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A case with mosaic di- tetra- and octacentric ring Y chromosomes

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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
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[81]/46,X,+r(Y)[19], 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:
Received 6 September 1996; Accepted 3 March 1997
© 1997 Wiley-Liss, Inc.
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[81]/
46,X,+r[19]. 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.
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)
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-
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.
Karyotypic analysis of the peripheral blood of the
infant indicated the presence of 45,X[81]/46,X,+r[19]
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
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O (1993): Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is
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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.
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mosaic, ring, tetra, case, chromosome, octacentric
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