Am J Hum Genet 27:441-453, 1975
Human X-Autosome Translocations: Differential Inactivation of the X Chromosome in a Kindred with an X-9 Translocation JAAKKO T. LEISTI,' MICHAEL M. KABACK, AND DAVID L. RIMOIN
A number of X-autosome translocations have been reported in the last few years, reflecting the expanded capability of new staining techniques to accurately identify structural anomalies in the group C (6-12,X) chromosomes. Autoradiographic analyses of these translocations have provided interesting data on the replication patterns of the X chromosome and, in some cases, on the inactivation of the involved autosomal chromosomes as well ([1-24]; R. E. Tipton, personal communication). We have studied a kindred in which a translocation between the long arm of an X chromosome and the long arm of a chromosome 9 has occurred. The involved chromosomes show different late replication patterns in the mother and the proposita. The family, which also illustrates a rare adjacent-two type of segregation, had been previously reported by Rohde and Catz as a possible t(6;9) translocation [25]. The clinical and cytogenetic features of this kindred are compared with those of the previously reported cases of X-autosome translocation, and the potential mechanisms of selective X inactivation are discussed. CASE REPORT
The proposita is a 28-year-old black female who was referred because of primary amenorrhea, developmental retardation, and multiple minor anomalies. She was a term product of a 27-year-old mother and a 33-year-old father. The pregnancy was uneventful except for a threatened abortion during the seventh month. The birth weight was 2,590 g, and no anomalies were noted at birth. Both mental and motor development were slow, language developed slowly, and she attended a school and workshop for the mentally handicapped. She has also grown slowly, and her height has remained considerably below the third percentile for sex and age. Her general health has been generally good. Hypothyroidism was suspected at age 11, and she was treated with desiccated thyroid until age 16. At age 17, goiter and hypothyroidism with low protein-bound iodine and elevated thyroglobulin titers were verified, and the treatment was reinstituted. Because of primary amenorrhea, lack of secondary sexual development, and hypoplastic genitalia [25], cyclic Received October 21, 1974; revised December 20, 1974. This project was supported in part by U.S. Public Health Service Research Grant HD-05624, Graduate Research Training Grant HD-00417, Clinical Research Center Grant RR-00425, and National Foundation-March of Dimes Research and Birth Defects Center grants. 1 All authors: Division of Medical Genetics, Harbor General Hospital, UCLA School of Medicine, Torrance, California 90509. © 1975 by the American Society of Human Genetics. All rights reserved.
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estrogen-progesterone therapy was started at age 18. At age 27, elevated blood pressure and hyperglycemia were found. The patient has three brothers, all in good health. The mother has had two miscarriages, occurring in the first and second trimester of pregnancy, respectively. Otherwise the family history is noncontributory. Physical examination revealed a short, obese black female with sexual characteristics secondary to substitution therapy (fig. 1). Her height was 136 cm and weight 50 kg. Her
F7G. 1.-Patient at age 27 head was small, with a circumference of 50 cm; a flattened occiput was noted. The facial features were slightly coarse, and the palpebral fissures were asymmetric, the left being narrower than the right. Both pupils were slightly displaced nasally, and there was exotropia of the right eye. The eyes appeared deep set, although not microphthalmic. The face was covered with abundant lanugolike hair. The oral cavity showed no abnormalities, but there was malocclusion of teeth. The posterior hairline, neck, and external ears appeared normal. The shoulders and chest appeared wide, and the extremities were sturdy. There was bilateral cubitus valgus, and both the hands and feet appeared short and broad. Both fourth metacarpals were short, and the fifth fingers were short and clinodactylic. The fingernails were hyperconvex and deep set (fig. 2). Both palms had a Simian crease, the axial triradii were proximally located, and interdigital loops were found in both the third and fourth interspaces. Total ridge count was 192 (99 + 93). The first and second toes were widely spaced, and the second and third toes showed a slight cutaneous syndactyly. The fourth and fifth metatarsals and toes were short bilaterally. METHODS
The presence of sex chromatin was analyzed from buccal epithelial cells stained with basic fuchsin. The karyotypes were established from lymphocyte metaphases after 3-day
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FIG. 2.-Right hand of patient. Note short fingers, short fourth metacarpal, and deep set hyperconvex nails.
culture according to a modified method of Moorhead et al. [26]. Chromosomes were stained with conventional Giemsa or with the acetic-saline-Giemsa method of Sumner et al. [27]. Autoradiography was performed after the addition of 3H-thymidine to lymphocyte cultures to a final activity of 1 RCi/ml 5 hr before the cells were fixed. RESULTS
Both the mother and the proposita had a single sex chromatin mass in 42%o and 41%o of the buccal epithelial cells, respectively. The sex chromatin appeared to be roughly of normal size in both individuals. The mother's karyotype had 46 chromosomes with a reciprocal translocation involving the long arm of an X chromosome and a chromosome 9 in all analyzed cells (fig. 3a). In the X chromosome, the breakpoint was located in the long arm, most probably very close to the centromere in band q 11, and thus almost the entire long arm was translocated to the distal region of the long arm of chromosome 9. Here the breakpoint was considered to be at band q32, and the mother's karyotype was interpreted as 46,X,rcp (X; 9) (qil ; q32). Labeling patterns were analyzed from 24 metaphases in the late S phase, and in all of these the normal X was the latereplicating one. In four additional cells, much less intense late replication could be shown on the X portion of the larger abnormal chromosome der(9). The patient also had 46 chromosomes, but her karyotype was unbalanced. According to the banding patterns of the affected chromosomes, she had only one intact X chromosome, two intact no. 9 chromosomes, and the long translocation chromosome der(9) (fig. 3b). She thus had three centromeres of chromosome 9 origin and only one of X origin, indicating the occurrence of an adjacent-two type of segregation in the mother. The proposita's karyotype was interpreted as 46,X,-X,+der(9),rcp(X;9)(qll;q32)mat. The labeling pattern of her X chromosomes was different from that of the mother's. Nineteen cells of the late S phase consistently showed the translocation chromosome der(9) to be the late-replicating one, while in only one cell could a late-replicating C-group chromosome be found. Interestingly, the late label on the der(9) was not confined to the translocated X portion of the chromosome but was consistently spread over the autosomal portion,
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FIG. 3.-a, Karyotype of mother showing reciprocal translocation between an X chromosome and chromosome 9; b, karyotype of proposita, 46,X,-X,+der(9),rcp(X;9) (qil ;q32)mat. Note presence of only one X chromosome and three no. 9 chromosomes, one of which has long arm material of X translocated to distal long arm.
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FIG. 4.-Late replication patterns of der(9) chromosome of nine cells from proposita shown with (right) and without (eft) autoradiographic grains. All other chromosomes in these nine cells had little or no label.
covering almost the entire chromosome (fig. 4). In several cells, however, the very distal part of the short arm of this chromosome did not show a heavy label. The chromosomes of the father and the three brothers had previously been found to be normal [2 5 ]. DISCUSSION
Cytogenetic analysis in this family demonstrated that the mother is a carrier of a reciprocal translocation between an X chromosome and chromosome 9, while her daughter has inherited only one structurally abnormal chromosome with a subsequently unbalanced karyotype. Segregation in the mother's X-9 quadrivalent has apparently followed the adjacent-two pattern, where the adjacent homologous centromeres pass to the same pole. Thus the patient has three centromeres of chromosome 9 origin and is trisomic for almost the entire chromosome 9, while having only one centromere of X origin. The adjacent-two type of segregation is uncommon in human reciprocal translocations, as pointed out by Hamerton [28] who found only two such cases in a series of 75 families. The mother's reproductive history and normal phenotype suggest that her translocation is balanced. While her normal X chromosome is selectively inactivated in all cells, the two translocated fragments have remained active and apparently have not interfered with the function of their adjacent autosomal segments. The patient, on the other hand, is phenotypically abnormal and has an unbalanced karyotype which is further modified by the pattern of chromosomal inactivation. Her phenotype, which is suggestive of Turner syndrome, can be readily accounted for by her
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sex chromosome complement, for she has a deletion of the short arm of an X chromosome with selective inactivation of the long arm of the abnormal X. This constellation is usually found in Turner syndrome patients with deletion of the short arm of an X chromosome [29, 30]. The less typical clinical features, such as hypoplastic phalanges and microcephaly with associated mental retardation, could be explained by the small extra active short arm segment of chromosome 9 in the der(9) chromosome which did not show constant late replication. Inactivation of the major part of the chromosome 9 material in this chromosome probably explains why the full phenotype of trisomy 9 [31] or trisomy of the short arm of chromosome 9 [32] did not become clinically manifest. Thus, in this patient, the phenotypic features along with the late-replicating characteristics of the der(9) chromosome indicate the presence of autosomal genetic inactivation. The findings in the present family and the previously reported cases of X-autosome translocations bring out several interesting points regarding inactivation of the human X chromosome and adjacent autosomal genetic material and the related phenotypic changes. Tables 1 and 2 outline the main cytogenetic and clinical data in the human X-autosome translocations reported to date. The cases are divided into "balanced" (table 1) or unbalanced (table 2) categories, since these criteria appear to be important from the standpoint of both phenotypic changes and the pattern of X inactivation. Most of the cases are thought to represent reciprocal translocations, although one case apparently is nonreciprocal [12]. There are five families in which some individuals have balanced and other individuals have unbalanced forms of translocation (present family; [5, 9, 11]; R. E. Tipton, personal communication). These kindreds also show differential inactivation of the X chromosome in different family members. Comparison of the cytogenetic features of the balanced translocations shows that all are different, with at least 10 different autosomes involved and an even larger number of breakpoints. The main direction of exchange has been a translocation of a long arm segment of an X chromosome to an autosome [1-10], the short arm was affected in one case [ 11 ], and in four cases ( [ 12, ? 13, 14]; R. E. Tipton, personal communication) the main direction has been the translocation of an autosomal segment to either the short or long arm of an X chromosome. As to the breakpoints in the affected X chromosome, both proximal, median, and distal segments of the arms have been involved. The pattern of X inactivation has been fairly constant in the balanced translocation cases, the normal X having been selectively inactivated in almost all cases (table 1). The phenotypes of the individuals with apparently balanced X-autosome translocations show marked variability, which does not seem to be associated with either the particular autosome involved, the site of the breakpoint in the X chromosome, or the direction of the exchange (table 1). This clinical variability ranges from normal female to primary amenorrhea and hypogonadism or mental retardation with multiple anomalies. A normal phenotype would be expected in the presence of a balanced karyotype and apparently normally functional X genes, even if they were separated into two translocated segments. The presence of the abnormal
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phenotype in the case of Thelen et al. [14] can be explained by the cytogenetic findings, for inactivation of the autosomal segment in most of the analyzed cells has apparently led to effective monosomy of the homologous segment. In the other cases with abnormal phenotypes, however, no such logical explanations are apparent. The frequency of primary amenorrhea in this series is high and may, as pointed out by Sarto et al. [4], be directly related to the X-autosome translocation. In view of the apparent similarity of the cytogenetic features in the normal and amenorrheic females, however, explanations such as effective hemizygosity of a recessive gene in the active X chromosome or disruption of chromosomal continuity [4] seem highly improbable. It is, of course, possible that the amenorrhea could be due to a minor chromosomal imbalance which was not detected. The unbalanced cases show even greater cytogenetic and phenotypic variability (table 2). The karyotypic heterogeneity is further complicated by the observed variation in the late replication or inactivation patterns of X chromosomes and affected autosomes. Thus the inactivation may be completely random [23] or affect selectively the normal X [15], the X portion of the translocation X [3], the entire translocation X including the autosomal segment (R. E. Tipton, personal communication), or almost the entire translocation X (present case). To make assessment of the phenotype-genotype relationship even more difficult, many of the unbalanced cases are further characterized by mosaicism [11, 20, 21], presence of an extra chromosome [5], loss of a centric fragment [22, 23], or even a ring formation [24]. In some cases the phenotypic changes can be explained on the basis of the observed cytogenetic abnormalities, especially when autosomal late replication is taken to represent genetic inactivation. For example, the patient presented here represents a good example of autosomal inactivation with functional elimination of an apparent trisomic state. In a similar way, the patient of Crandall et al. [16, 17] may represent a partial trisomy of chromosome 13, and the patient of Summitt et al. [9], an effective monosomy of chromosome 21. In many cases of X-autosome translocation where autosomal inactivation has occurred, however, this autosomal inactivation has not "corrected" an unbalanced karyotype by some kind of dosage compensation but has led to an unbalanced hemizygous state, as in the case of Summitt et al. [9] and in the balanced case of Thelen et al. [14]. Furthermore, in some cases the inactivation has not spread over the autosomal segment, resulting in a partial trisomy of a chromosomal segment [3, 15]. Understanding the selectivity of chromosomal inactivation in X-autosome translocations becomes difficult if the basic purpose of inactivation is to maintain dosage compensation and a balance of the X chromosomal complement in the male and female [33]. In the normal female this is accomplished by a random inactivation of the paternal and maternal X chromosome, in X polysomies by inactivation of all but one X, and in simple structural anomalies by the selective inactivation of the structurally abnormal X chromosome. However, only the balanced X-autosome translocations show some consistency in the X chromosome that is inactivated and the tendency to restore a balanced karyotype; that is, selective inactivation of the normal X was observed in 11 of 14 cases. In the unbalanced cases the in-
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activation patterns appear more or less coincidental, both from the point of view of selection of which X chromosomes are inactivated and the extent of adjacent autosomal involvement. Even when autosomal material becomes inactivated, the primary event appears to be the initiation of the inactivation process in the X chromosome, with subsequent autosomal involvement, possibly by a "spreading" mechanism [34]. The mechanism of initiation is not known, although several hypotheses have been presented (see [35, 36]). Several authors have suggested that the determining factor in the selection of the X to be inactivated would be the direction of the exchange of chromosomal material in the translocation process [3, 17]. Thus in cases where X material would be translocated to an autosome, the normal X would be preferentially inactivated, and in cases of translocation from autosome to X, inactivation would be variable in nature. This has been ruled out definitively, as evidenced by the presence of different types of inactivation patterns both among the X-to-autosome and autosome-to-X translocations and by the kindreds with differential inactivation of X in different family members. The latter kindreds also rule out the possibility that the choice of inactivation would be an inherent property of the X chromosome per se. The most plausible explanation for the variety of inactivation patterns would be the existence of a control mechanism whose purpose is to maintain a balanced genetic constitution in the cells. Such a mechanism could operate before or during the initiation phase, leading to predetermined selection of the X chromosome to be inactivated or following random inactivation by selection against the more genetically unbalanced cells [28]. In both possibilities the risk or presence of autosomal inactivation would play a crucial role in the final outcome. It is apparent from evidence derived from both human and mouse translocations [37] that autosomal segments tend to become inactivated when in close physical contact with inactive X material. Thus the preferential inactivation of the normal X in balanced translocations appears rational, since if the translocated X were inactivated the adjacent autosomal segments would be at risk of becoming inactivated, as actually happened in the case of Thelen et al. [14]. The inactivation pattern in the unbalanced cases is again more difficult to explain. One might assume that the opposite choice in the pattern of X inactivation would lead to even more severe phenotypic consequences. This appears to be true in certain cases, such as in the present patient. In other patients (e.g., [15]) inactivation of the other X chromosome would appear to have resulted in a more balanced state. Thus the complexity of chromosomal rearrangements, the variation in the initial selection of the inactive X,-and the unpredictability of the presence and extent of autosomal involvement in unbalanced X-autosome translocations make it impossible to explain all cases by a simple unified hypothesis. Significantly, mosaicism of the inactivation pattern has been observed in many cases, which may indicate randomness in the initial X inactivation with later selection of the most viable cell line.
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SUMMARY
A kindred with an X-autosome translocation and differential inactivation of the X chromosome is described. The phenotypically normal mother has a reciprocal translocation [46,X,rcp(X;9)(qll;q32)] while the daughter's karyotype is unbalanced [46,X,-X,+der(9) ,rcp(X;9) (ql 1 ;q32)mat], indicating adjacent-two type of segregation in the mother. In the mother's cells the normal X is late replicating, while in the daughter's cells almost the entire der(9) is late replicating, indicating the presence of autosomal inactivation. The daughter's abnormal phenotype can be explained by her sex chromosomal complement and the absence of effective trisomy 9. At this stage there is no simple explanation to account for all types of inactivation patterns encountered in the 14 balanced and 15 unbalanced cases of Xautosome translocations reported to date. Selection of X inactivation is not an inherent characteristic of the X chromosome per se, and it is not dependent on the direction of chromosomal exchange, as was suggested previously. Correlation of the phenotypic and cytogenetic features of these patients suggests a pattern of X and autosomal inactivation consistent with the least amount of genotypic and phenotypic imbalance in most cases. The data are most consistent with random X inactivation followed by selection of the most viable cell line. ACKNOWLEDGMENTS We are indebted to Mrs. D. Thornbury and Mrs. Florence DeNice for valuable assistance in evaluating this family. 1. 2. 3.
4.
5.
6. 7. 8.
9.
REFERENCES MANN JD, VALDMANIS A, CAPPS SC, PUITE RH: A case of primary amenorrhea with a translocation involving chromosomes of groups B and C. Am J Hum Genet 17:377-383, 1965 MANN JD, HIGGINS J: A case of primary amenorrhea associated with X-autosomal translocation [46,X,t(Xq-;5q+)]. Am J Hum Genet 26:416, 1974 COHEN MM, LIN C-C, SYBERT V, ORECCHIo EJ: Two human X-autosome translocations identified by autoradiography and fluorescence. Am J Hum Genet 24:583597, 1972 SARTO GE, THERMAN E, PATAU K: X inactivation in man: a woman with t(Xq-; 12q+). Am J Hum Genet 25:262-270, 1973 ALLDERDICE PW, MILLER OJ, KLINGER HP, PALLISTER PD, OPITZ JM: Demonstration of a spreading effect in an X-autosome translocation by combined autoradiographic and quinacrine-fluorescence studies. Excerpta Medica Int Congr Ser 233:1415, 1971 SUJANSKY E, Hsu LY, LucAs M, HIRSCHHORN K: Nature of X-autosome translocation and choice of X-inactivation (abstr.). Pediatr Res 7:343, 1973 LuCAS M, SMITHIES A: Banding patterns and autoradiographic studies of cells with an X-autosome translocation. Ann Hum Genet 37:9-12, 1973 GERALD P, BRUNS G, MONEDJIKOVA V: Localization of genes on the X chromosome by somatic cell hybridization (abstr.). Pediatr Res 7:344, 1973 SUMMITT RL, MARTENS PR, WILROY RS: X-autosome translocation in normal mother and effectively 21-monosomic daughter. J Pediatr 84:539-546, 1974
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10. KALLIO H: Cytogenetic and clinical study
on 100
cases of primary amenorrhea. Acta
Obstet Gynecol Scand [Suppl] 24:1-79, 1973 11. BUCKTON KE, JACOBS PA, RAE LA, NEWTON MS, SANGER R: An inherited X-autosome translocation in man. Ann Hum Genet 35:171-178, 1971 12. DE LA CHAPELLE A, SCHRODER J: Apparently non-reciprocal balanced human (3q-; Xq+) translocation: late replication of structurally normal X. Chromosomes Today 4:261-265, 1973 13. THORBURN MJ, MARTIN PA, PATHAK UN: Possible X/autosomal translocation in a girl with gonadal dysgenesis. J Med Genet 7:402-406, 1970 14. THELEN TH, ABRAMS DJ, FISCH RO: Multiple abnormalities due to possible genetic
inactivation in an X/autosome translocation. Am J Hum Genet 23:410-418, 1971 15. MIKKELSEN M, DAHL G: Unbalanced X/autosomal translocation with inactivation of the normal X chromosome. Cytogenet Cell Genet 12 :357-366, 1973 16. CRANDALL BF, CARREL RE, MULLER H: Trisomy 13 syndrome resulting from a 13-X translocation [46,X,t(Xql3q)] (abstr.). Am J Hum Genet 25 :22A, 1973 17. CRANDALL BF, CARREL RE, HOWARD J, SCHROEDER WA, MULLER H: Trisomy 13 with a 13-X translocation. Am J Hum Genet 26:385-392, 1974 18. WIE LIE G, COENEGRACHT JM, STALDER G: A very large metacentric chromosome in a woman with symptoms of Turner's syndrome. Cytogenetics 3:427-440, 1964 19. GERMAN J: Autoradiographic studies on human chromosomes. I. A review, in Proceedings 3d International Congress of Human Genetics, edited by CROW JF, NEEL JV, Baltimore, Johns Hopkins Press, 1967, pp 123-136 20. HUGH-JONES K, WALLACE SJ, THORNBER JM, ATKIN NB: Gonadal dysgenesis with unusual abnormalities. Arch Dis Child 40:274-279, 1965 21. THORBURN MJ, MILLER CG, DoVEY P: Anomalies of development in a girl with unusual sex chromosomal mosaicism. J Med Genet 4:283-287, 1967 22. NEUHAUSER G, BACK F: X-Autosom-Translokation bei einem Kind mit multiplen Missbildungen. Humangenetik 3:300-311, 1967 23. ENGEL W, VOGEL W, REINWEIN H: Autoradiographische Untersuchungen an einer X-Autosomentranslokation beim Menschen: 45,X,1 5-,tan( 1 5qXq+) +. Cytogenetics 10:87-98, 1971 24. MUKERJEE D, BURDETTE WJ: Multiple congenital anomalies associated with a ring 3 chromosome and translocated 3/X chromosome. Nature (Lond) 212:153-155, 1966 25. ROHDE RA, CATZ B: Maternal transmission of a new group-C(6/9) chromosomal syndrome. Lancet 2:838-840, 1964 26. MOORHEAD PS, NOWELL PC, MELLMAN WJ, BATTIPS DM, HUNGERFORD DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613-616, 1960 2 7. SUMNER AT, EVANS HJ, BUCKLAND RA: A new technique for distinguishing between human chromosomes. Nature [New Biol] 232 :31-32, 1971 28. HAMERTON JL: Human Cytogenetics. Vol 1: General Cytogenetics. New York, Academic Press, 1971 29. FERGUSON-SMITH MA: Karyotype-phenotype correlations in gonadal dysgenesis and their bearing on the pathogenesis of malformations. J Med Genet 2 :142-155, 1965 30. GRUMBACH MM, VAN WYK JJ: Disorders of sex differentiation, in Textbook of Endocrinology, 5th ed, edited by WILLIAMS RH, Philadelphia, Saunders, 1974, pp 423-501 31. FEINGOLD M, ATKINS L: A case of trisomy 9. J Med Genet 10:184-187, 1973 32. RETHORE MO, HOEHN H, ROTT HD, COUTURIER J, DUTRILLAUX B, LEJEUNE J: Analyse de la trisomie 9p par denaturation menagee. A propos d'un nouveau cas. Humangenetik 18:129-138, 1973 33. LYON MF: X-chromosome inactivation in mammals. Adv Teratol 1:25-54, 1966
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34. RUSSEL LB: Mammalian X-chromosome action: inactivation limited in spread and in region of origin. Science 140:976-978, 1963 35. LYON MF: Possible mechanisms of X chromosome inactivation. Nature [New Biol] 232 :229-232, 1971 36. BROWN SW, CHANDRA HS: Inactivation of the mammalian X chromosome. Proc Natl Acad Sci USA 70:195-199, 1973 37. RusSEL LB, MONTGOMERY CS: Comparative studies on X-autosome translocations in the mouse. II. Inactivation of autosomal loci, segregation, and the mapping of autosomal breakpoints in five T(X; 1)'s. Genetics 64:281-312, 1970
Human X-Autosome Translocations: Differential Inactivation of the X Chromosome in a Kindred with an X-9 Translocation JAAKKO T. LEISTI,' MICHAEL M. KABACK, AND DAVID L. RIMOIN
A number of X-autosome translocations have been reported in the last few years, reflecting the expanded capability of new staining techniques to accurately identify structural anomalies in the group C (6-12,X) chromosomes. Autoradiographic analyses of these translocations have provided interesting data on the replication patterns of the X chromosome and, in some cases, on the inactivation of the involved autosomal chromosomes as well ([1-24]; R. E. Tipton, personal communication). We have studied a kindred in which a translocation between the long arm of an X chromosome and the long arm of a chromosome 9 has occurred. The involved chromosomes show different late replication patterns in the mother and the proposita. The family, which also illustrates a rare adjacent-two type of segregation, had been previously reported by Rohde and Catz as a possible t(6;9) translocation [25]. The clinical and cytogenetic features of this kindred are compared with those of the previously reported cases of X-autosome translocation, and the potential mechanisms of selective X inactivation are discussed. CASE REPORT
The proposita is a 28-year-old black female who was referred because of primary amenorrhea, developmental retardation, and multiple minor anomalies. She was a term product of a 27-year-old mother and a 33-year-old father. The pregnancy was uneventful except for a threatened abortion during the seventh month. The birth weight was 2,590 g, and no anomalies were noted at birth. Both mental and motor development were slow, language developed slowly, and she attended a school and workshop for the mentally handicapped. She has also grown slowly, and her height has remained considerably below the third percentile for sex and age. Her general health has been generally good. Hypothyroidism was suspected at age 11, and she was treated with desiccated thyroid until age 16. At age 17, goiter and hypothyroidism with low protein-bound iodine and elevated thyroglobulin titers were verified, and the treatment was reinstituted. Because of primary amenorrhea, lack of secondary sexual development, and hypoplastic genitalia [25], cyclic Received October 21, 1974; revised December 20, 1974. This project was supported in part by U.S. Public Health Service Research Grant HD-05624, Graduate Research Training Grant HD-00417, Clinical Research Center Grant RR-00425, and National Foundation-March of Dimes Research and Birth Defects Center grants. 1 All authors: Division of Medical Genetics, Harbor General Hospital, UCLA School of Medicine, Torrance, California 90509. © 1975 by the American Society of Human Genetics. All rights reserved.
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estrogen-progesterone therapy was started at age 18. At age 27, elevated blood pressure and hyperglycemia were found. The patient has three brothers, all in good health. The mother has had two miscarriages, occurring in the first and second trimester of pregnancy, respectively. Otherwise the family history is noncontributory. Physical examination revealed a short, obese black female with sexual characteristics secondary to substitution therapy (fig. 1). Her height was 136 cm and weight 50 kg. Her
F7G. 1.-Patient at age 27 head was small, with a circumference of 50 cm; a flattened occiput was noted. The facial features were slightly coarse, and the palpebral fissures were asymmetric, the left being narrower than the right. Both pupils were slightly displaced nasally, and there was exotropia of the right eye. The eyes appeared deep set, although not microphthalmic. The face was covered with abundant lanugolike hair. The oral cavity showed no abnormalities, but there was malocclusion of teeth. The posterior hairline, neck, and external ears appeared normal. The shoulders and chest appeared wide, and the extremities were sturdy. There was bilateral cubitus valgus, and both the hands and feet appeared short and broad. Both fourth metacarpals were short, and the fifth fingers were short and clinodactylic. The fingernails were hyperconvex and deep set (fig. 2). Both palms had a Simian crease, the axial triradii were proximally located, and interdigital loops were found in both the third and fourth interspaces. Total ridge count was 192 (99 + 93). The first and second toes were widely spaced, and the second and third toes showed a slight cutaneous syndactyly. The fourth and fifth metatarsals and toes were short bilaterally. METHODS
The presence of sex chromatin was analyzed from buccal epithelial cells stained with basic fuchsin. The karyotypes were established from lymphocyte metaphases after 3-day
INACTIVATION IN X-9 TRANSLOCATION
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FIG. 2.-Right hand of patient. Note short fingers, short fourth metacarpal, and deep set hyperconvex nails.
culture according to a modified method of Moorhead et al. [26]. Chromosomes were stained with conventional Giemsa or with the acetic-saline-Giemsa method of Sumner et al. [27]. Autoradiography was performed after the addition of 3H-thymidine to lymphocyte cultures to a final activity of 1 RCi/ml 5 hr before the cells were fixed. RESULTS
Both the mother and the proposita had a single sex chromatin mass in 42%o and 41%o of the buccal epithelial cells, respectively. The sex chromatin appeared to be roughly of normal size in both individuals. The mother's karyotype had 46 chromosomes with a reciprocal translocation involving the long arm of an X chromosome and a chromosome 9 in all analyzed cells (fig. 3a). In the X chromosome, the breakpoint was located in the long arm, most probably very close to the centromere in band q 11, and thus almost the entire long arm was translocated to the distal region of the long arm of chromosome 9. Here the breakpoint was considered to be at band q32, and the mother's karyotype was interpreted as 46,X,rcp (X; 9) (qil ; q32). Labeling patterns were analyzed from 24 metaphases in the late S phase, and in all of these the normal X was the latereplicating one. In four additional cells, much less intense late replication could be shown on the X portion of the larger abnormal chromosome der(9). The patient also had 46 chromosomes, but her karyotype was unbalanced. According to the banding patterns of the affected chromosomes, she had only one intact X chromosome, two intact no. 9 chromosomes, and the long translocation chromosome der(9) (fig. 3b). She thus had three centromeres of chromosome 9 origin and only one of X origin, indicating the occurrence of an adjacent-two type of segregation in the mother. The proposita's karyotype was interpreted as 46,X,-X,+der(9),rcp(X;9)(qll;q32)mat. The labeling pattern of her X chromosomes was different from that of the mother's. Nineteen cells of the late S phase consistently showed the translocation chromosome der(9) to be the late-replicating one, while in only one cell could a late-replicating C-group chromosome be found. Interestingly, the late label on the der(9) was not confined to the translocated X portion of the chromosome but was consistently spread over the autosomal portion,
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FIG. 3.-a, Karyotype of mother showing reciprocal translocation between an X chromosome and chromosome 9; b, karyotype of proposita, 46,X,-X,+der(9),rcp(X;9) (qil ;q32)mat. Note presence of only one X chromosome and three no. 9 chromosomes, one of which has long arm material of X translocated to distal long arm.
INACTIVATION IN X-9 TRANSLOCATION
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FIG. 4.-Late replication patterns of der(9) chromosome of nine cells from proposita shown with (right) and without (eft) autoradiographic grains. All other chromosomes in these nine cells had little or no label.
covering almost the entire chromosome (fig. 4). In several cells, however, the very distal part of the short arm of this chromosome did not show a heavy label. The chromosomes of the father and the three brothers had previously been found to be normal [2 5 ]. DISCUSSION
Cytogenetic analysis in this family demonstrated that the mother is a carrier of a reciprocal translocation between an X chromosome and chromosome 9, while her daughter has inherited only one structurally abnormal chromosome with a subsequently unbalanced karyotype. Segregation in the mother's X-9 quadrivalent has apparently followed the adjacent-two pattern, where the adjacent homologous centromeres pass to the same pole. Thus the patient has three centromeres of chromosome 9 origin and is trisomic for almost the entire chromosome 9, while having only one centromere of X origin. The adjacent-two type of segregation is uncommon in human reciprocal translocations, as pointed out by Hamerton [28] who found only two such cases in a series of 75 families. The mother's reproductive history and normal phenotype suggest that her translocation is balanced. While her normal X chromosome is selectively inactivated in all cells, the two translocated fragments have remained active and apparently have not interfered with the function of their adjacent autosomal segments. The patient, on the other hand, is phenotypically abnormal and has an unbalanced karyotype which is further modified by the pattern of chromosomal inactivation. Her phenotype, which is suggestive of Turner syndrome, can be readily accounted for by her
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sex chromosome complement, for she has a deletion of the short arm of an X chromosome with selective inactivation of the long arm of the abnormal X. This constellation is usually found in Turner syndrome patients with deletion of the short arm of an X chromosome [29, 30]. The less typical clinical features, such as hypoplastic phalanges and microcephaly with associated mental retardation, could be explained by the small extra active short arm segment of chromosome 9 in the der(9) chromosome which did not show constant late replication. Inactivation of the major part of the chromosome 9 material in this chromosome probably explains why the full phenotype of trisomy 9 [31] or trisomy of the short arm of chromosome 9 [32] did not become clinically manifest. Thus, in this patient, the phenotypic features along with the late-replicating characteristics of the der(9) chromosome indicate the presence of autosomal genetic inactivation. The findings in the present family and the previously reported cases of X-autosome translocations bring out several interesting points regarding inactivation of the human X chromosome and adjacent autosomal genetic material and the related phenotypic changes. Tables 1 and 2 outline the main cytogenetic and clinical data in the human X-autosome translocations reported to date. The cases are divided into "balanced" (table 1) or unbalanced (table 2) categories, since these criteria appear to be important from the standpoint of both phenotypic changes and the pattern of X inactivation. Most of the cases are thought to represent reciprocal translocations, although one case apparently is nonreciprocal [12]. There are five families in which some individuals have balanced and other individuals have unbalanced forms of translocation (present family; [5, 9, 11]; R. E. Tipton, personal communication). These kindreds also show differential inactivation of the X chromosome in different family members. Comparison of the cytogenetic features of the balanced translocations shows that all are different, with at least 10 different autosomes involved and an even larger number of breakpoints. The main direction of exchange has been a translocation of a long arm segment of an X chromosome to an autosome [1-10], the short arm was affected in one case [ 11 ], and in four cases ( [ 12, ? 13, 14]; R. E. Tipton, personal communication) the main direction has been the translocation of an autosomal segment to either the short or long arm of an X chromosome. As to the breakpoints in the affected X chromosome, both proximal, median, and distal segments of the arms have been involved. The pattern of X inactivation has been fairly constant in the balanced translocation cases, the normal X having been selectively inactivated in almost all cases (table 1). The phenotypes of the individuals with apparently balanced X-autosome translocations show marked variability, which does not seem to be associated with either the particular autosome involved, the site of the breakpoint in the X chromosome, or the direction of the exchange (table 1). This clinical variability ranges from normal female to primary amenorrhea and hypogonadism or mental retardation with multiple anomalies. A normal phenotype would be expected in the presence of a balanced karyotype and apparently normally functional X genes, even if they were separated into two translocated segments. The presence of the abnormal
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phenotype in the case of Thelen et al. [14] can be explained by the cytogenetic findings, for inactivation of the autosomal segment in most of the analyzed cells has apparently led to effective monosomy of the homologous segment. In the other cases with abnormal phenotypes, however, no such logical explanations are apparent. The frequency of primary amenorrhea in this series is high and may, as pointed out by Sarto et al. [4], be directly related to the X-autosome translocation. In view of the apparent similarity of the cytogenetic features in the normal and amenorrheic females, however, explanations such as effective hemizygosity of a recessive gene in the active X chromosome or disruption of chromosomal continuity [4] seem highly improbable. It is, of course, possible that the amenorrhea could be due to a minor chromosomal imbalance which was not detected. The unbalanced cases show even greater cytogenetic and phenotypic variability (table 2). The karyotypic heterogeneity is further complicated by the observed variation in the late replication or inactivation patterns of X chromosomes and affected autosomes. Thus the inactivation may be completely random [23] or affect selectively the normal X [15], the X portion of the translocation X [3], the entire translocation X including the autosomal segment (R. E. Tipton, personal communication), or almost the entire translocation X (present case). To make assessment of the phenotype-genotype relationship even more difficult, many of the unbalanced cases are further characterized by mosaicism [11, 20, 21], presence of an extra chromosome [5], loss of a centric fragment [22, 23], or even a ring formation [24]. In some cases the phenotypic changes can be explained on the basis of the observed cytogenetic abnormalities, especially when autosomal late replication is taken to represent genetic inactivation. For example, the patient presented here represents a good example of autosomal inactivation with functional elimination of an apparent trisomic state. In a similar way, the patient of Crandall et al. [16, 17] may represent a partial trisomy of chromosome 13, and the patient of Summitt et al. [9], an effective monosomy of chromosome 21. In many cases of X-autosome translocation where autosomal inactivation has occurred, however, this autosomal inactivation has not "corrected" an unbalanced karyotype by some kind of dosage compensation but has led to an unbalanced hemizygous state, as in the case of Summitt et al. [9] and in the balanced case of Thelen et al. [14]. Furthermore, in some cases the inactivation has not spread over the autosomal segment, resulting in a partial trisomy of a chromosomal segment [3, 15]. Understanding the selectivity of chromosomal inactivation in X-autosome translocations becomes difficult if the basic purpose of inactivation is to maintain dosage compensation and a balance of the X chromosomal complement in the male and female [33]. In the normal female this is accomplished by a random inactivation of the paternal and maternal X chromosome, in X polysomies by inactivation of all but one X, and in simple structural anomalies by the selective inactivation of the structurally abnormal X chromosome. However, only the balanced X-autosome translocations show some consistency in the X chromosome that is inactivated and the tendency to restore a balanced karyotype; that is, selective inactivation of the normal X was observed in 11 of 14 cases. In the unbalanced cases the in-
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activation patterns appear more or less coincidental, both from the point of view of selection of which X chromosomes are inactivated and the extent of adjacent autosomal involvement. Even when autosomal material becomes inactivated, the primary event appears to be the initiation of the inactivation process in the X chromosome, with subsequent autosomal involvement, possibly by a "spreading" mechanism [34]. The mechanism of initiation is not known, although several hypotheses have been presented (see [35, 36]). Several authors have suggested that the determining factor in the selection of the X to be inactivated would be the direction of the exchange of chromosomal material in the translocation process [3, 17]. Thus in cases where X material would be translocated to an autosome, the normal X would be preferentially inactivated, and in cases of translocation from autosome to X, inactivation would be variable in nature. This has been ruled out definitively, as evidenced by the presence of different types of inactivation patterns both among the X-to-autosome and autosome-to-X translocations and by the kindreds with differential inactivation of X in different family members. The latter kindreds also rule out the possibility that the choice of inactivation would be an inherent property of the X chromosome per se. The most plausible explanation for the variety of inactivation patterns would be the existence of a control mechanism whose purpose is to maintain a balanced genetic constitution in the cells. Such a mechanism could operate before or during the initiation phase, leading to predetermined selection of the X chromosome to be inactivated or following random inactivation by selection against the more genetically unbalanced cells [28]. In both possibilities the risk or presence of autosomal inactivation would play a crucial role in the final outcome. It is apparent from evidence derived from both human and mouse translocations [37] that autosomal segments tend to become inactivated when in close physical contact with inactive X material. Thus the preferential inactivation of the normal X in balanced translocations appears rational, since if the translocated X were inactivated the adjacent autosomal segments would be at risk of becoming inactivated, as actually happened in the case of Thelen et al. [14]. The inactivation pattern in the unbalanced cases is again more difficult to explain. One might assume that the opposite choice in the pattern of X inactivation would lead to even more severe phenotypic consequences. This appears to be true in certain cases, such as in the present patient. In other patients (e.g., [15]) inactivation of the other X chromosome would appear to have resulted in a more balanced state. Thus the complexity of chromosomal rearrangements, the variation in the initial selection of the inactive X,-and the unpredictability of the presence and extent of autosomal involvement in unbalanced X-autosome translocations make it impossible to explain all cases by a simple unified hypothesis. Significantly, mosaicism of the inactivation pattern has been observed in many cases, which may indicate randomness in the initial X inactivation with later selection of the most viable cell line.
INACTIVATION IN X-9 TRANSLOCATION
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SUMMARY
A kindred with an X-autosome translocation and differential inactivation of the X chromosome is described. The phenotypically normal mother has a reciprocal translocation [46,X,rcp(X;9)(qll;q32)] while the daughter's karyotype is unbalanced [46,X,-X,+der(9) ,rcp(X;9) (ql 1 ;q32)mat], indicating adjacent-two type of segregation in the mother. In the mother's cells the normal X is late replicating, while in the daughter's cells almost the entire der(9) is late replicating, indicating the presence of autosomal inactivation. The daughter's abnormal phenotype can be explained by her sex chromosomal complement and the absence of effective trisomy 9. At this stage there is no simple explanation to account for all types of inactivation patterns encountered in the 14 balanced and 15 unbalanced cases of Xautosome translocations reported to date. Selection of X inactivation is not an inherent characteristic of the X chromosome per se, and it is not dependent on the direction of chromosomal exchange, as was suggested previously. Correlation of the phenotypic and cytogenetic features of these patients suggests a pattern of X and autosomal inactivation consistent with the least amount of genotypic and phenotypic imbalance in most cases. The data are most consistent with random X inactivation followed by selection of the most viable cell line. ACKNOWLEDGMENTS We are indebted to Mrs. D. Thornbury and Mrs. Florence DeNice for valuable assistance in evaluating this family. 1. 2. 3.
4.
5.
6. 7. 8.
9.
REFERENCES MANN JD, VALDMANIS A, CAPPS SC, PUITE RH: A case of primary amenorrhea with a translocation involving chromosomes of groups B and C. Am J Hum Genet 17:377-383, 1965 MANN JD, HIGGINS J: A case of primary amenorrhea associated with X-autosomal translocation [46,X,t(Xq-;5q+)]. Am J Hum Genet 26:416, 1974 COHEN MM, LIN C-C, SYBERT V, ORECCHIo EJ: Two human X-autosome translocations identified by autoradiography and fluorescence. Am J Hum Genet 24:583597, 1972 SARTO GE, THERMAN E, PATAU K: X inactivation in man: a woman with t(Xq-; 12q+). Am J Hum Genet 25:262-270, 1973 ALLDERDICE PW, MILLER OJ, KLINGER HP, PALLISTER PD, OPITZ JM: Demonstration of a spreading effect in an X-autosome translocation by combined autoradiographic and quinacrine-fluorescence studies. Excerpta Medica Int Congr Ser 233:1415, 1971 SUJANSKY E, Hsu LY, LucAs M, HIRSCHHORN K: Nature of X-autosome translocation and choice of X-inactivation (abstr.). Pediatr Res 7:343, 1973 LuCAS M, SMITHIES A: Banding patterns and autoradiographic studies of cells with an X-autosome translocation. Ann Hum Genet 37:9-12, 1973 GERALD P, BRUNS G, MONEDJIKOVA V: Localization of genes on the X chromosome by somatic cell hybridization (abstr.). Pediatr Res 7:344, 1973 SUMMITT RL, MARTENS PR, WILROY RS: X-autosome translocation in normal mother and effectively 21-monosomic daughter. J Pediatr 84:539-546, 1974
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10. KALLIO H: Cytogenetic and clinical study
on 100
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Obstet Gynecol Scand [Suppl] 24:1-79, 1973 11. BUCKTON KE, JACOBS PA, RAE LA, NEWTON MS, SANGER R: An inherited X-autosome translocation in man. Ann Hum Genet 35:171-178, 1971 12. DE LA CHAPELLE A, SCHRODER J: Apparently non-reciprocal balanced human (3q-; Xq+) translocation: late replication of structurally normal X. Chromosomes Today 4:261-265, 1973 13. THORBURN MJ, MARTIN PA, PATHAK UN: Possible X/autosomal translocation in a girl with gonadal dysgenesis. J Med Genet 7:402-406, 1970 14. THELEN TH, ABRAMS DJ, FISCH RO: Multiple abnormalities due to possible genetic
inactivation in an X/autosome translocation. Am J Hum Genet 23:410-418, 1971 15. MIKKELSEN M, DAHL G: Unbalanced X/autosomal translocation with inactivation of the normal X chromosome. Cytogenet Cell Genet 12 :357-366, 1973 16. CRANDALL BF, CARREL RE, MULLER H: Trisomy 13 syndrome resulting from a 13-X translocation [46,X,t(Xql3q)] (abstr.). Am J Hum Genet 25 :22A, 1973 17. CRANDALL BF, CARREL RE, HOWARD J, SCHROEDER WA, MULLER H: Trisomy 13 with a 13-X translocation. Am J Hum Genet 26:385-392, 1974 18. WIE LIE G, COENEGRACHT JM, STALDER G: A very large metacentric chromosome in a woman with symptoms of Turner's syndrome. Cytogenetics 3:427-440, 1964 19. GERMAN J: Autoradiographic studies on human chromosomes. I. A review, in Proceedings 3d International Congress of Human Genetics, edited by CROW JF, NEEL JV, Baltimore, Johns Hopkins Press, 1967, pp 123-136 20. HUGH-JONES K, WALLACE SJ, THORNBER JM, ATKIN NB: Gonadal dysgenesis with unusual abnormalities. Arch Dis Child 40:274-279, 1965 21. THORBURN MJ, MILLER CG, DoVEY P: Anomalies of development in a girl with unusual sex chromosomal mosaicism. J Med Genet 4:283-287, 1967 22. NEUHAUSER G, BACK F: X-Autosom-Translokation bei einem Kind mit multiplen Missbildungen. Humangenetik 3:300-311, 1967 23. ENGEL W, VOGEL W, REINWEIN H: Autoradiographische Untersuchungen an einer X-Autosomentranslokation beim Menschen: 45,X,1 5-,tan( 1 5qXq+) +. Cytogenetics 10:87-98, 1971 24. MUKERJEE D, BURDETTE WJ: Multiple congenital anomalies associated with a ring 3 chromosome and translocated 3/X chromosome. Nature (Lond) 212:153-155, 1966 25. ROHDE RA, CATZ B: Maternal transmission of a new group-C(6/9) chromosomal syndrome. Lancet 2:838-840, 1964 26. MOORHEAD PS, NOWELL PC, MELLMAN WJ, BATTIPS DM, HUNGERFORD DA: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613-616, 1960 2 7. SUMNER AT, EVANS HJ, BUCKLAND RA: A new technique for distinguishing between human chromosomes. Nature [New Biol] 232 :31-32, 1971 28. HAMERTON JL: Human Cytogenetics. Vol 1: General Cytogenetics. New York, Academic Press, 1971 29. FERGUSON-SMITH MA: Karyotype-phenotype correlations in gonadal dysgenesis and their bearing on the pathogenesis of malformations. J Med Genet 2 :142-155, 1965 30. GRUMBACH MM, VAN WYK JJ: Disorders of sex differentiation, in Textbook of Endocrinology, 5th ed, edited by WILLIAMS RH, Philadelphia, Saunders, 1974, pp 423-501 31. FEINGOLD M, ATKINS L: A case of trisomy 9. J Med Genet 10:184-187, 1973 32. RETHORE MO, HOEHN H, ROTT HD, COUTURIER J, DUTRILLAUX B, LEJEUNE J: Analyse de la trisomie 9p par denaturation menagee. A propos d'un nouveau cas. Humangenetik 18:129-138, 1973 33. LYON MF: X-chromosome inactivation in mammals. Adv Teratol 1:25-54, 1966
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34. RUSSEL LB: Mammalian X-chromosome action: inactivation limited in spread and in region of origin. Science 140:976-978, 1963 35. LYON MF: Possible mechanisms of X chromosome inactivation. Nature [New Biol] 232 :229-232, 1971 36. BROWN SW, CHANDRA HS: Inactivation of the mammalian X chromosome. Proc Natl Acad Sci USA 70:195-199, 1973 37. RusSEL LB, MONTGOMERY CS: Comparative studies on X-autosome translocations in the mouse. II. Inactivation of autosomal loci, segregation, and the mapping of autosomal breakpoints in five T(X; 1)'s. Genetics 64:281-312, 1970
