Hum. Genet. 38, 15--23 (1977) © by Springer-Verlag 1977
Partial Trisomy 21 Further Evidence that Trisomy of Band 21q22 is Essential for Down's Phenotype
A. Hagemeijer and E. M. E. Smit Department of Cell Biology and Genetics, Medical Faculty, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands
Summary. Cytogenetic analysis of a 6-year-old girl with moderate mental retardation revealed 46 chromosomes with a tandem translocation (21;21) resulting in a partial trisomy 21. Only the terminal band 21q22 was not in triplicate. G-, Q-, R-, and C-banding techniques and silver nitrate staining of the nucleolus organizer regions (NORs) were used to identify this chromosome fully. The phenotype of the patient was not typical for Down's syndrome, providing additional evidence that trisomy of band 21q22 is pathogenetic for the phenotype of Down's syndrome. This is also a new example in human pathology of a stable 'dicentric' chromosome in which one of the centromeric constrictions appears to be nonfunctional.
Introduction Recently a few cases of familial reciprocal translocations involving chromosome 21 were studied by Williams et al. (1975), Sinet et al. (1976), and Wahrman et al. (1976). They related the phenotype of Down's syndrome to the trisomy of the distal part of the long arm of chromosome 21. Niebuhr (1974) reviewing the different cases of tandem translocation (21;21) had already reached a similar conclusion but without cytologic demonstration. The patient presented here has an almost complete trisomy 21 except for the band 21q22 which is not triplicated; her phenotype is different from the one in Down's syndrome. Her abnormal chromosome is the result of a tandem translocation (21;21) with centromeric heterochromatin present at two sites. It provides a new example of a stable dicentric chromosome with a latent or nonfunctional centromere. Similar cases have been reported, mainly X-to-X fusions (Dist~che et al., 1972; Therman et al., 1974; Ruthner and Golob, 1974; de la Chapelle and Stenstrand, 1974; Laurent et al., 1975; Fraisse et al., 1975) or 21 to 21 fusions (Bartsch-Sandhoff and Schade, 1973; Niebuhr, 1974).
16
A. Hagemeijer and E. M. E. Smit
Case Report The proband is a 6-year-old girl, the product of an uncomplicated full-term pregnancy. Her birth weight was 3400 g and length 53 cm. There was no history of irradiation or viral diseases during pregnancy. Her father and mother were both 28 years old at that time. She is the second child. Her brother, who is 4 years older, is physically and mentally normal. Somatic development was perfectly normal. Her growth and weight curves run between the 50th and the 75th percentile. Her first tooth appeared at 6 months of age. Dentition is normal and healthy, without decays or cavities. Her psychomotor development was slightly delayed. She could sit at 6 months of age, stay upright at 11 months, walk without help at 14.5 months. Her speech was clearly retarded: she could say a few words at 13 months but was 4 years old before she could make a short sentence. During childhood bronchitis and upper respiratory tract infections were frequent. Tonsillectomy was performed at 4 years and subsequently her speech and social contact improved very rapidly. Physical examination at 6 years old shows an active girl of normal height and weight, 118 cm and 22kg respectively. Her head circumference is 50 cm. The occipital bone is not flattened. She has thick straight hair. The face is oval, the palpebral fissures are horizontal. She has bilateral epicanthic folds and a flattened nose bridge but no hypertelorism. The interpupillary distance is 55 mm, normal for her age. She has an intermittent strabismus of the left eye. The nose is short and slightly stubbed. The mouth is wide and usually closed but is open when she is interviewed or when some task requires all her attention. The tongue is normal, not protruding and not furrowed. T h e palate is normal, not narrow or highly arched. The neck is not particularly short, the posterior hairline is normal. The hands are short and plump, very similar to the hands of both parents. The fifth finger is not particularly short or incurved. The dermatoglyphs are not characteristic for Down's syndrome. Simian creases and patterns on the thenar or hypothenar area are absent. Dermatoglyphs show 10 ulnar loops on the fingertips, palmar triradii in proximal position t, a distal loop in the third interdigital space, and a large distal loop on both hallux areas. The toes are small with hypotrophic nails; there is no wide gap between toes I and II. Her gait is normal. W h e n she stands she shows a small valgus deformity of the knee (X-shaped legs). There is no hyperlaxity of the joints. Roentgenograms were not made, so it was not possible to evaluate her skeletal age, the iliac and acetabular angles of the pelvis, or the interorbital distance. T h e r e is no heart m u r m u r and the rest of the physical examination is normal. She appears alert, sometimes restless. Her power of concentration is low, her memory is poor and she still communicates in short sentences. Cytogenetic investigation was initiated because of her intellectual immaturity when reaching school age.
Material and Methods Karyotypes were performed on standard PHA-stimulated blood cultures. Chromosome identification was achieved using different banding techniques: the A S G technique, Q-banding with atebrine and R-banding with acridine orange after heat denaturation (Verma and Lubs, 1975) or after a 5-bromodeoxyuridine (BrdU) pulse during the last 5 or 6 h of the culture (Hagemeijer et al., 1976). C-banding was carried out with a combination of different published techniques: airdried slides, a few weeks old, were stained with atebrine, dried, hydrolyzed for 10 min in HC1 0.2N, rinsed, dried, denaturated for 15 min in a 0.5% Ba(OH)2 solution at 50°C, rinsed again, incubated during 1 h in 2 × SSC at 60°C, then stained with Giemsa as in the A S G technique. Specific staining of the nucleolus organizer regions (NORs) with silver nitrate was performed according to Denton et al. (1976).
Trisomy of Band 21q22 Essential for Down's Phenotype
17
Results Thirty metaphases of the proband were fully analyzed: her karyotype was 46,XX, 21q+ (Fig. 1). The elongated 21 appeared as an acrocentric chromosome larger than a G but definitely smaller than a D-group chromosome (Figs. 2--5). G- and Q-banding suggested a 21 with a repetition of the clearly stained band 21q21 (Figs. 2 a and 5 a). The end of the long arm was stubbed but occasionally seen in association with chromosomes of the D or G group (Fig. 2b). R-banding after BrdU pulse showed a much more detailed pattern: the elongated chromosome appeared symmetrical around band 21q22 (Fig. 3). We interpreted this rearrangement as a long-arm fusion of two chromosomes 21, in opposition. The band 21q22 is clearly not duplicated (Fig. 4) which indicates that at the time of the formation of this chromosome a deletion of this band in one of the two partners has occurred. Evidence was sought for the presence of a latent centromere and of NORs. Cbanding demonstrated the presence of centromeric heterochromatin at both ends of this chromosome (Figs. 5b and c). The two C bands showed a similar rather weak staining intensity. Silver nitrate staining provided excellent images of the NORs which appeared as black knobs on top of the D and G group chromosomes. The abnormal 21 showed four black dots, two at each extremity (Fig. 6).
Fig. 1. Karyotype of the proband: 46,XX,21q+. Acridine orange (AO) staining after BrdU pulse
18
A. Hagemeijer and E. M. E. Smit
Fig. 2. a Chromosomal pairs 13, 21, and 22 of the proband. Q-banding technique, b Examples of satellite associations between the abnormal chromosome 21 and other acrocentrics of the proband
Fig. 3. Chromosomal pairs 13, 21, and 22 of the proband and of her parents. Reverse banding techniques Fig. 4. Composite arrangement of the normal and abnormal chromosome 21 demonstrating our interpretation of the rearrangement, a The abnormal 21 with on either side the normal 21 of the same cell in upright and in upside down position, b The abnormal 21 and next to it two chromosomes 21 placed above each other, in opposition demonstrating that fusion of two complete chromosomes 21 would result in a much larger R-band region t h a t the one we observed
Trisomy of Band 21q22 Essential for Down's Phenotype
19
Fig. 5. a Chromosomal pairs 13, 21, and 22 of the proband G-banding technique, b Idem with C-banding technique, e C-banded metaphase of the proband; the abnormal 21 is indicated by cIFrOW
Fig. 6. a Metaphase of the proband stained with silver nitrate to demonstrate the NORs; a r r o w indicates the abnormal chromosome 21 with 4 silver stained knobs, two at each extremity. b Example of the abnormal chromosome 21 next to a representative D-group chromosome from 5 different cells of the proband; the last one shows satellite association
20
A. Hagemeijer and E. M. E. Smit
This is consistent with our interpretation of this chromosomal rearrangement. The four dots appeared so similar that with this technique both ends of the chromosome were indistinguishable. According to the Paris Conference (1971) the full description of this chromosome will be: (21) ( p t e r - c e n - q 2 2 : : q 2 1 ~ l a t e n t cen-pter). Twenty metaphases of each parent were analyzed and showed normal karyotypes.
Discussion
A tandem fusion is a 'whole-arm' translocation resulting from one break in the vicinity of the centromere in one chromosome and another break near the end of a second chromosome (White, 1957). In human pathology Lejeune et al. (1965) used this term to describe a nonrobertsonian translocation between two acrocentric chromosomes. Except for the case of translocation (21;21) described by Poissonnier et al. (1976) most of the reported observations of tandem (or rather nonrobertsonian) translocations (21;21), studied with the new banding techniques, proved to be bisatellited pseudodicentric chromosomes. They result from the fusion, in opposition, of the long arm of two chromosomes 21, both having the break near the end of the long arm (Bartsch-Sandhoff and Schade, 1973; Niebuhr, 1974). Fusion of two complete chromosomes 21 would result in a D-size chromosome, which indeed was observed in most of the cases of tandem translocations in patients with typical Down's syndrome (Lejeune et al., 1965; Kadotani et al., 1970; Vogel, 1972; Bartsch-Sandhoff and Schade, 1973; Jernigan et al., 1974). The chromosomal aberration we describe here is also a translocation (21;21), in opposition, but without duplication of the distal band 21q22. This is particularly manifest with the R-banding technique. Such a dicentric chromosome originates from an interchange between the two arms of one chromosome or between two homologous chromosomes. These different mechanisms were extensively discussed by Therman and P~itau (1974) for X-X dicentrics, but can be extrapolated to a 21-21 dicentric. The centromeric and short-arm regions of acrocentric chromosomes are highly polymorphic. In our case, C bands and silver staining of the NORs are quite identical at both chromosomal ends, which suggests a rearrangement between the two arms of the same chromosome 21. This could have happened during gametogenesis in one of the parents or at the first zygotic division, with, in the latter case, secondary loss of a cell line with monosomy 21. The mechanism by which one of the centromeres became latent or nonfunctional is unknown. The karyotype of the patient in vivo and in vitro appears very stable, clearly indicating that this abnormal chromosome behaves as a monocentric. Our interpretation is an asymmetrical rearrangement with deletion of band 21q22 in one of the partners. Cytologically we cannot exclude a symmetrical rearrangement with duplication of band 21q22.1 and absence of band 21q22.3. This would restrict the phenotype of Down's syndrome to a trisomy of band
Trisomy of Band 21q22 Essential for Down's Phenotype
21
PARTIAL TRISOMY 21 With Down's Phenotype
Without Down's Phenotype
© 22
qll
(1) t(15;21)(p;q21) AuIa e t a [ .
1973
(2) t(10;21)(q26;q21) WitIiams et cd.
1975
(3) tan.(21;21) Poisonnier et al.
1976
(&) t(2;21)(q;q21) Wahrman et (at.
1976
(5~ t(15;21)(q26.2; q21) Sinet et (al.
1976
(6) t(12;21)(q2/.;q21) Chen et o.I.
1976
(2)(6) q21
q221
(5)(7)
q222
(3) (4)(5)
q223
(1)(2)
(7) t(an.(21;21) Our Observation
Fig. 7. Schematic representation of chromosome 21 (R-banding). The bands involved in the different case reports of partial trisomy 21 are represented by straight lines parallel to the chromosome: on the left side the cases with the phenotype of Down's syndrome and on the right side the cases without the Down's syndrome phenotype. The numbers in brackets refer to the different
publications
21q22.3, which is in contradiction with the observation of Poissonnier et al. (1976). The absence of clinical signs of monosomy 21 in our patient makes this interpretation rather unlikely. Cytoplasmic superoxide dismutase-1 (SOD-l) was tentatively mapped on band 21q22.1 (Sinet et al., 1976). Reliable measurement of the concentration of this enzyme may indicate the presence in two- or threefold of band 21q22.1 in this karyotype. Physically, our patient does not at all impress one as a mongoloid child. Looking at the check list of 25 clinical signs of Down's syndrome established by Lee and Jackson (1972), our patient has only 4 of them (epicanthic folds, flat nasal bridge, open mouth, short, broad hands) placing her in the group of nontrisomic patients. Cytogenetically we found an almost complete trisomy 21. Only band 21q22 is not trisomic. This suggests that band 21q22 is pathogenetic for the phenotype of Down's syndrome and provides a cytologic demonstration of the hypothesis of Niebuhr (1974). Reviewing 15 cases of Down's syndrome due to tandem translocations (G-G), Niebuhr emphasized the fact that some of them presented with a mild form of mongolism and he put forward the hypothesis that in this type of rearrangement a deletion of the terminal band of 21q was likely to happen, whereas in centric fusion the long arms of both chromosomes involved remained intact. The clinical importance of the terminal part of 21q was also suggested by Williams et al. (1975), Wahrman et al. (1976), Sinet et al. (1976), and Chen et al. (1976), who studied different cases of partial trisomy 21 in families with reciprocal translocations. The different cases of partial trisomy 21 reported
22
A. Hagemeijer and E. M. E. Smit
i n the literature a n d studied with the m o d e r n b a n d i n g techniques are s u m m a r i z e d in F i g u r e 7. The p h e n o t y p e of D o w n ' s s y n d r o m e appears here also to be clearly related with t r i s o m y o f b a n d 21q22.
Acknowledgements. We sincerely thank Prof. D. Bootsma for critically reviewing the manuscript, Dr. E. S. Sachs for the analysis of the dermatoglyphs, and Dr. J. Toledo for referring his patient to us.
References Aula, P., Leisti, J., von Koskull, H.: Partial trisomy 21. Clin. Genet. 4, 241--251 (1973) Bartsch-Sandhoff, M., Schade, H.: Zwei subterminale Heterochromatinregionen bei einer seltenen Form einer 21/2l-Translokation. Humangenetik 18, 329--336 (1973) Chen, H., Tyrkus, M., Woolley, jr., P. V.: Partial trisomy 21 due to maternal t(12;21) translocation: Further evidence that the Down phenotype is related to trisomy of the distal segment of chromosome 21. Proc. of the Vth Intern. Congr. Hum. Genet., Mexico, 1976. Excerpta Medica, int. congr, series 397, 116 (1976) de la Chapelle, A., Stenstrand, K.: Dicentric human X chromosomes. Hereditas 76, 259--268 (1974) Denton, T. E., Howell, W. M., Barrett, J. V.: Human nucleolar organizer chromosomes: Satellite associations. Chromosoma (Berl.) 55, 81--84 (1976) Distbche, C., Hagemeijer, A., Frederic, J., Progneaux, D.: 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 microdensitometry. Clin. Genet. 3, 388--395 (1972) Fraisse, J., Laurent, C., Collard, N., Biemont, M. C., Dutrillaux, B.: Un deuxi~me exemple de fusion t61omerique de deux chromosomes X. Ann. G6n&. 18, 243--245 (1975) Hagemeijer, A., Hoovers, J., Hasper-Voogt, I., Von Ruhe-Zurcher, T., Bootsma, D.: Latereplicating ring X-chromosomes identified by R-banding after BrdU pulse. Three new examples of mosaicism 45, XO/46, Xr(X). Hum. Genet. 34, 45--52 (1976) Jernigan, D., Curl, N., Keeler, C.: Milledgeville mongoloid: a rare karyotype of Down's syndrome. J. Hered. 65, 254--257 (1974) Kadotani, T., Ohama, K., Takahara, H., Nagai, I., Shimizu, B., Makino, S.: A case of Down's syndrome associated with G / G translocation. Proc. Japan Acad. 46, 858--862 (1970) Laurent, C., Biemont, M. C., Dutrillaux, B.: Sur quatre nouveaux cas de translocation du chromosome X chez l'homme. Humangenetik 26, 35--46 (1975) Lee, L. G., Jackson, J. F.: Diagnosis of Down's syndrome: clinical vs. Laboratory. Clin. Pediat. (Phila.) 11, 353--356 (1972) Lejeune, J., Berger, R., Vidal, O. R., Rethor6, M. O.: Un cas de translocation G - G en tandem. Ann. G6n&. 8, 60--62 (1965) Niebuhr, E.: Down's syndrome. The possibility of a pathogenetic segment on chromosome No. 21. Humangenetik 21, 99--101 (1974) Poissonnier, M., Saint-Paul, B., DutriUaux, B., Chassaigne, M., Gruyer, P., de Bligni~res-Strouk, G.: Trisomie 21 partielle (21q21-21q22.2). Ann. G6n&. 19, 69--73 (1976) Ruthner, U°, Golob, E.: Fusion of the short arms of one X-chromosome in a patient with gonadal dysgenesis. Humangenetik 24, 159--160 (1974) Sinet, P. M., Couturier, J., Dutrillaux, B., Poissonnier, M., Raoul, O., Rethor6, M. O., Allard, D., Lejeune, J., J6rome, H.: Trisomie 21 et superoxyde dismutase-1 (IPO-A). Tentative de localisation sur la sous bande 21q22.1. Exp. Cell Res. 97, 47--55 (1976) Therman, E., Sarto, G. E., Patau, K.: Apparently isodicentric but functionally monocentric X chromosome in man. Amer. J. hum. Genet. 26, 83--92 (1974) Therman, E., P~itau, K.: Abnormal X chromosomes in man: Origin, behavior and effects. Humangenetik 25, 1--16 (1974) Verma, R. S., Lubs, H. A.: A simple R banding technic. Amer. J. hum. Genet. 27, 110--117 (1975)
Trisomy of Band 21q22 Essential for Down's Phenotype
23
Vogel, W.: Identification of G-group chromosomes involved in a G / G tandem-translocationby the Giemsa-banding technique. Humangenetik 14, 255--256 (1972) Wahrman, J., Goitein, R., Richler, C., Goldman, B., Akstein, E., Chaki, R.: The mongoloid phenotype in man is due to trisomy of the distal pale G-band of chromosome 21. In: Chromosomes today, P. L. Pearson, K. R. Lewis, eds., Vol. 5, pp. 241--248. Isra61 Universities Press 1976 Warkany, J., Soukup, S. W.: A chromosomal abnormality in a girl with some features of Down's syndrome (mongolism). J. Pediat. 62, 890--894 (1963) White, M. J. D.: Some general problems of chromosomal evolution and speciation in animals. Survey Biol. Progr. (N.Y.) 3, 109--147 (1957) Williams, J. D., Summitt, R. L., Martens, P. R., Kimbrell, R. A.: Familial Down syndrome due to t(10;21) translocation: Evidence that the Down phenotype is related to trisomy of a specific segment of chromosome 21. Amer. J. hum. Genet. 27, 478--485 (1975)
Received January 20, 1977
Partial Trisomy 21 Further Evidence that Trisomy of Band 21q22 is Essential for Down's Phenotype
A. Hagemeijer and E. M. E. Smit Department of Cell Biology and Genetics, Medical Faculty, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands
Summary. Cytogenetic analysis of a 6-year-old girl with moderate mental retardation revealed 46 chromosomes with a tandem translocation (21;21) resulting in a partial trisomy 21. Only the terminal band 21q22 was not in triplicate. G-, Q-, R-, and C-banding techniques and silver nitrate staining of the nucleolus organizer regions (NORs) were used to identify this chromosome fully. The phenotype of the patient was not typical for Down's syndrome, providing additional evidence that trisomy of band 21q22 is pathogenetic for the phenotype of Down's syndrome. This is also a new example in human pathology of a stable 'dicentric' chromosome in which one of the centromeric constrictions appears to be nonfunctional.
Introduction Recently a few cases of familial reciprocal translocations involving chromosome 21 were studied by Williams et al. (1975), Sinet et al. (1976), and Wahrman et al. (1976). They related the phenotype of Down's syndrome to the trisomy of the distal part of the long arm of chromosome 21. Niebuhr (1974) reviewing the different cases of tandem translocation (21;21) had already reached a similar conclusion but without cytologic demonstration. The patient presented here has an almost complete trisomy 21 except for the band 21q22 which is not triplicated; her phenotype is different from the one in Down's syndrome. Her abnormal chromosome is the result of a tandem translocation (21;21) with centromeric heterochromatin present at two sites. It provides a new example of a stable dicentric chromosome with a latent or nonfunctional centromere. Similar cases have been reported, mainly X-to-X fusions (Dist~che et al., 1972; Therman et al., 1974; Ruthner and Golob, 1974; de la Chapelle and Stenstrand, 1974; Laurent et al., 1975; Fraisse et al., 1975) or 21 to 21 fusions (Bartsch-Sandhoff and Schade, 1973; Niebuhr, 1974).
16
A. Hagemeijer and E. M. E. Smit
Case Report The proband is a 6-year-old girl, the product of an uncomplicated full-term pregnancy. Her birth weight was 3400 g and length 53 cm. There was no history of irradiation or viral diseases during pregnancy. Her father and mother were both 28 years old at that time. She is the second child. Her brother, who is 4 years older, is physically and mentally normal. Somatic development was perfectly normal. Her growth and weight curves run between the 50th and the 75th percentile. Her first tooth appeared at 6 months of age. Dentition is normal and healthy, without decays or cavities. Her psychomotor development was slightly delayed. She could sit at 6 months of age, stay upright at 11 months, walk without help at 14.5 months. Her speech was clearly retarded: she could say a few words at 13 months but was 4 years old before she could make a short sentence. During childhood bronchitis and upper respiratory tract infections were frequent. Tonsillectomy was performed at 4 years and subsequently her speech and social contact improved very rapidly. Physical examination at 6 years old shows an active girl of normal height and weight, 118 cm and 22kg respectively. Her head circumference is 50 cm. The occipital bone is not flattened. She has thick straight hair. The face is oval, the palpebral fissures are horizontal. She has bilateral epicanthic folds and a flattened nose bridge but no hypertelorism. The interpupillary distance is 55 mm, normal for her age. She has an intermittent strabismus of the left eye. The nose is short and slightly stubbed. The mouth is wide and usually closed but is open when she is interviewed or when some task requires all her attention. The tongue is normal, not protruding and not furrowed. T h e palate is normal, not narrow or highly arched. The neck is not particularly short, the posterior hairline is normal. The hands are short and plump, very similar to the hands of both parents. The fifth finger is not particularly short or incurved. The dermatoglyphs are not characteristic for Down's syndrome. Simian creases and patterns on the thenar or hypothenar area are absent. Dermatoglyphs show 10 ulnar loops on the fingertips, palmar triradii in proximal position t, a distal loop in the third interdigital space, and a large distal loop on both hallux areas. The toes are small with hypotrophic nails; there is no wide gap between toes I and II. Her gait is normal. W h e n she stands she shows a small valgus deformity of the knee (X-shaped legs). There is no hyperlaxity of the joints. Roentgenograms were not made, so it was not possible to evaluate her skeletal age, the iliac and acetabular angles of the pelvis, or the interorbital distance. T h e r e is no heart m u r m u r and the rest of the physical examination is normal. She appears alert, sometimes restless. Her power of concentration is low, her memory is poor and she still communicates in short sentences. Cytogenetic investigation was initiated because of her intellectual immaturity when reaching school age.
Material and Methods Karyotypes were performed on standard PHA-stimulated blood cultures. Chromosome identification was achieved using different banding techniques: the A S G technique, Q-banding with atebrine and R-banding with acridine orange after heat denaturation (Verma and Lubs, 1975) or after a 5-bromodeoxyuridine (BrdU) pulse during the last 5 or 6 h of the culture (Hagemeijer et al., 1976). C-banding was carried out with a combination of different published techniques: airdried slides, a few weeks old, were stained with atebrine, dried, hydrolyzed for 10 min in HC1 0.2N, rinsed, dried, denaturated for 15 min in a 0.5% Ba(OH)2 solution at 50°C, rinsed again, incubated during 1 h in 2 × SSC at 60°C, then stained with Giemsa as in the A S G technique. Specific staining of the nucleolus organizer regions (NORs) with silver nitrate was performed according to Denton et al. (1976).
Trisomy of Band 21q22 Essential for Down's Phenotype
17
Results Thirty metaphases of the proband were fully analyzed: her karyotype was 46,XX, 21q+ (Fig. 1). The elongated 21 appeared as an acrocentric chromosome larger than a G but definitely smaller than a D-group chromosome (Figs. 2--5). G- and Q-banding suggested a 21 with a repetition of the clearly stained band 21q21 (Figs. 2 a and 5 a). The end of the long arm was stubbed but occasionally seen in association with chromosomes of the D or G group (Fig. 2b). R-banding after BrdU pulse showed a much more detailed pattern: the elongated chromosome appeared symmetrical around band 21q22 (Fig. 3). We interpreted this rearrangement as a long-arm fusion of two chromosomes 21, in opposition. The band 21q22 is clearly not duplicated (Fig. 4) which indicates that at the time of the formation of this chromosome a deletion of this band in one of the two partners has occurred. Evidence was sought for the presence of a latent centromere and of NORs. Cbanding demonstrated the presence of centromeric heterochromatin at both ends of this chromosome (Figs. 5b and c). The two C bands showed a similar rather weak staining intensity. Silver nitrate staining provided excellent images of the NORs which appeared as black knobs on top of the D and G group chromosomes. The abnormal 21 showed four black dots, two at each extremity (Fig. 6).
Fig. 1. Karyotype of the proband: 46,XX,21q+. Acridine orange (AO) staining after BrdU pulse
18
A. Hagemeijer and E. M. E. Smit
Fig. 2. a Chromosomal pairs 13, 21, and 22 of the proband. Q-banding technique, b Examples of satellite associations between the abnormal chromosome 21 and other acrocentrics of the proband
Fig. 3. Chromosomal pairs 13, 21, and 22 of the proband and of her parents. Reverse banding techniques Fig. 4. Composite arrangement of the normal and abnormal chromosome 21 demonstrating our interpretation of the rearrangement, a The abnormal 21 with on either side the normal 21 of the same cell in upright and in upside down position, b The abnormal 21 and next to it two chromosomes 21 placed above each other, in opposition demonstrating that fusion of two complete chromosomes 21 would result in a much larger R-band region t h a t the one we observed
Trisomy of Band 21q22 Essential for Down's Phenotype
19
Fig. 5. a Chromosomal pairs 13, 21, and 22 of the proband G-banding technique, b Idem with C-banding technique, e C-banded metaphase of the proband; the abnormal 21 is indicated by cIFrOW
Fig. 6. a Metaphase of the proband stained with silver nitrate to demonstrate the NORs; a r r o w indicates the abnormal chromosome 21 with 4 silver stained knobs, two at each extremity. b Example of the abnormal chromosome 21 next to a representative D-group chromosome from 5 different cells of the proband; the last one shows satellite association
20
A. Hagemeijer and E. M. E. Smit
This is consistent with our interpretation of this chromosomal rearrangement. The four dots appeared so similar that with this technique both ends of the chromosome were indistinguishable. According to the Paris Conference (1971) the full description of this chromosome will be: (21) ( p t e r - c e n - q 2 2 : : q 2 1 ~ l a t e n t cen-pter). Twenty metaphases of each parent were analyzed and showed normal karyotypes.
Discussion
A tandem fusion is a 'whole-arm' translocation resulting from one break in the vicinity of the centromere in one chromosome and another break near the end of a second chromosome (White, 1957). In human pathology Lejeune et al. (1965) used this term to describe a nonrobertsonian translocation between two acrocentric chromosomes. Except for the case of translocation (21;21) described by Poissonnier et al. (1976) most of the reported observations of tandem (or rather nonrobertsonian) translocations (21;21), studied with the new banding techniques, proved to be bisatellited pseudodicentric chromosomes. They result from the fusion, in opposition, of the long arm of two chromosomes 21, both having the break near the end of the long arm (Bartsch-Sandhoff and Schade, 1973; Niebuhr, 1974). Fusion of two complete chromosomes 21 would result in a D-size chromosome, which indeed was observed in most of the cases of tandem translocations in patients with typical Down's syndrome (Lejeune et al., 1965; Kadotani et al., 1970; Vogel, 1972; Bartsch-Sandhoff and Schade, 1973; Jernigan et al., 1974). The chromosomal aberration we describe here is also a translocation (21;21), in opposition, but without duplication of the distal band 21q22. This is particularly manifest with the R-banding technique. Such a dicentric chromosome originates from an interchange between the two arms of one chromosome or between two homologous chromosomes. These different mechanisms were extensively discussed by Therman and P~itau (1974) for X-X dicentrics, but can be extrapolated to a 21-21 dicentric. The centromeric and short-arm regions of acrocentric chromosomes are highly polymorphic. In our case, C bands and silver staining of the NORs are quite identical at both chromosomal ends, which suggests a rearrangement between the two arms of the same chromosome 21. This could have happened during gametogenesis in one of the parents or at the first zygotic division, with, in the latter case, secondary loss of a cell line with monosomy 21. The mechanism by which one of the centromeres became latent or nonfunctional is unknown. The karyotype of the patient in vivo and in vitro appears very stable, clearly indicating that this abnormal chromosome behaves as a monocentric. Our interpretation is an asymmetrical rearrangement with deletion of band 21q22 in one of the partners. Cytologically we cannot exclude a symmetrical rearrangement with duplication of band 21q22.1 and absence of band 21q22.3. This would restrict the phenotype of Down's syndrome to a trisomy of band
Trisomy of Band 21q22 Essential for Down's Phenotype
21
PARTIAL TRISOMY 21 With Down's Phenotype
Without Down's Phenotype
© 22
qll
(1) t(15;21)(p;q21) AuIa e t a [ .
1973
(2) t(10;21)(q26;q21) WitIiams et cd.
1975
(3) tan.(21;21) Poisonnier et al.
1976
(&) t(2;21)(q;q21) Wahrman et (at.
1976
(5~ t(15;21)(q26.2; q21) Sinet et (al.
1976
(6) t(12;21)(q2/.;q21) Chen et o.I.
1976
(2)(6) q21
q221
(5)(7)
q222
(3) (4)(5)
q223
(1)(2)
(7) t(an.(21;21) Our Observation
Fig. 7. Schematic representation of chromosome 21 (R-banding). The bands involved in the different case reports of partial trisomy 21 are represented by straight lines parallel to the chromosome: on the left side the cases with the phenotype of Down's syndrome and on the right side the cases without the Down's syndrome phenotype. The numbers in brackets refer to the different
publications
21q22.3, which is in contradiction with the observation of Poissonnier et al. (1976). The absence of clinical signs of monosomy 21 in our patient makes this interpretation rather unlikely. Cytoplasmic superoxide dismutase-1 (SOD-l) was tentatively mapped on band 21q22.1 (Sinet et al., 1976). Reliable measurement of the concentration of this enzyme may indicate the presence in two- or threefold of band 21q22.1 in this karyotype. Physically, our patient does not at all impress one as a mongoloid child. Looking at the check list of 25 clinical signs of Down's syndrome established by Lee and Jackson (1972), our patient has only 4 of them (epicanthic folds, flat nasal bridge, open mouth, short, broad hands) placing her in the group of nontrisomic patients. Cytogenetically we found an almost complete trisomy 21. Only band 21q22 is not trisomic. This suggests that band 21q22 is pathogenetic for the phenotype of Down's syndrome and provides a cytologic demonstration of the hypothesis of Niebuhr (1974). Reviewing 15 cases of Down's syndrome due to tandem translocations (G-G), Niebuhr emphasized the fact that some of them presented with a mild form of mongolism and he put forward the hypothesis that in this type of rearrangement a deletion of the terminal band of 21q was likely to happen, whereas in centric fusion the long arms of both chromosomes involved remained intact. The clinical importance of the terminal part of 21q was also suggested by Williams et al. (1975), Wahrman et al. (1976), Sinet et al. (1976), and Chen et al. (1976), who studied different cases of partial trisomy 21 in families with reciprocal translocations. The different cases of partial trisomy 21 reported
22
A. Hagemeijer and E. M. E. Smit
i n the literature a n d studied with the m o d e r n b a n d i n g techniques are s u m m a r i z e d in F i g u r e 7. The p h e n o t y p e of D o w n ' s s y n d r o m e appears here also to be clearly related with t r i s o m y o f b a n d 21q22.
Acknowledgements. We sincerely thank Prof. D. Bootsma for critically reviewing the manuscript, Dr. E. S. Sachs for the analysis of the dermatoglyphs, and Dr. J. Toledo for referring his patient to us.
References Aula, P., Leisti, J., von Koskull, H.: Partial trisomy 21. Clin. Genet. 4, 241--251 (1973) Bartsch-Sandhoff, M., Schade, H.: Zwei subterminale Heterochromatinregionen bei einer seltenen Form einer 21/2l-Translokation. Humangenetik 18, 329--336 (1973) Chen, H., Tyrkus, M., Woolley, jr., P. V.: Partial trisomy 21 due to maternal t(12;21) translocation: Further evidence that the Down phenotype is related to trisomy of the distal segment of chromosome 21. Proc. of the Vth Intern. Congr. Hum. Genet., Mexico, 1976. Excerpta Medica, int. congr, series 397, 116 (1976) de la Chapelle, A., Stenstrand, K.: Dicentric human X chromosomes. Hereditas 76, 259--268 (1974) Denton, T. E., Howell, W. M., Barrett, J. V.: Human nucleolar organizer chromosomes: Satellite associations. Chromosoma (Berl.) 55, 81--84 (1976) Distbche, C., Hagemeijer, A., Frederic, J., Progneaux, D.: 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 microdensitometry. Clin. Genet. 3, 388--395 (1972) Fraisse, J., Laurent, C., Collard, N., Biemont, M. C., Dutrillaux, B.: Un deuxi~me exemple de fusion t61omerique de deux chromosomes X. Ann. G6n&. 18, 243--245 (1975) Hagemeijer, A., Hoovers, J., Hasper-Voogt, I., Von Ruhe-Zurcher, T., Bootsma, D.: Latereplicating ring X-chromosomes identified by R-banding after BrdU pulse. Three new examples of mosaicism 45, XO/46, Xr(X). Hum. Genet. 34, 45--52 (1976) Jernigan, D., Curl, N., Keeler, C.: Milledgeville mongoloid: a rare karyotype of Down's syndrome. J. Hered. 65, 254--257 (1974) Kadotani, T., Ohama, K., Takahara, H., Nagai, I., Shimizu, B., Makino, S.: A case of Down's syndrome associated with G / G translocation. Proc. Japan Acad. 46, 858--862 (1970) Laurent, C., Biemont, M. C., Dutrillaux, B.: Sur quatre nouveaux cas de translocation du chromosome X chez l'homme. Humangenetik 26, 35--46 (1975) Lee, L. G., Jackson, J. F.: Diagnosis of Down's syndrome: clinical vs. Laboratory. Clin. Pediat. (Phila.) 11, 353--356 (1972) Lejeune, J., Berger, R., Vidal, O. R., Rethor6, M. O.: Un cas de translocation G - G en tandem. Ann. G6n&. 8, 60--62 (1965) Niebuhr, E.: Down's syndrome. The possibility of a pathogenetic segment on chromosome No. 21. Humangenetik 21, 99--101 (1974) Poissonnier, M., Saint-Paul, B., DutriUaux, B., Chassaigne, M., Gruyer, P., de Bligni~res-Strouk, G.: Trisomie 21 partielle (21q21-21q22.2). Ann. G6n&. 19, 69--73 (1976) Ruthner, U°, Golob, E.: Fusion of the short arms of one X-chromosome in a patient with gonadal dysgenesis. Humangenetik 24, 159--160 (1974) Sinet, P. M., Couturier, J., Dutrillaux, B., Poissonnier, M., Raoul, O., Rethor6, M. O., Allard, D., Lejeune, J., J6rome, H.: Trisomie 21 et superoxyde dismutase-1 (IPO-A). Tentative de localisation sur la sous bande 21q22.1. Exp. Cell Res. 97, 47--55 (1976) Therman, E., Sarto, G. E., Patau, K.: Apparently isodicentric but functionally monocentric X chromosome in man. Amer. J. hum. Genet. 26, 83--92 (1974) Therman, E., P~itau, K.: Abnormal X chromosomes in man: Origin, behavior and effects. Humangenetik 25, 1--16 (1974) Verma, R. S., Lubs, H. A.: A simple R banding technic. Amer. J. hum. Genet. 27, 110--117 (1975)
Trisomy of Band 21q22 Essential for Down's Phenotype
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Vogel, W.: Identification of G-group chromosomes involved in a G / G tandem-translocationby the Giemsa-banding technique. Humangenetik 14, 255--256 (1972) Wahrman, J., Goitein, R., Richler, C., Goldman, B., Akstein, E., Chaki, R.: The mongoloid phenotype in man is due to trisomy of the distal pale G-band of chromosome 21. In: Chromosomes today, P. L. Pearson, K. R. Lewis, eds., Vol. 5, pp. 241--248. Isra61 Universities Press 1976 Warkany, J., Soukup, S. W.: A chromosomal abnormality in a girl with some features of Down's syndrome (mongolism). J. Pediat. 62, 890--894 (1963) White, M. J. D.: Some general problems of chromosomal evolution and speciation in animals. Survey Biol. Progr. (N.Y.) 3, 109--147 (1957) Williams, J. D., Summitt, R. L., Martens, P. R., Kimbrell, R. A.: Familial Down syndrome due to t(10;21) translocation: Evidence that the Down phenotype is related to trisomy of a specific segment of chromosome 21. Amer. J. hum. Genet. 27, 478--485 (1975)
Received January 20, 1977
