicentric isochromosomes occurred at the short arm chromatids (point 4, Fig. 5 ) as suggested by DE LA CHAPELLE et al. (1966). According to the Paris Conference (1971) nomenclature the designation of case 1 and 2 is 46,X,i(X) (qter-cen-qter) and that ofcases 3-7: 46,X,dic(X),
Hereditas 86 (1977)
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(qter-rpl 1 :: p l l -+qter).It was not possible to determine whether these cases in fact d o represent isochromosomes or translocations. The nomenclature suggested at the Paris Conference (1971) and suppl. (1975) is, however, not adequate to convey the information about the different types of i(Xq) chromosomes which can be differentiated with heterochromatin staining. A nomenclature describing the different parts of the centromere is necessary to allow a more precise designation of isochromosomes due to breaks within or close to the centromere.
I(XQ)KARYOTYPE
127
It is known from studies in Agropyron scabium with a large dicentric isochromosome (HAIR 1953) that the distance between the two centromeres influences the stability of the abnormal chromosome. Chromosomes with a long intercentric segment are unstable due to criss-cross separations. Repetition of a breakage-fusion-bridge cycle tends to shorten the distance with production of stable dicentric chromosomes or apparently monocentrics. A similar phenomenon has been observed in man (NIEBUHR1972a, cases 1 and 2). The absence of constriction in one of the two centromeres in a dicentric chromosome could be due to destruction of the juxtacentromeric 3 . Centromere suppression regions by criss-cross separation. Likewise, the stabiIn spite of two separated heterochromatic blocks lity could be due to destruction of the point of (cases 3-7) indicating the presence of two centroattachment for the spindle apparatus. The smaller meres, iso-chromosomes with a monocentric appear- size of one centromere with separated heterochromatance were occasionally observed in the same patient ic blocks in some metaphase plates from case 7 may with techniques other than C-banding. A similar be due to loss of part of one centromere, and thus monocentric appearance has been described e.g. in a an example of juxtacentromeric destruction. Howlarge idic(Xq) (DE LA CHAPELLE and STENSTRANDever, the demonstration of very stable dicentric 1974; THERMAN et al. 1974), in five dicentric Roberthuman chromosomes with a large intercentric dissonian translocations (NIEBUHR1972a) and in a tance (e.g. DISTECHEet al. 1972; DE LA CHAPELLE dicentric t(5;13) (NIEBUHR 1972b). and STENSTRAND 1974; THERMAN et al. 1974) suggests The origin of the human No. 2 chromosome has an inactivation mechanism, not dependent upon been shown to be two acrocentric chromosomes from destruction of the chromatids. The observation of a primate progenitor fused short arm-short arm dicentric translocation chromosomes with non-ran1972). Thus, No. 2 is phylogenetically dom separation of the chromatids in one centromere (DE GROUCHY a dicentric chromosome, but only one centromere and the normal function of the other (NIEBUHR functions under normal circumstances. The other 1972a, case 4; WARBURTON et al. 1973) may support the existence of such a mechanism. However, before centromere (2q22) has even lost its C-staining ability; occasionally it is active as a starting point of chroma- the ultrastructure of the centromere in man is known tid replication in cases of selective endoreduplication in more detail, the mechanisms behind centromere inactivation are unlikely to be disclosed. (LEJEUNE et al. 1973). According to LIMA-DE-FARIA (l956), association Acknowledgments. We are indebted to Mrs. Lizzie Kristenbetween sister chromatids is maintained by the sen and Mrs. Anita Niebuhr for skilful technical assistance juxtacentromeric regions (Fig. 5 , point 3). Thus, and to Mrs. Lone Quedens for preparing the manuscript. what is generally taken as an indication of a cen- This investigation was supported in part by grants from the P. Carl Petersens Foundation (B. 885) and the Danish Medical tromere in conventionally stained chromosomes Research Council (5 12-4276). that the chromatids are associated is no proof of centromere activity with regard to the chromosome movements in the spindle. Even if both “spindle activity” and “constriction activity” are lost in one Literature cited centromere (as in stable dicentric chromosomes with BAHNER, F., SCHWARZ, G., HARNDEN, D. G., JACOBS, P. A , , HIENZ,H. A . and WALTER,K. 1960. A fertile female with a monocentric appearance), “C-staining activity” XO sex chromosome constitution. - Lancet (2): 100-101 may remain. The last remnant of an active centromere J . L., HAZAEL-MASSIEUX. P., LEONARD, seems to be “synthesis activity”. as seen in selective BouE, J . , TAILLEMITE, C. and BouE, A. 1975. Association of pericentric inversion endoreduplication. of chromosome 9 and reproductive failure in ten unrelated At present it is obscure how these different levels families. -- Humangenetik 30: 21 7--224 T., ZECH,L., JOHANSSON, C. and MODEST,E. J., of centromere function are regulated. Terms such as CASPERSSON, 1970. Identification o f human chromosomes by D N A “dominating” (SEARSand CAMARA 1952), “suppresbinding fluorescent agents. - Chromosoma 30: 21 5-227 sed” (NIEBUHR1972c) and “inactivated” centromere DE LA CHAPELLE, A. and STENSTRAND, K . 1974. Dicentric (THERMAN et al., 1974) have been used to describe human X chromosomes. - Hereditas 76: 259-268 J., HORTHING, H. and DE LA CHAPELLE, A , , WENNSTROM, the phenomenon in dicentric chromosomes. -
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OCKEY.C. H. 1966. Isochromosome-X in man. Part I. Hereditas 54: 260-276 COHEN,M. M., ROSENMANN, A,, HACHAM-ZADEK. S. and DAHAN,S. 1975. Dicentric X-isochromosome (Xqi dic) and pericentric inversion of No. 2 (inv(2)(p15q21) in a patient Clin. Genet. 8: 1 1 -I7 with gonadal dysgenesis. DARLINGTON, C. D. 1939. Misdivision and the genetics of the centromere. J . Genel. 37: 341--364 DARLINGTON, C. D. and WYLIE,A. P. 1953. A dicentric cycle in Narcissus. - Heredity 6 (Suppl.): 197--213 DISTECHE,C., HAGEMEIJER. A., FREDERIC, J. and PROGENEAUX, D. 1972. An abnormal large human chromosome identified as an end-to-end fusion of two X’s by combined results of the new banding techniques and microdensitoClin. Genet. 3: 388-395 metry. ~DUTRILLAUX, B. and LEJEUNE,J. 1973. Sur une nouvelle technique d’analyse du caryotype humain. C. R. Acad. Sci. (Paris) 272: 2638 -2640 FRACCARO, M.. IKKOS. D., LINDSTEN.J., LUFT. R. and KAIJSER,K. 1960. A new type of chromosomal abnormality in gonadal dysgenesis. Lancet ( 2 ) : 1144-1 145 GILBOA, Y. and ROSENBERG, T . 1975. Typical Turner’s syndrome with 45x0 karyotype and normal menstruation. Actu Pediat. Helv. 30: 281 --288 DE GROUCHY,J . , TURLEAU,C., ROBIN,M. and KLEIN,M. 1972. Evolutions caryotypiques de I’homme et d u chimpanze. Etude comparative des topographies de bandes apres denaturation menagee. -- Ann. GPnPt. I S : 79-84 HAIR,J . B. 1953. The origin of new chromosomes in AgroHeredity 6 (Suppl.):215 --233 pyron. HAMERTON, J . L. 1971. Human Cytogenetics. General Cytogenetics. Acad. Press, New York. (Vol. I ) HOWELL,R. T., ROBERTS.S. H. and BEARD,R. J. 1976. Dicentric X isochromosomes in man. J. Med. Genet. 13: 496-500 LEJEUNE, J., DUTRILLAUX, B., RETHORE,M. 0. and PRIEUR, M. 1973. Comparaison de la structure fine des chromatides d’ Homo sapiens et de Pan troglodytes. - Chromosoma 43: 42 3 -444 LIMA-DE-FARIA. A. 1956. The role of the kinetochore in chromosome organization. Hereditas 42: 85- I60 -
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NIEBUHR, E. 1972a. Dicentric and monocentric Robertsonian translocations in man. - Humangenetik 16: 217 -226 NIEBUHR.E. 1972b. A 45,XX,5-, 13-, dic+ karyotype in a case of cri-du-chat syndrome. - - Cytogenrtics 11: 165-177 NIEBUHR, E. 1972~.Unusual findings by fluorescence microscopy of a t(13q14q). - Humangenetik 15: 90-92 Paris Conference I97 I , Standardization in human cytogenetics. - Birth Defects: Original Article Series, VIII: 7 , 1972. The National Foundation, New York Paris Conference 1971. Suppl. 1975. Standardization in human cytogenetics. - Birth Defects: Original Article Series, XI: 9. 1975. The National Foundation, New York PHILIP,J. and SELE,V. 1976.45,XOTurner’s syndrome without evidence of mosaicism in a patient with two pregnancies. Acta Obstet. Gynecol. Scand. 55: 283--286 PRIEST,J . H., BLACKSTON, R. D.. Au, K. S. and RAY,S. L. 1975. Differences in human X isochromosomes. J . Med. Genet. 12: 378-389 SCHMID,W., NAEF. E., MURSET,G. and PRADER,A. 1974. Cytogenetic findings in 89 cases of Turner’s syndrome with abnormal karyotypes. ~ -Humangenetik . 24: 93- 104 SEARS,E. R. and CAMARA, A. 1952. A transmissible dicentric chromosome. - Genetics 3 7 125--135 SUMNER, A. T . 1972. A simple technique for demonstrating centromeric heterochromatin. - Exp. Cell Res. 75: 304-306 THERMAN, E., SARTO.G. E. and PATAIJ,K. 1974. Apparently isodicentric but functionally monocentric X chromosome in man. - A m . J. Hum. Genet. 26: 83-92 WARBURTON. D., HENDERSON, A. S., SHAPIRO. L. R. and Hsu, L. Y. F. 1973. A stable human dicentric chromosome, t dic( 12:14)(pl3;pl3) including an intercalary satellite region between centromeres. - A m . J. Hum. Genet. 25: 439 --445 YANAGISAWA, S. 1973. lsochromosome X associated with paracentric inversion. - Lancet ( 2 ) : 1448 ZARATE,A., GARCIA-REYES, J. A,, CASTELAZO-AYALA. L., ESTEVEZ,R. and SILVA,J. 1969. Turner’s phenotype with menstruation, XO karyotype and germ cells in the ovary: Ohstet. Gynecol. 33: 818--821 Report of a case. ~
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Cytogenetic studies in seven individuals with an i(Xq) karyotype E. NIEBUHR AND F. SKOVBY’ University Institute of Medical Genetics, Copenhagen,Denmark
NIEBUHR, E. and SKOVBY, F. 1977. Cytogenetic studies in seven individuals with an i(Xq) karyotype. 86: 121-128. Lund, Sweden. ISSN 0018-0661. Received March 19, 1977
- Herediras
Seven individuals with stigmata of the Turner syndrome, large Barr bodies and a late replicating, structurally abnormal X-chromosome, have been reinvestigated with different banding techniques to elucidate the mechanisms leading to formation of isochromosomes. Dicentric chromosomes With separated C-blocks were found in five of these individuals. The two remaining patients had a slightly larger and a nearly double sized block of centromeric heterochromatin. Using the centromeric model proposed by Lima-de-Faria in 1956, the size of these blocks can be related to different locations of break points within the centromere or in the short arm. The presence of a 45,X cell line in all cases is probably due to anaphase lag secondary to instability of an abnormal isochromosome centromere. The mechanisms behind centromere inactivation in dicentrics are still obscure and not likely to be disclosed before the ultrastructure of these abnormal centromeres is known in more detail, The necessity of a more specific centromere nomenclature is emphasized. Additional rare or unexpected findings were normal menstruation in one individual, bipartite Barr bodies in two cases and one pericentric inversion of chromosome 9. Erik Niehuhr, University Institute of Medical Genetics, Tagensvej 14, DK-2200 Copenhagen, Denmark
Misdivision of a centromere may lead to an unstable, telocentric chromosome and subsequently to formation of an isochromosome (DARLINGTON 1939). Such a mechanism has been favoured in most reports of subjects with an abnormal X chromosome resembling an autosome No. 3. DE LA CHAPELLE et al. (1966) suggested an alternative mechanism of misdivision in the centromeric region near the short arm or in the short arm itself, leading to formation of a dicentric X isochromosome by fusion of the sister chromatids. This possibility was predicted by DARLINGTON (1939) and the terms “dicentric isochromosome” or “isodicentric” (DARLINGTON and WYLIE 1953) were proposed. In order to test these two mechanisms we have reexamined the chromosomes in ten females with a missing C-group chromosome and an extra No. 3-like chromosome. A brief clinical description and the results of different banding techniques, autoradiography and sex chromatin studies are presented and discussed in view of the morphology of the centromere as proposed by LIMA-DE-FARIA (1 956). Case reports
The patients reported on were referred for chromosome analysis because of a Turner-like phenotype
and/or short stature. We were able to repeat the cytogenetic study on ten of eleven patients with presumptive i(Xq) chromosomes. Three of these cases will not be considered further because the abnormal chromosomes were identified as two Xautosome translocations and one 6p + . Case 1. - BGH 24/73 was referred at age 25 for infertility and short stature. She had never menstruated. Her height was 142 cm. Pubic and axillary hair was absent. The uterus was small and the adnexae were not palpated at pelvic examination. She was of normal intelligence. X-rays showed a slightly android pelvis and open iliac epiphyses. Case 2. - HBA 119/73 was referred at the age of 11 for short stature and obesity. Birth weight was 2400 gm. She had had generalized seizures since age 1. Her height at referral was 119 cm. The extremities were short with cubitus valgus. The intelligence was within normal limits. X-rays showed a horseshoeshaped kidney. Case 3. - DAS 242173 was referred at age 16 for obesity, short stature and amenorrhea. Her height
’
Present address: Department of Obstetrics and Gynecology, Johns Hopkins Hospital, Baltimore, Maryland 21205, U.S.A.
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was 134 cm. Webbing of the neck was present. The breast development was poor with widely spaced nipples. The extremities were short with cubitus valgus. Pubic and axillary hair was scant. The uterus and the adnexae were not felt at pelvic examination. The intelligence was normal. Case 4 . - LES 29/74 was referred at age 45 for short stature. She had entered puberty normally and had menstruated until the age of 32. Laparotomy at age 36 showed a small uterus. Microscopy of ovarian tissue revealed normal stroma with corpora albicantia but without primordial follicles and without corpora lutea. Her height at referral was 135 cm and the lower extremities were disproportionally short. Breast development and pubic hair were both within normal limits. She was of normal intelligence. X-rays showed a small sella turcica and brachymetacarpalism. Case 5. - HRM 30/74 was referred at age 10 because of obesity, impaired hearing and short stature. Actual height was 117 cm. Her nipples were widely spaced, and the labia majora and the clitoris were absent. She had cubitus valgus. The intelligence was normal. Case 6. - KIM 91/74 was referred at the age of 12 for short stature and obesity. When seen at age 16, she had not menstruated. Her height was then 135 cm. There was no webbing of the neck but the hair line was low. A shield chest and a large number of pigmented nevi were present. Breast development was absent and the nipples were widely spaced. Pubic and axillary hair was scant. The uterus and the adnexae were not palpable at pelvic examination. Her intelligence was normal. X-rays showed a small sella turcica but no brachymetacarpalism. Case 7. - M K P 332/75 was referred at the age of 8 for precocious puberty on the basis of incipient pubic hair. Family history was negative. Her height was 123 cm. The neck was short. X-rays showed a normal sella turcica; skeletal age was two years advanced.
Material and methods Buccal smears were stained with Feulgen. Five hundred cells from each proband were examined for the presence of X-chromatin. Peripheral blood leucocytes were cultured for three days and prepared for chromosomal analysis. Conventional staining was performed with lactoacetic Orcein. Cultures for autoradiography were labelled with
3H-thymidine (Amersham, specific activity 1,9 pCi/mM) at a final concentration of 0.5 pCi/ml of culture medium for the last four hours of incubation. The slides were stained with Orcein, coated with photographic emulsion (Ilford K 2) and exposed for 11-13 days. Fifty metaphase plates were photographed in each case. Q-, R- and C-banding patterns were obtained according to the techniques described by CASPERSSON et al. (1970), DUTRILLAUX and LEIEUNE(1972) and (1972), respectively. SUMNER
Results All patients were X-chromatin positive with lower mean values than our routine counts from normal females (Table I). The Barr bodies were larger than normal. Cells with two large Barr bodies were observed in case 5, 6 and 7. Bipartite Barr bodies were observed on buccal smears in cases 6 and 7. Two cell lines were found in all seven cases: a cell line with a modal count of 45 in which a C-group chromosome was missing, and a cell line with 46 chromosomes in which a C-group chromosome was replaced by a metacentric or nearly metacentric chromosome resembling a No. 3. A third cell line with a chromosome number of 47 and two such chromosomes was observed in cases 5 , 6 and 7. The frequencies of the different cell lines are shown in Table 1. A wide centromeric constriction or a dicentric morphology was often observed in cases 3-7. A monocentric appearance was always observed in case 1 and 2 and occasionally in cases 3-7 (e.g. Fig. 1, cases 5 and 7). Autoradiography of all seven probands showed a late replicating chromosome resembling a No. 3. Its pattern of DNA synthesis appeared symmetrical around the centromere. Q- and R-banding patterns in all patients showed one normal X chromosome only. In most cells with a chromosome count of 46, the banding pattern of both arms of the abnormal C-group chromosome was identical to that of the long arm of a normal X chromosome (Fig. 1). However, in some cells (cases 3-7) the banding pattern was asymmetric around the centromere (e.g. Fig. I , case 7). C-banding of cases 1 and 2 showed only one block of heterochromatin in the isochromosome. The size of the block in case 1 was slightly larger than that of a normal X-chromosome and it was nearly of double size in case 2 (Fig. 2). One C-group chromosome in case 1 was abnormal
INDIVIDUALS WITH AN I(XQ) KARYOTYPE
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Table I . Chromosome number and X-chromatin counts Case No.
Blood cultures
X-chromatin' Percentage frequencies of the different cell lines (n = 100) Percentage frequencies of Barr bodies (n = 500)
45,XO
46,XX
46,X,i(Xq)
47,X,i(Xq),i(Xq) 0
1
2
Bipartite
0
92 94 90 74 78 25 46
0 0 0 0 4 3 7
22 24 19 13 18 11 8
0
3 4 5 6 1
8 6 10 26 18 12 47
0 0 0 0 0 3 4
'
normal range 20-32%
1
7
0 0 0
0 0 0
with a large heterochromatic block located on the short arm. Q- and C-bands revealed a pericentric inversion of chromosome No. 9: inv(9)(p12;q21) (Fig. 3). Twice as much centromeric heterochromatin as in a normal X was observed in the isochromosomes of cases 3-7. The heterochromatin was observed as two average sized and clearly separated blocks (Fig. 2). In some metaphase plates from case 7, one of the centromeric blocks appeared smaller and more weakly stained than the other (Fig. 4b). The distance between the blocks varied from case to case; the largest distance was observed in case 7. Intrapersonal variance was not observed. The identity of the large metacentric chromosomes containing the additional C-band material was in all cases verified by successive application of Q- and C-band staining to the same metaphase plates. (Fig. 3, 4) - Isochromosomes with only one average sized heterochromatic block were never observed in cases 3-7.
Discussion 1. General considerations
The first description of the Turner syndrome associated with an isochromosome for the long arm of et al. the human X was published by FRACCARO (1960). According to SCHMIDet al. (1974), this chromosomal abnormality accounts for about 13% of patients with the Turner syndrome. Patients with i(Xq) are reported to be clinically indistinguishable from those in whom an entire X is missing. This held true in the present material (because of the
78 76 81 87 80 84 85
0
0 0 2 2 3
mode of ascertainment) with exception of case 4 who menstruated regularly for 18 years. A few cases of the Turner syndrome with regular menstruation and ROSENBERG (e.g. ZARATEet al. 1969; GILBOA 1975) or even pregnancy (e.g. BAHNERet al. 1960; PHILIPand SELE1976) have been reported. In 1966 DE LA CHAPELLE et al. suggested that some i(Xq) were dicentric, but the cytological methods available at that time did not allow any confirmatory investigations. More recently, DE LA CHAPELLE and STENSTRAND (1974) were able to demonstrate twice the normal amount of centromeric heterochromatin in two out of four patients with i(Xq), consistent with the previous interpretation as idic(Xq). Similar cases have been reported by YANACISAWA (1973), COHENet al. (1975), PRIESTet al. (1979, and HOWELL et al. (1976). At least five of the seven individuals (cases 3-7) in the present study had dicentric X-chromosomes, suggesting that this type is more frequent than estimated by DE LA CHAPELLE and STENSTRAND (1974). Case 2 might also be a dicentric. The true incidence of idic(Xq) can only be determined by applying a C-band technique to each case of i(Xq), particularly since dicentric chromosomes sometimes appear monocentric by conventional staining methods (NIEBUHR 1972a, e.g. case 4). A monocentric appearance in 6 out of 11 i(Xq) cases studied without heterochromatin staining (SCHMIDet al. 1974) does not give any additional information concerning the frequency of dicentric i(Xq) chromosomes. The presence of a 45,X cell line in all patients is most likely due to anaphase lag of an isochromosome present in the zygote. If the formation of the isochromosome was a postzygotic event, a 46,XX cell line should also be present which was not the case.
124
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3
3
i.(Xq) X
Case 1
Case 2
Case 3
Case 3
Case 4
Case 5
P
Case 6
Case 7 I
2
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INDIVIDUALSWITH AN I(XQ) KARYOTYPE
125
k
*
"I
0-
4b Fig. 3 and 4. Details of two mitoses successively stained with quinacrine mustard (a) and C-band technique (b). x 2700. - Fig. 3. Case 1; pair No. 9 with a pericentric inversion, the normal X and the i(Xq) with a somewhat larger heterochromatic block. - Fig. 4. Case 7; arrows indicating two abnormal X-chromosomes (47,X,i(Xq), i(Xq)); note the smaller size and less intense staining of one centromere.
It is difficult to assess the significance of a pericentric inversion of chromosome 9 in case 1. According to BouE et al. (1979, the estimated incidence of this inversion is about 1%. COHENet al. (1975) reported a pericentric inversion involving chromo-
some 2 in a patient with another dicentric X-isochromosome. Preferential inactivation of the abnormal X and large Barr bodies are common features of cases with i(Xq) chromosomes. The relatively low frequency of
Fig. 1 and 2. - Fig. I . Partial karyotypes showing pair No. 3, abnormal and normal X stained with quinacrine mustard (case 3 also with acridine orange). Note asymmetry around the centromere (case 3, acridine orange and case 7). the dicentric appearance in cases 3 , 4 and 6, and the monocentric appearance in cases 1,2,5 and 7. x 2500. - Fig. 2. C-band stained partial karyotypes showing pair No. 3, abnormal X and in case 1 and 2 also the normal X chromosome. Note the larger appearance of the heterochromatic block on the isochromosome than that of the normal X (case 1 and 2) and the two separated blocks in cases 3 7. ~ 2 5 0 0 .
126
E. NIEBUHR AND F. SKOVBY
A
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u B
C
D
E
Fig. 5. A: the centromere model as proposed by LIMA-DE-FARIA (1956) with possible break points and centromeric heterochromatin. B, C, D, E: centromere structure after breakage at points 1-4, sister strand fusion and replication.
Barr bodies in our study may be due to a 45,X cell line in all our patients. The 47,i(Xq), i(Xq) cell line in cases 5, 6 and 7 was probably responsible for the occasional presence of cells with two large Barr bodies. Bipartite Barr bodies as seen in cases 6 and 7 are a rare, but not unusual observation in patients with dicentric i(Xq) chromosomes (THERMAN et al. 1974). 2. Formation of isochromosomes Monocentrics. In the centromere model, as proposed by LIMA-DE-FARIA (l956), breakage may occur at different points (Fig. 5). A break at point 1 (inner zone) at the G , or G2 stage will produce a chromosome which is unstable during meiosis or mitosis because of insufficient centromeric material to which the spindle apparatus may attach. However, the sister parts of the centromere may heal, creating a monocentric isochromosome which behaves like a normal chromosome during meiosis and mitosis. In such isochromosomes the amount of centromeric heterochromatin would be similar to that of a normal X. Two of the patients studied by DE LA CHAPELLE and STENSTRAND (1 974) showed a single, normal sized heterochromatic block. Our case 1 might be another example of a true isochromosome, but the somewhat larger heterochromatic block seems to indicate a more distal break in the centromere. ~
Dicentrics. Breaks at point 2, 3 or 4 (Fig. 5) will produce a telocentric chromosome or an acrocentric. ~
If fusion between sister chromatids occur, a breakagefusion cycle will be established during mitosis or the second meiotic division due to the presence of two functioning centromeres. The outcome will usually be elimination of the cell or loss of the chromosome. Little attention has been paid to the sequence of events which makes isochromosome formation possible when a chromosome break has occurred at point (1939, p. 357) and HAMERTON 2 , 3 or 4. DARLINGTON (1971) named this “second misdivision” but it does not at all imply a division. The essentiel events are a centromere non-disjunction, allowing the chromatids to pass to the same cell, preceded or followed by a fusion of the chromatids at the point of breakage. As discussed below, the inactivation of one of the two centromeres in the spindle apparatus is necessary in order to secure the transmission of the isochromosome in future cell divisions. If an isochromosome is due to a break at point 2 or 3, the centromeric heterochromatin should maximally be twice as large as that of a normal X. It would appear as one larger block, as demonstrated in case 2. However, the cases of DE LA CHAPELLE and STENSTRAND and our cases 3-7 showed a clear separation of the two heterochromatic blocks in most cells. This indicates that the breaks leading to the dicentric isochromosomes occurred at the short arm chromatids (point 4, Fig. 5 ) as suggested by DE LA CHAPELLE et al. (1966). According to the Paris Conference (1971) nomenclature the designation of case 1 and 2 is 46,X,i(X) (qter-cen-qter) and that ofcases 3-7: 46,X,dic(X),
Hereditas 86 (1977)
INDIVIDUALS WITH AN
(qter-rpl 1 :: p l l -+qter).It was not possible to determine whether these cases in fact d o represent isochromosomes or translocations. The nomenclature suggested at the Paris Conference (1971) and suppl. (1975) is, however, not adequate to convey the information about the different types of i(Xq) chromosomes which can be differentiated with heterochromatin staining. A nomenclature describing the different parts of the centromere is necessary to allow a more precise designation of isochromosomes due to breaks within or close to the centromere.
I(XQ)KARYOTYPE
127
It is known from studies in Agropyron scabium with a large dicentric isochromosome (HAIR 1953) that the distance between the two centromeres influences the stability of the abnormal chromosome. Chromosomes with a long intercentric segment are unstable due to criss-cross separations. Repetition of a breakage-fusion-bridge cycle tends to shorten the distance with production of stable dicentric chromosomes or apparently monocentrics. A similar phenomenon has been observed in man (NIEBUHR1972a, cases 1 and 2). The absence of constriction in one of the two centromeres in a dicentric chromosome could be due to destruction of the juxtacentromeric 3 . Centromere suppression regions by criss-cross separation. Likewise, the stabiIn spite of two separated heterochromatic blocks lity could be due to destruction of the point of (cases 3-7) indicating the presence of two centroattachment for the spindle apparatus. The smaller meres, iso-chromosomes with a monocentric appear- size of one centromere with separated heterochromatance were occasionally observed in the same patient ic blocks in some metaphase plates from case 7 may with techniques other than C-banding. A similar be due to loss of part of one centromere, and thus monocentric appearance has been described e.g. in a an example of juxtacentromeric destruction. Howlarge idic(Xq) (DE LA CHAPELLE and STENSTRANDever, the demonstration of very stable dicentric 1974; THERMAN et al. 1974), in five dicentric Roberthuman chromosomes with a large intercentric dissonian translocations (NIEBUHR1972a) and in a tance (e.g. DISTECHEet al. 1972; DE LA CHAPELLE dicentric t(5;13) (NIEBUHR 1972b). and STENSTRAND 1974; THERMAN et al. 1974) suggests The origin of the human No. 2 chromosome has an inactivation mechanism, not dependent upon been shown to be two acrocentric chromosomes from destruction of the chromatids. The observation of a primate progenitor fused short arm-short arm dicentric translocation chromosomes with non-ran1972). Thus, No. 2 is phylogenetically dom separation of the chromatids in one centromere (DE GROUCHY a dicentric chromosome, but only one centromere and the normal function of the other (NIEBUHR functions under normal circumstances. The other 1972a, case 4; WARBURTON et al. 1973) may support the existence of such a mechanism. However, before centromere (2q22) has even lost its C-staining ability; occasionally it is active as a starting point of chroma- the ultrastructure of the centromere in man is known tid replication in cases of selective endoreduplication in more detail, the mechanisms behind centromere inactivation are unlikely to be disclosed. (LEJEUNE et al. 1973). According to LIMA-DE-FARIA (l956), association Acknowledgments. We are indebted to Mrs. Lizzie Kristenbetween sister chromatids is maintained by the sen and Mrs. Anita Niebuhr for skilful technical assistance juxtacentromeric regions (Fig. 5 , point 3). Thus, and to Mrs. Lone Quedens for preparing the manuscript. what is generally taken as an indication of a cen- This investigation was supported in part by grants from the P. Carl Petersens Foundation (B. 885) and the Danish Medical tromere in conventionally stained chromosomes Research Council (5 12-4276). that the chromatids are associated is no proof of centromere activity with regard to the chromosome movements in the spindle. Even if both “spindle activity” and “constriction activity” are lost in one Literature cited centromere (as in stable dicentric chromosomes with BAHNER, F., SCHWARZ, G., HARNDEN, D. G., JACOBS, P. A , , HIENZ,H. A . and WALTER,K. 1960. A fertile female with a monocentric appearance), “C-staining activity” XO sex chromosome constitution. - Lancet (2): 100-101 may remain. The last remnant of an active centromere J . L., HAZAEL-MASSIEUX. P., LEONARD, seems to be “synthesis activity”. as seen in selective BouE, J . , TAILLEMITE, C. and BouE, A. 1975. Association of pericentric inversion endoreduplication. of chromosome 9 and reproductive failure in ten unrelated At present it is obscure how these different levels families. -- Humangenetik 30: 21 7--224 T., ZECH,L., JOHANSSON, C. and MODEST,E. J., of centromere function are regulated. Terms such as CASPERSSON, 1970. Identification o f human chromosomes by D N A “dominating” (SEARSand CAMARA 1952), “suppresbinding fluorescent agents. - Chromosoma 30: 21 5-227 sed” (NIEBUHR1972c) and “inactivated” centromere DE LA CHAPELLE, A. and STENSTRAND, K . 1974. Dicentric (THERMAN et al., 1974) have been used to describe human X chromosomes. - Hereditas 76: 259-268 J., HORTHING, H. and DE LA CHAPELLE, A , , WENNSTROM, the phenomenon in dicentric chromosomes. -
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OCKEY.C. H. 1966. Isochromosome-X in man. Part I. Hereditas 54: 260-276 COHEN,M. M., ROSENMANN, A,, HACHAM-ZADEK. S. and DAHAN,S. 1975. Dicentric X-isochromosome (Xqi dic) and pericentric inversion of No. 2 (inv(2)(p15q21) in a patient Clin. Genet. 8: 1 1 -I7 with gonadal dysgenesis. DARLINGTON, C. D. 1939. Misdivision and the genetics of the centromere. J . Genel. 37: 341--364 DARLINGTON, C. D. and WYLIE,A. P. 1953. A dicentric cycle in Narcissus. - Heredity 6 (Suppl.): 197--213 DISTECHE,C., HAGEMEIJER. A., FREDERIC, J. and PROGENEAUX, D. 1972. An abnormal large human chromosome identified as an end-to-end fusion of two X’s by combined results of the new banding techniques and microdensitoClin. Genet. 3: 388-395 metry. ~DUTRILLAUX, B. and LEJEUNE,J. 1973. Sur une nouvelle technique d’analyse du caryotype humain. C. R. Acad. Sci. (Paris) 272: 2638 -2640 FRACCARO, M.. IKKOS. D., LINDSTEN.J., LUFT. R. and KAIJSER,K. 1960. A new type of chromosomal abnormality in gonadal dysgenesis. Lancet ( 2 ) : 1144-1 145 GILBOA, Y. and ROSENBERG, T . 1975. Typical Turner’s syndrome with 45x0 karyotype and normal menstruation. Actu Pediat. Helv. 30: 281 --288 DE GROUCHY,J . , TURLEAU,C., ROBIN,M. and KLEIN,M. 1972. Evolutions caryotypiques de I’homme et d u chimpanze. Etude comparative des topographies de bandes apres denaturation menagee. -- Ann. GPnPt. I S : 79-84 HAIR,J . B. 1953. The origin of new chromosomes in AgroHeredity 6 (Suppl.):215 --233 pyron. HAMERTON, J . L. 1971. Human Cytogenetics. General Cytogenetics. Acad. Press, New York. (Vol. I ) HOWELL,R. T., ROBERTS.S. H. and BEARD,R. J. 1976. Dicentric X isochromosomes in man. J. Med. Genet. 13: 496-500 LEJEUNE, J., DUTRILLAUX, B., RETHORE,M. 0. and PRIEUR, M. 1973. Comparaison de la structure fine des chromatides d’ Homo sapiens et de Pan troglodytes. - Chromosoma 43: 42 3 -444 LIMA-DE-FARIA. A. 1956. The role of the kinetochore in chromosome organization. Hereditas 42: 85- I60 -
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NIEBUHR, E. 1972a. Dicentric and monocentric Robertsonian translocations in man. - Humangenetik 16: 217 -226 NIEBUHR.E. 1972b. A 45,XX,5-, 13-, dic+ karyotype in a case of cri-du-chat syndrome. - - Cytogenrtics 11: 165-177 NIEBUHR, E. 1972~.Unusual findings by fluorescence microscopy of a t(13q14q). - Humangenetik 15: 90-92 Paris Conference I97 I , Standardization in human cytogenetics. - Birth Defects: Original Article Series, VIII: 7 , 1972. The National Foundation, New York Paris Conference 1971. Suppl. 1975. Standardization in human cytogenetics. - Birth Defects: Original Article Series, XI: 9. 1975. The National Foundation, New York PHILIP,J. and SELE,V. 1976.45,XOTurner’s syndrome without evidence of mosaicism in a patient with two pregnancies. Acta Obstet. Gynecol. Scand. 55: 283--286 PRIEST,J . H., BLACKSTON, R. D.. Au, K. S. and RAY,S. L. 1975. Differences in human X isochromosomes. J . Med. Genet. 12: 378-389 SCHMID,W., NAEF. E., MURSET,G. and PRADER,A. 1974. Cytogenetic findings in 89 cases of Turner’s syndrome with abnormal karyotypes. ~ -Humangenetik . 24: 93- 104 SEARS,E. R. and CAMARA, A. 1952. A transmissible dicentric chromosome. - Genetics 3 7 125--135 SUMNER, A. T . 1972. A simple technique for demonstrating centromeric heterochromatin. - Exp. Cell Res. 75: 304-306 THERMAN, E., SARTO.G. E. and PATAIJ,K. 1974. Apparently isodicentric but functionally monocentric X chromosome in man. - A m . J. Hum. Genet. 26: 83-92 WARBURTON. D., HENDERSON, A. S., SHAPIRO. L. R. and Hsu, L. Y. F. 1973. A stable human dicentric chromosome, t dic( 12:14)(pl3;pl3) including an intercalary satellite region between centromeres. - A m . J. Hum. Genet. 25: 439 --445 YANAGISAWA, S. 1973. lsochromosome X associated with paracentric inversion. - Lancet ( 2 ) : 1448 ZARATE,A., GARCIA-REYES, J. A,, CASTELAZO-AYALA. L., ESTEVEZ,R. and SILVA,J. 1969. Turner’s phenotype with menstruation, XO karyotype and germ cells in the ovary: Ohstet. Gynecol. 33: 818--821 Report of a case. ~
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