Humangenetik 28, 269--272 (1975) © by Springcr-Verlag 1975
Clinical Case Reports Ring 17 Chromosome Detected by Amniocentesis A r t h u r G. W e i n b e r g Departments of Pathology and Pediatrics, University of Texas Health Science Center at Dallas and Children's Medical Center, Dallas, Texas J a n i c e L. B a i r Department of Pathology, Children's Medical Center, Dallas, Texas Mary Jo Harrod Department of Medicine, Division of Medical Genetics, University of Texas Health Science Center at Dallas, Dallas, Texas Received February 3, 1975/March 28, 1975
Summary. A fetus with a 46,XX,r(17) karyotype identified initially by amniocentesis and confirmed by banding studies is described. No significant phenotypic abnormalities were identified despite isolation of the ring chromosome from multiple fetal tissues. This paucity of abnormal features of gross development is consistent with findings in 3 previously reported patients with ring 17 chromosomes. Chromosome analysis of fetal cells o b t a i n e d b y amniocentesis has become a n e s t a b l i s h e d p r o c e d u r e for t h e a n t e n a t a l d e t e c t i o n of b i r t h defects in older m a t e r n a l age groups. This p r o c e d u r e has p r o v e n to be a c c u r a t e in p r e d i c t i n g t h e fetal k a r y o t y p e w i t h t h e recognized difficulties of failure to i d e n t i f y m.osaicism (Hsu et al., 1973), o v e r i n t e r p r e t a t i o n of mosaicism (Cox et al., 1974) a n d p o l y p l o i d y ( K o h n a n d I~obinson, 1970). I n several large series a v a r i e t y of c h r o m o s o m a l abn o r m a l i t i e s h a v e been d e t e c t e d b y this m e t h o d (Hsu et al., i973; M i l u n s k y a n d A t k i n s , 1974 ; Nadler, 1972). These h a v e included deletion, t r i s o m y , mosaic monos o m y a n d t r a n s l o c a t i o n . To our k n o w l e d g e a ring chromosome has n o t been prev i o u s l y described in this context. The following r e p o r t describes such an occurrence.
Case Report A 41-year-old Gravida IV, Para III, AB 0 female was referred for diagnostic amniocentesis because of her age. The pregnancy had been uncomplicated. The patient was receiving no medication. Her other children were living and well. There was no family history of congenital anomalies or excessive miscarriages. There was no known exposure of either parent to teratogens or X-ray during the pregnancy. Transabdominal amnioeentesis was performed at 16 weeks gestation, following placenta localization by ultrasonography. 16 cm 3 of clear amniotic fluid was obtained and cultured according to the method of Cedarqvist et al. (1973). Ham's F-10 medium 1, containing 30~o fetal calf serum was used. 1 Grand Island Biological Company.
270
A . G . Weinberg et al.
Fig. 1. E group chromosomes from cultured amniotic fluid cells
The cultures were harvested at 14 days, and 20 metaphase spreads were examined with routine Giemsa staining. In 16 of 20 cells examined an E group chromosome was replaced by a ring. In the remaining 4 cells an E group chromosome was missing, presumably the less stable ring. Giemsa banding by a modification of the technique of Sumner et al. (1971) was performed. Freshly prepared, air-dried slides were placed in a 60°C incubator overnight. The slides were then incubated in 2 X SSC for 21/2 hrs at 60 ° C, rinsed in deionized 1120, and stained in a 2~o solution of Giemsa 2 for 10--12 rain. After rinsing in deionized tt20, the slides were air-dried and mounted in Permount. The banding studies indicated that the ring replaced a normal 17 chromosome: 46,XX,r(17)/45,XX,--17 (Fig. 1). Other E group chromosomes were morphologically normal. After consultation with the patient and her husband and a discussion of the anomalies previously reported to be associated with ring chromosomes of the E group, a decision was made to terminate the pregnancy. A therapeutic abortion by hysterotomy was performed at 20 weeks of gestation. The female fetus weighed 290 g with a crown-rump length of 16.5 cm, crown-heel length of 23.5 cm, and head circumference of 17 cm. The 150 g placenta was unremarkable and the cord contained three vessels. A complete autopsy examination was performed upon the fetus. Careful examination revealed no external phenotypic abnormalities. Neither were there developmental abnormalities of any of the internal organs, t~epresentative microscopic sections from each of the fetal tissues were entirely normal and consistent with the gestational age. All fetal cells examined (15--20 per tissue) from cord blood, kidney, skin and amniotic fluid obtained at the time of the hysterotomy showed a single cell line: 46,XX,r(17). Thymic tissue was also cultured, but failed to yield any analysable metaphases. Analysis of 30 peripheral blood lymphocytes from the father showed a normal male karyotype. Analysis of 50 peripheral blood lymphocytes from the mother showed a normal female karyotype in 46. Two ceils showed what appeared to be an extra X chromosome, two cells showed loss of a C group chromosome, and two cells contained a large chromosome interpreted as a possible isochromosome X.
Discussion R i n g c h r o m o s o m e s are n o t c o m m o n b u t h a v e b e e n d e s c r i b e d in a s s o c i a t i o n with a variety of malformation syndromes. They most frequently involve members o f t h e D, E a n d G g r o u p s ( H a m e r t o n , 197i). P h e n o t y p i e f e a t u r e s a s s o c i a t e d w i t h ring chromosomes are usually similar to the features associated with deletion a n o m a l i e s o f t h e s e s a m e c h r o m o s o m e s . T h i s is w h a t m i g h t be e x p e c t e d if b r e a k a g e a n d loss o f t e l o m e r i c c h r o m a t i n m a t e r i a l w e r e i m p o r t a n t in f o r m a t i o n o f t h e r i n g ( H a m e r t o n , 1971). M o s t rings f o r m e d b y E g r o u p c h r o m o s o m e s h a v e b e e n r e f e r r e d t o as r i n g 18 or 1 7 - - 1 8 c h r o m o s o m e s in t h e a b s e n c e o£ b a n d i n g studies. 2 Gurr's R66, buffered ca. pH 6.8.
Ring 17 Chromosome
271
Two r e p o r t s h a v e described ring f o r m a t i o n involving c h r o m o s o m e 17. B u r d e a
et al. (1973) described a 46,XY,r(17) k a r y o t y p e in a f a t h e r a n d son. The child was r e t a r d e d b u t e x h i b i t e d r e l a t i v e l y m i n o r p h e n o t y p i e a b n o r m a l i t i e s (hypotonia, obesity, slight m i e r o c e p h a l y , epieanthus, h y p e r t e l o r i s m a n d e r y p t o r c h i d i s m ) . The f a t h e r was said to be normal. H o w e v e r , t h e e x a c t i d e n t i t y of t h e a b n o r m a l chromosome was n o t confirmed b y a u t o r a d i o g r a p h i c or b a n d i n g studies. P e t i t a n d K o u lischer (1971) described a y o u n g female w i t h a 4 6 , X X / 4 6 , X X , r ( 1 7 ) k a r y o t y p e confirmed b y a u t o r a d i o g r a p h y . This child e x h i b i t e d s h o r t stature, low weight a n d m e n t a l r e t a r d a t i o n b u t was otherwise p h e n o t y p i c a l l y normal. The a u t h o r s caut i o n e d t h a t t h e p a t i e n t should n o t be a c c e p t e d as an e x a m p l e of full p h e n o t y p i c expression of a ring 17 c h r o m o s o m e because of t h e presence of mosaieism. The fetus delivered b y h y s t e r o t o m y a t 20 weeks g e s t a t i o n in t h e p r e s e n t ease d i d n o t show a n y significant a b n o r m a l p h e n o t y p i e features, either grossly or microscopically. H o w e v e r , s u b t l e a b n o r m a l i t i e s of facial d e v e l o p m e n t m i g h t n o t be a p p a r e n t a t this stage of gestation. Mosaicism was e x c l u d e d b y e x a m i n a t i o n of m u l t i p l e fetal tissues in a d d i t i o n to blood l y m p h o c y t e s , all of which showed a single 46,XX,r(17) cell line. This suggests t h a t ring f o r m a t i o n in t h e 17 chromosome m a y n o t result in m a r k e d p h e n o t y p i c a b n o r m a l i t i e s . The absence of m a j o r associated anomalies m a y in p a r t e x p l a i n t h e failure to i d e n t i f y p a t i e n t s w i t h deletion a b n o r m a l i t i e s of chromosome 17. R e c e n t l y a t t e n t i o n has been focused u p o n t h e pitfalls of i n t e r p r e t a t i o n of a m n i o t i e fluid cell k a r y o t y p e s (IIsu et al., 1973), p a r t i c u l a r l y w i t h respect to mosaic cell lines (Cox et al., 1974). I n t e r p r e t a t i o n of t h e k a r y o t y p e in t h e p r e s e n t ease was difficult as ring chromosomes h a d not, to our knowledge, been p r e v i o u s l y d e s c r i b e d in a m n i o t i c fluid cell culture. H y s t e r o t o m y was p e r f o r m e d because of t h e u n i f o r m i t y of t h e a b n o r m a l i t y in all of t h e cells studied. R e c e n t l y , M i l u n s k y a n d A t k i n s (1974) h a v e identified i s o l a t e d cells w i t h ring chromosomes in a m n i o t i c fluid samples from 3 p a t i e n t s . I n none of these eases was t h e ring f o u n d in m o r e t h a n one cell. I n each i n s t a n c e a n o r m a l fetus was delivered.
Acknowledgement. We wish to express our appreciation to Stuart Stone, M.D. for referring the patient, Juan Jimenez, )/I.D. for performing the amniocentesis, and to Joann Fleming, B.A. and Kathy Faust, MT(ASCP) for assistance in performing the chromosome analyses. References Burdea, M., Lupascu, E., ~argineanu, L. : Un caz familial de ehromozom din grupa e in inel (i7)r cu transmitere de la rata la flu. Rev. med.-chir. Iasi 77, 353--357 (1973) Cedarqvist, L. L., Wennerstrom, C., Scnterfit, L. B., Baldridge, P. B., Rothe, I. J. : Simplified method for the accelerated growth of amniotie fluid cell cultures. Amer. J. Obstet. Gynee. 116, 871--874 (1973) Cox, D.M., Niewizas-Late, V., Riffell, M. I., Hamerton, J. L.: Chromosomal mosaicism in diagnostic amniotic fluid cell cultures. Pedlar. Res. 8, 679--683 (1974) Hamerton, J. L. : Human cytogeneties. New York: Academic Press 1971 Hsu, L. Y. F., Dubin, E.C., Kerenyi, T., Hirschhorn, K. : Results and pitfalls in prenatal eytogenetie diagnosis. J. med. Genet. 10, 112--119 (1973) Kohn, G., Robinson, A. : Tetraploidy in cells cultured from amniotic fluid. Lancet 1970 II, 778 Milunsky, A., Atkins, A. : Prenatal diagnosis of genetic disorders - - an analysis of experience with 600 cases. J. Amer. med. Ass. $30, 232--236 (1974)
272
A . G . Weinberg et al.
Nadler, H. L. : Prenatal detection of genetic disorders. In: Advances in human genetics (eds. H. Harris, K. Hirschhorn), pp. 1--38. New York: Plenum Press 1972 Petit, P., Koulischer, L.: Etude d'une mosaique 46,XX/46,XX,17r. Ann. G~n~t. 14, 55--58 (1971) Sumner, A. T., Evans, H . J . , Buckland, R . A . : New technique for distinguishing between human chromosomes. Nature (Lond.) New Biol. 282, 31--32 (1971) A. G. Weinberg, M.D. Director of Laboratories Children's Medical Center Dallas, Tx. 75235, USA Note Added in Proo]. Following the therapeutic abortion the mother again became pregnant. Amniocentesis at 15 weeks gestation showed a 47,XY,-]-21 karyotype. A therapeutic abortion was performed. The fetus had left clinodactyly, a left simian crease, excessive soft tissue at the back of the neck and slight brachycephaly. No other developmental anomalies were evident. The abnormal karyotype was confirmed in amniotic fluid, cord blood, fetal skin and kidney.
Clinical Case Reports Ring 17 Chromosome Detected by Amniocentesis A r t h u r G. W e i n b e r g Departments of Pathology and Pediatrics, University of Texas Health Science Center at Dallas and Children's Medical Center, Dallas, Texas J a n i c e L. B a i r Department of Pathology, Children's Medical Center, Dallas, Texas Mary Jo Harrod Department of Medicine, Division of Medical Genetics, University of Texas Health Science Center at Dallas, Dallas, Texas Received February 3, 1975/March 28, 1975
Summary. A fetus with a 46,XX,r(17) karyotype identified initially by amniocentesis and confirmed by banding studies is described. No significant phenotypic abnormalities were identified despite isolation of the ring chromosome from multiple fetal tissues. This paucity of abnormal features of gross development is consistent with findings in 3 previously reported patients with ring 17 chromosomes. Chromosome analysis of fetal cells o b t a i n e d b y amniocentesis has become a n e s t a b l i s h e d p r o c e d u r e for t h e a n t e n a t a l d e t e c t i o n of b i r t h defects in older m a t e r n a l age groups. This p r o c e d u r e has p r o v e n to be a c c u r a t e in p r e d i c t i n g t h e fetal k a r y o t y p e w i t h t h e recognized difficulties of failure to i d e n t i f y m.osaicism (Hsu et al., 1973), o v e r i n t e r p r e t a t i o n of mosaicism (Cox et al., 1974) a n d p o l y p l o i d y ( K o h n a n d I~obinson, 1970). I n several large series a v a r i e t y of c h r o m o s o m a l abn o r m a l i t i e s h a v e been d e t e c t e d b y this m e t h o d (Hsu et al., i973; M i l u n s k y a n d A t k i n s , 1974 ; Nadler, 1972). These h a v e included deletion, t r i s o m y , mosaic monos o m y a n d t r a n s l o c a t i o n . To our k n o w l e d g e a ring chromosome has n o t been prev i o u s l y described in this context. The following r e p o r t describes such an occurrence.
Case Report A 41-year-old Gravida IV, Para III, AB 0 female was referred for diagnostic amniocentesis because of her age. The pregnancy had been uncomplicated. The patient was receiving no medication. Her other children were living and well. There was no family history of congenital anomalies or excessive miscarriages. There was no known exposure of either parent to teratogens or X-ray during the pregnancy. Transabdominal amnioeentesis was performed at 16 weeks gestation, following placenta localization by ultrasonography. 16 cm 3 of clear amniotic fluid was obtained and cultured according to the method of Cedarqvist et al. (1973). Ham's F-10 medium 1, containing 30~o fetal calf serum was used. 1 Grand Island Biological Company.
270
A . G . Weinberg et al.
Fig. 1. E group chromosomes from cultured amniotic fluid cells
The cultures were harvested at 14 days, and 20 metaphase spreads were examined with routine Giemsa staining. In 16 of 20 cells examined an E group chromosome was replaced by a ring. In the remaining 4 cells an E group chromosome was missing, presumably the less stable ring. Giemsa banding by a modification of the technique of Sumner et al. (1971) was performed. Freshly prepared, air-dried slides were placed in a 60°C incubator overnight. The slides were then incubated in 2 X SSC for 21/2 hrs at 60 ° C, rinsed in deionized 1120, and stained in a 2~o solution of Giemsa 2 for 10--12 rain. After rinsing in deionized tt20, the slides were air-dried and mounted in Permount. The banding studies indicated that the ring replaced a normal 17 chromosome: 46,XX,r(17)/45,XX,--17 (Fig. 1). Other E group chromosomes were morphologically normal. After consultation with the patient and her husband and a discussion of the anomalies previously reported to be associated with ring chromosomes of the E group, a decision was made to terminate the pregnancy. A therapeutic abortion by hysterotomy was performed at 20 weeks of gestation. The female fetus weighed 290 g with a crown-rump length of 16.5 cm, crown-heel length of 23.5 cm, and head circumference of 17 cm. The 150 g placenta was unremarkable and the cord contained three vessels. A complete autopsy examination was performed upon the fetus. Careful examination revealed no external phenotypic abnormalities. Neither were there developmental abnormalities of any of the internal organs, t~epresentative microscopic sections from each of the fetal tissues were entirely normal and consistent with the gestational age. All fetal cells examined (15--20 per tissue) from cord blood, kidney, skin and amniotic fluid obtained at the time of the hysterotomy showed a single cell line: 46,XX,r(17). Thymic tissue was also cultured, but failed to yield any analysable metaphases. Analysis of 30 peripheral blood lymphocytes from the father showed a normal male karyotype. Analysis of 50 peripheral blood lymphocytes from the mother showed a normal female karyotype in 46. Two ceils showed what appeared to be an extra X chromosome, two cells showed loss of a C group chromosome, and two cells contained a large chromosome interpreted as a possible isochromosome X.
Discussion R i n g c h r o m o s o m e s are n o t c o m m o n b u t h a v e b e e n d e s c r i b e d in a s s o c i a t i o n with a variety of malformation syndromes. They most frequently involve members o f t h e D, E a n d G g r o u p s ( H a m e r t o n , 197i). P h e n o t y p i e f e a t u r e s a s s o c i a t e d w i t h ring chromosomes are usually similar to the features associated with deletion a n o m a l i e s o f t h e s e s a m e c h r o m o s o m e s . T h i s is w h a t m i g h t be e x p e c t e d if b r e a k a g e a n d loss o f t e l o m e r i c c h r o m a t i n m a t e r i a l w e r e i m p o r t a n t in f o r m a t i o n o f t h e r i n g ( H a m e r t o n , 1971). M o s t rings f o r m e d b y E g r o u p c h r o m o s o m e s h a v e b e e n r e f e r r e d t o as r i n g 18 or 1 7 - - 1 8 c h r o m o s o m e s in t h e a b s e n c e o£ b a n d i n g studies. 2 Gurr's R66, buffered ca. pH 6.8.
Ring 17 Chromosome
271
Two r e p o r t s h a v e described ring f o r m a t i o n involving c h r o m o s o m e 17. B u r d e a
et al. (1973) described a 46,XY,r(17) k a r y o t y p e in a f a t h e r a n d son. The child was r e t a r d e d b u t e x h i b i t e d r e l a t i v e l y m i n o r p h e n o t y p i e a b n o r m a l i t i e s (hypotonia, obesity, slight m i e r o c e p h a l y , epieanthus, h y p e r t e l o r i s m a n d e r y p t o r c h i d i s m ) . The f a t h e r was said to be normal. H o w e v e r , t h e e x a c t i d e n t i t y of t h e a b n o r m a l chromosome was n o t confirmed b y a u t o r a d i o g r a p h i c or b a n d i n g studies. P e t i t a n d K o u lischer (1971) described a y o u n g female w i t h a 4 6 , X X / 4 6 , X X , r ( 1 7 ) k a r y o t y p e confirmed b y a u t o r a d i o g r a p h y . This child e x h i b i t e d s h o r t stature, low weight a n d m e n t a l r e t a r d a t i o n b u t was otherwise p h e n o t y p i c a l l y normal. The a u t h o r s caut i o n e d t h a t t h e p a t i e n t should n o t be a c c e p t e d as an e x a m p l e of full p h e n o t y p i c expression of a ring 17 c h r o m o s o m e because of t h e presence of mosaieism. The fetus delivered b y h y s t e r o t o m y a t 20 weeks g e s t a t i o n in t h e p r e s e n t ease d i d n o t show a n y significant a b n o r m a l p h e n o t y p i e features, either grossly or microscopically. H o w e v e r , s u b t l e a b n o r m a l i t i e s of facial d e v e l o p m e n t m i g h t n o t be a p p a r e n t a t this stage of gestation. Mosaicism was e x c l u d e d b y e x a m i n a t i o n of m u l t i p l e fetal tissues in a d d i t i o n to blood l y m p h o c y t e s , all of which showed a single 46,XX,r(17) cell line. This suggests t h a t ring f o r m a t i o n in t h e 17 chromosome m a y n o t result in m a r k e d p h e n o t y p i c a b n o r m a l i t i e s . The absence of m a j o r associated anomalies m a y in p a r t e x p l a i n t h e failure to i d e n t i f y p a t i e n t s w i t h deletion a b n o r m a l i t i e s of chromosome 17. R e c e n t l y a t t e n t i o n has been focused u p o n t h e pitfalls of i n t e r p r e t a t i o n of a m n i o t i e fluid cell k a r y o t y p e s (IIsu et al., 1973), p a r t i c u l a r l y w i t h respect to mosaic cell lines (Cox et al., 1974). I n t e r p r e t a t i o n of t h e k a r y o t y p e in t h e p r e s e n t ease was difficult as ring chromosomes h a d not, to our knowledge, been p r e v i o u s l y d e s c r i b e d in a m n i o t i c fluid cell culture. H y s t e r o t o m y was p e r f o r m e d because of t h e u n i f o r m i t y of t h e a b n o r m a l i t y in all of t h e cells studied. R e c e n t l y , M i l u n s k y a n d A t k i n s (1974) h a v e identified i s o l a t e d cells w i t h ring chromosomes in a m n i o t i c fluid samples from 3 p a t i e n t s . I n none of these eases was t h e ring f o u n d in m o r e t h a n one cell. I n each i n s t a n c e a n o r m a l fetus was delivered.
Acknowledgement. We wish to express our appreciation to Stuart Stone, M.D. for referring the patient, Juan Jimenez, )/I.D. for performing the amniocentesis, and to Joann Fleming, B.A. and Kathy Faust, MT(ASCP) for assistance in performing the chromosome analyses. References Burdea, M., Lupascu, E., ~argineanu, L. : Un caz familial de ehromozom din grupa e in inel (i7)r cu transmitere de la rata la flu. Rev. med.-chir. Iasi 77, 353--357 (1973) Cedarqvist, L. L., Wennerstrom, C., Scnterfit, L. B., Baldridge, P. B., Rothe, I. J. : Simplified method for the accelerated growth of amniotie fluid cell cultures. Amer. J. Obstet. Gynee. 116, 871--874 (1973) Cox, D.M., Niewizas-Late, V., Riffell, M. I., Hamerton, J. L.: Chromosomal mosaicism in diagnostic amniotic fluid cell cultures. Pedlar. Res. 8, 679--683 (1974) Hamerton, J. L. : Human cytogeneties. New York: Academic Press 1971 Hsu, L. Y. F., Dubin, E.C., Kerenyi, T., Hirschhorn, K. : Results and pitfalls in prenatal eytogenetie diagnosis. J. med. Genet. 10, 112--119 (1973) Kohn, G., Robinson, A. : Tetraploidy in cells cultured from amniotic fluid. Lancet 1970 II, 778 Milunsky, A., Atkins, A. : Prenatal diagnosis of genetic disorders - - an analysis of experience with 600 cases. J. Amer. med. Ass. $30, 232--236 (1974)
272
A . G . Weinberg et al.
Nadler, H. L. : Prenatal detection of genetic disorders. In: Advances in human genetics (eds. H. Harris, K. Hirschhorn), pp. 1--38. New York: Plenum Press 1972 Petit, P., Koulischer, L.: Etude d'une mosaique 46,XX/46,XX,17r. Ann. G~n~t. 14, 55--58 (1971) Sumner, A. T., Evans, H . J . , Buckland, R . A . : New technique for distinguishing between human chromosomes. Nature (Lond.) New Biol. 282, 31--32 (1971) A. G. Weinberg, M.D. Director of Laboratories Children's Medical Center Dallas, Tx. 75235, USA Note Added in Proo]. Following the therapeutic abortion the mother again became pregnant. Amniocentesis at 15 weeks gestation showed a 47,XY,-]-21 karyotype. A therapeutic abortion was performed. The fetus had left clinodactyly, a left simian crease, excessive soft tissue at the back of the neck and slight brachycephaly. No other developmental anomalies were evident. The abnormal karyotype was confirmed in amniotic fluid, cord blood, fetal skin and kidney.
