American Journal of Medical Genetics 71:426–429 (1997) Brief Clinical Report A Case With Mosaic Di-, Tetra-, and Octacentric Ring Y Chromosomes Octavian Henegariu,1* Ora Hirsch Pescovitz,2 Gail H. Vance,1 Jennifer Verbrugge,1 and Nyla A. Heerema1 1 2 Department of Medical and Molecular Genetics, Indiana University, Indianapolis Department of Pediatrics and Physiology, Indiana University, Indianapolis A newborn female infant presented with abnormalities of the external genitalia including a 3 × 1 cm phallic structure, a perineal urethral opening, bifid scrotum, and a single urogenital opening. Peripheral blood karyotype was 45,X/46,X,+r(Y), however, there were no signs of Ullrich-Turner syndrome. High resolution G-banding as well as C-and Q-banding did not demonstrate any specific banding pattern or presence of heterochromatin on the ring. However, it was noticed that some of the rings were larger than others. FISH with a probe for Yq12 was negative in all metaphases studied. A Y-specific paint probe hybridized to the entire ring chromosome, confirming its origin. PCR analysis showed the presence of the SRY locus and of proximal Yq locus DYS271. Triple color FISH with probes for the Y centromere, DYZ5 (Yp), and all human telomeres showed the existence of different types of rings, some dicentric, some tetracentric, and some probably octacentric. Owing to the increased risk for gonadoblastoma, a surgical removal of the gonads was performed. Am. J. Med. Genet. 71:426– 429, 1997. © 1997 Wiley-Liss, Inc. KEY WORDS: dicentric; tetracentric; octacentric; mosaic ring Y; single urogenital opening *Correspondence to: Dr. Octavian Henegariu, Department of Medical and Molecular Genetics, Indiana University School of Medicine, Medical Research and Library Building, 975 West Walnut Street, Indianapolis, IN 46202-5251. E-mail: firstname.lastname@example.org Received 6 September 1996; Accepted 3 March 1997 © 1997 Wiley-Liss, Inc. INTRODUCTION The Y chromosome is essential in inducing maleness in mammals and other species due to the presence of the SRY gene [Goodfellow and Lovell-Badge, 1993]. Few genes have been identified or postulated on the Y; among these, there are Yq gene(s) involved in spermatogenesis [Kobayashi et al., 1994; Page, 1994] and a gene responsible for gonadoblastoma in proximal Yp or Yq [Tsuchiya et al., 1995], especially in patients with Ullrich-Turner phenotype and streak gonads [Barbosa et al., 1995]. Structural abnormalities of the Y chromosome include deletions, translocations, dicentric chromosomes, isochromosomes, and rings [Hsu, 1994]. Owing to the ‘‘instability’’ of ring and dicentric chromosomes in mitosis, the karyotype of many reported cases included a 45,X cell line. The phenotype of the patients usually depends on the presence or absence of the SRY gene and the proportion of the cells carrying the ring Y in different tissues, especially in the gonads, where more cells carrying the SRY gene result in more testicular structure and increased virilization due to higher production of testosterone by the Leydig cells. We report on a female infant with genital abnormalities and a mosaic peripheral blood karyotype 45,X/ 46,X,+r. The ring was presumed to be of Y origin because of the significant degree of virilization at the physical examination. As the size of the ring chromosome was fairly large in some of the metaphases, FISH and PCR analyses were performed to define the origin and the structure of the ring and for karyotype/ phenotype correlations. MATERIALS AND METHODS Patient Report A term black female infant was noted to have ambiguous genitalia at birth. Prenatal history and labor were unremarkable. Physical findings were entirely normal with the exception of the external genitalia, Octacentric Ring Y which consisted of a 3 × 1 cm phallic structure, a perineal urethral opening, a bifid scrotum with rugated folds, a common urogenital opening, a dimple posterior to the urethral folds, and a more posterior anal opening. A genito-urogram showed the presence of a short urogenital sinus, a well-developed vagina with a normal cervical impression, and an elongated (female-like) urethra. The significant virilization was not accompanied by any detectable hormonal or enzymatic abnormalities (21-hydroxylase, 11-hydroxylase, or 3-betahydroxysteroid dehydrogenase deficiency), and an hCG stimulation test was performed to investigate the presence of testicular material. The testosterone level increased from 80 ng/dl to 287 ng/dl following hCG stimulation, leading to the suspicion of an intra-abdominal testis. An exploratory laparotomy and pathologic examination showed a dysgenetic testis with marked germ cell hypoplasia, embryonal tubular remnant and adrenal cortical rests, a vas deferens, and a fallopian tube on the right side. On the left side, a fallopian tube was found next to a streak ovary. Both gonads were removed. No indication of gonadoblastoma was mentioned in the pathology report. The uterus appeared normal and a complex vaginoplasty was performed. Cytogenetic Studies Metaphases were prepared from PHA-stimulated peripheral blood lymphocytes according to usual procedures. GTG-banded metaphases were analyzed at the 550–600 band level. CBG and QFQ banding were performed according to standard laboratory procedures. FISH Analysis Commercial probes (Vysis, Downers Grove, IL) for dual color detection of the X centromere (DXZ1) and of Yq12 (DYZ1 locus), as well as painting probes for the X chromosome (Vysis) and the Y chromosome (Gibco, BRL, Gaithersburg, MD) were used to clarify the origin of the ring, following the protocols provided by the vendors. Plasmid probes Y-190 (DYZ5 locus, Yp11.1-11.2) and pDP97 (DYZ3 locus, Y centromere) were purchased from ATCC (Rockville, MD) and were used to detect the respective loci. Total human telomere probe was generated by PCR, by using the telomere repeat oligonucleotides (TTAGGG)5 and (CCCTAA)5 in the presence of Digoxigenin (Dig)-11-dUTP without a template [Ijdo et al., 1991]. Plasmid DNA was labeled by nick translation using dinitrophenyl (DNP)-11-dUTP (pY-190 probe) and Biotin (Bio)-11-dUTP (pDP97 probe); 5–10 ng labeled probes were used for each hybridization, with ×500 Cot1 DNA (Gibco BRL, Gaithersburg, MD). Probes were denatured, hybridized overnight, and detected with avidin conjugated with AMCA (BoehringerMannheim Biochemicals, Indianapolis, IN), or with Cy3-conjugated antibodies (Accurate Chemical & Scientific Co., Westbury, NY) against digoxigenin or with FITC-conjugated antibodies (Sigma Chemical Co., St. Louis, MO) against DNP. Analysis of FISH images was done using an imaging system developed by Oncor, Inc. (Gaithersburg, MD). Acquired gray images were pseu- 427 docolored and superimposed by the computer to give the final image. PCR Analysis Genomic DNA from blood cells was prepared using SDS and proteinase K, following standard laboratory procedures. Primers sY14 (SRY gene locus, 472 bp) and sY81 (locus DYS271 on Yq, 209 bp) [Vollrath et al., 1992] (see Fig. 2e) were used to test for the presence of the corresponding loci on the Y chromosome; 100–150 ng DNA (patient or control) were used in each reaction in a 9600 Thermocycler (Perkin Elmer Cetus, Norwalk, CT), denaturing for 40 sec at 94°C, annealing for 30 sec at 54°C, and extending for 90 sec at 68°C (35 cycles). Six to seven microliters of PCR products were separated on a 1.5% agarose gel and visualized after staining with ethidium bromide. RESULTS AND DISCUSSION Karyotypic analysis of the peripheral blood of the infant indicated the presence of 45,X/46,X,+r mosaicism. There was no specific banding pattern on the ring by standard GTG banding as well as CBG and QFQ banding and its origin remained unclear (Fig. 1). FISH with a probe for the X centromere (DXZ1) and a probe for the Yq12 (DYZ1 locus) showed no hybridization to the ring. A commercial X paint probe hybridized only to the X chromosome whereas a Y paint probe (Gibco BRL, Gaithersburg, MD) uniformly hybridized to the ring, indicating that the marker was of Y origin. The SRY and DYS271 loci were shown to be present on the ring by PCR amplification (Fig. 1). Preservation of the SRY locus and the mosaic genotype with a high proportion of 45,X cells in this patient could explain the abnormalities of the external genitalia as well as the predominance of the female phenotype. Preservation of the DYS271 and lack of hybridization of the DYZ1 locus indicated a Yq11 breakpoint, whereas amplification of the SRY gene locus indicated that most of Yp was present. Preservation of the Y pericentromeric region, where the GBY (gonadoblastoma) gene has been postulated, in female subjects with abnormalities of the genitalia, is known to increase the risk for developing this tumor. By triple color FISH with probes for the Ycentromere (pDP97), a proximal Yp locus (pY-190) and a total human telomere probe, more than two cell lines were identified in this patient. Some cells had one and some had two rings and the rings were of different sizes. Over 90% of the cells with a Y chromosome had either one or rarely two dicentric Y chromosomes. Therefore, the more precise karyotype should be 45,X/ 46,X,dic r(Y). Two pairs of (red-orange) telomere signals were always seen on these chromosomes, but at least one pair was always located within the chromosome mass, indicating that the dicentric had a ring and not a linear structure (Fig. 1). No linear dicentric chromosomes were seen. Less than 10% of all Y chromosomes were larger, with four hybridization signals for each of the probes used (tetracentric rings). Two cells carried two equally sized tetracentric rings each. Finally, a few cells had an even larger ring, with more Figs. 1 and 2. Octacentric Ring Y than four signals for each of the probes used and one metaphase showed two such large rings (Fig. 1). The number of signals on the larger rings was difficult to count with accuracy, but each probe appeared to produce 7–8 signals (octacentric rings, Figs. 1 and 2). The mosaicism in this patient is explained by the presence of the ring chromosome, as ring chromosomes are frequently unstable and may be lost in mitosis [Fang et al., 1995; Pezzolo et al., 1993]. Although rare, dicentric, tetracentric, or octacentric linear chromosomes and ring chromosomes have been reported previously [Daniel et al., 1994; HutchinsonCole et al., 1986; Levine et al., 1995; McGinniss et al. 1992; Vig et al., 1990], and a number of mechanisms have been proposed to explain their formation [Conte et al., 1995; Jager and Philippsen, 1989; McGinniss et al., 1992]. As no monocentric ring Y or linear dicentric Y was found in this patient, it is likely that the abnormal Y chromosome originated from a dicentric ring in the zygote. It is possible that during spermatogenesis a break occurred in Yq, and the break was ‘‘healed’’ by ring formation with the preservation of an intact telomeric sequence of Yp. Subsequently, sister chromatid exchange in the ring lead to a dicentric ring formation, explaining the two telomeric sequences always seen in the rings. Owing to ring instability, loss of the ring Y resulted in a 45, X cell line. The presence of two dicentric rings in the same cell can be explained by mitotic nondisjunction. The same mechanism of sister chromatid exchange within the dicentric ring might have led to a tetracentric ring and in the tetracentric ring to an octacentric ring. Nondisjunction again would result in two tetracentric or two octacentric rings in the same cells. REFERENCES Barbosa AS, Ferraz-Costa TE, Semer M, Liberman B, Moreira-Filho CA (1995): XY gonadal dysgenesis and gonadoblastoma: A study in two sisters with a cryptic deletion of the Y chromosome involving the SRY gene. Hum Genet 95:63–66. 429 Conte RA, Luke S, Verma RS (1995): Characterization of a ring chromosome 21 by FISH-technique. Clin Genet 48:188–191 Daniel A, Malafiej P, Preece K, Chia N, Nelson J, Smith M (1994): Identification of marker chromosomes in thirteen patients using FISH probing [Review]. Am J Med Genet 53:8–18. Fang YY, Eyre HJ, Bohlander SK, Estop A, McPherson E, Trager T, Riess O, Callen DF (1995): Mechanisms of small ring formation suggested by the molecular characterization of two small accessory ring chromosomes derived from chromosome 4. Am J Hum Genet 57:1137–1142. Goodfellow PN, Lovell-Badge R (1993): SRY and sex determination in mammals [Review]. Annu Rev Genet 27:71–92. Hsu LY (1994): Phenotype/karyotype correlations of Y chromosome aneuploidy with emphasis on structural aberrations in postnatally diagnosed cases. [Review]. Am J Med Genet 53:108–140. Hutchinson-Cole H, Aleck KA, Davis JR, Veomett IC, Hauck L (1986): Mosaicism for ring and isopseudodicentric chromosome 13. Clin Genet 29:88–91. Ijdo JW, Wells RA, Baldini A, Reeders ST (1991): Improved telomere detection using a telomere repeat probe (TTAGGG):n generated by PCR. Nucl Ac Res 19:4780. Jager D, Philippsen P (1989): Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion. EMBO 8:247–254. Kobayashi K, Mizuno K, Hida A, Komaki R, Tomita K, Matsushita I, Namiki M, Iwamoto T, Tamura S, Minowada S (1994): PCR analysis of the Y chromosome long arm in azoospermic patients: Evidence for a second locus required for spermatogenesis. Hum Mol Genet 3:1965– 1967. Levine JG, Rossi MR, Gadi JG, Papenhausen PR (1995): Dicentric, tetracentric and octocentric ring chromosome 15 in AML-M6. Am J Hum Genet 57:4:A70. McGinniss MJ, Kazazian HH, Jr., Stetten G, Petersen MB, Boman H, Engel E, Greenberg F, Hertz JM, Johnson A, Laca Z, Mikkelsen M, Patil SR, Schinzel AA, Tranebjaerg L, Antonarakis SE (1992): Mechanisms of ring chromosome formation in 11 cases of human ring chromosome 21. Am J Hum Genet 50:15–28. Page DC (1994): Y chromosome sequences in Turner’s syndrome and risk of gonadoblastoma or virilisation [letter; comment]. Lancet 343:240. Pezzolo A, Gimelli G, Cohen A, Lavaggetto A, Romano C, Fogu G, Zuffardi O (1993): Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability. Hum Genet 92:23–27. Tsuchiya K, Reijo R, Page DC, Disteche CM (1995): Gonadoblastoma: Molecular definition of the susceptibility region on the Y chromosome. Am J Hum Genet 57:1400–1407. Vollrath D, Foote S, Hilton A, Brown LG, Beer-Romero P, Bogan JS, Page DC (1992): The human Y chromosome: A 43-interval map based on naturally occurring deletions. Science 258:52–59. Vig BK, Schroeter D, Paweletz N (1990): Centromere separation. Early replication of repetitive DNA associated with inactive centromeres. Cancer Genet Cytogenet 50:57–65. < Fig. 1. sY14 and sY81 show PCR results using the primers for the SRY gene (sY14, 472bp) and DYS271 (sY81, 209 bp) loci, respectively. Their approximate position on the Y is shown in Figure 2e. Both loci were positive in the patient. P, F, and M indicate patient, female and male DNA, respectively. ‘‘+’’ and ‘‘-’’ indicate the result of the PCR test. MR is the 1 kb ladder (Gibco, Grand Island, NY). G-bands and C-bands: Partial metaphase with a ring chromosome (red arrows). The small bands seen in the C-banded ring are probably residual bands from the previous G-banding. The numbers indicate other human chromosomes, for size and banding comparisons. Dicentric rings a and b. Triple color FISH detection: pDP97 was detected blue, Y-190 green and the telomere probe orange or red. Both dicentrics show telomere signals located within the chromosomal structure, indicating ring formation. Some scattered red-orange signals indicate telomeres of other chromosomes (not visible without counterstain). Multicentric rings show triple color FISH detection on a metaphase with two large ring Y chromosomes (white arrows). Each probe is present in seven-eight copies, indicating a probable octacentric ring formation. Other chromosomes are also partially visible (dark blue DAPI counterstain and telomere hybridization). Fig. 2. Triple color FISH on a large ring Y chromosome (a). Red-orange signals show hybridization with the telomere probe (b), light-blue signals the hybridization with the Y centromere probe and green signals the hybridization with pY-190 probe (d). Gray images for (b), (c), and (d) were pseudocolored and superposed by the computer, giving (a). The relative position of the PCR and FISH probes on the Y chromosome is shown in (e). The colored dots indicate the colors assigned to each of the three probes used for FISH.