Proc. Nati. Acad. Sci. USA
Vol. 83, pp. 7841-7844, October 1986
Small deletions of the short arm of the Y chromosome in
(sex determination/chromosomal deletion)
CHRISTINE M. DISTECHE*, MIRIAM CASANOVAt, HOWARD SAAL*t, CYNTHIA FRIEDMAN*, VIRGINIA SYBERTt,
JOHN GRAHAMt§, HORACE THULINE0, DAVID C. PAGE'1, AND MARC FELLOUSt
*Department of Pathology, University of Washington, Seattle, WA 98195; tUniversite d'Immunogenetique Humaine, Institut Pasteur, 28 rue du Dr. Roux,
75105 Paris, France; tDepartments of Pediatrics and Medicine, University of Washington, and Children's Orthopedic Hospital, Seattle, WA 98195;
%Genetics Program, State of Washington, Department of Social and Health Services, Seattle, WA 98155; and IlWhitehead Institute,
Nine Cambridge Center, Cambridge, MA 02142
Communicated by Arno G. Motulsky, July 1, 1986
provide a means to study the location of genes responsible for
sex determination. Recently, a type of sex reversal in humans,
the 46,XX male, was shown to result in some cases from
translocation ofY chromosome material to the X chromosome.
In the present report, another type of sex reversal, the 46,XY
female, is shown to result, in two cases, from small deletions of
the short arm of the Y chromosome. Prometaphase chromo-
some analysis showed a 46,X,Yp- karyotype. Several Y
chromosome-specific DNA probes were found to be deleted in
the two female patients. DNA analysis showed that the two
deletions were different but included a common overlapping
region likely to be essential for male determination.
Structural anomalies of the sex chromosomes
Genes located on the Y chromosome play an essential role in
human male sexual development and the presence of a Y
chromosome usually correlates with testis development.
However, there are males with an apparently normal 46,XX
karyotype and females with an apparently normal 46,XY
karyotype. These two conditions may have several causes,
including gene mutations and small chromosomal abnormal-
ities. For example, some 46,XX males appear to have a small
translocation of Y short arm material to the X chromosome
(1-4), which is evidence for the X-Y interchange hypothesis
(5). This hypothesis stated that at meiosis in the father,
exchanges between the pairing regions of the X and Y
chromosomes could produce an X chromosome carrying Y
chromosome material. DNA hybridization studies of 46,XX
males map the male-determining function of the Y chromo-
some to a small region of the short arm (4).
Females with a 46,XY karyotype have gonadal dysgenesis
and many have features of Turner syndrome, a condition
often associated with a45,X chromosomal constitution. Most
46,XY females with the Turner phenotype are mosaic, with
a 45,X cell line in addition to the 46,XY cell line. Two 46,XY
patients with features ofTurner syndrome and no evidence of
mosaicism were shown to have a deletion ofthe short arm of
the Y chromosome (6, 7). This and other more complex
chromosomal rearrangements confirm that functions essen-
tial to normal male differentiation are found on the short arm
of the Y chromosome. In addition, presence of the euchro-
matic portion of the long arm of the Y chromosome is
important for normal male meiosis (8, 9).
This report describes two female patients with features of
Turner syndrome and a 46,XY karyotype (by metaphase
analysis). Evaluation of these individuals by prometaphase
analysis and with seven DNA probes for regions on the short
and long arms ofthe Y chromosome revealed the presence of
different, but overlapping, small deletions ofthe short arm of
the Y chromosome.
MATERIALS AND METHODS
Patient 1 has some Turner stigmata. She is of normal height
at 4 years of age (75th percentile). At birth she was noted to
have lymphedema, especially of the feet. Her external
genitalia are normal. Her gonads, which were removed at 17
months, were streaks that consisted of dense ovarian stroma
with no primordial follicles or testicular tissue. Patient 1 and
her mother were both Xga-positive.
Patient 2 has several features of Turner syndrome. At age
15, she has a short neck, wide-spaced nipples, and very small
breasts but is of normal height. She had congenital
lymphedema. She had primary amenorrhea and developed
bilateral gonadoblastoma. Histological examination of the
gonads showed gonadoblastoma and streaks with no primor-
dial follicles. Patient 2 and her mother were both Xga-
positive; her father was Xga-negative.
Cytogenetic analysis was performed on peripheral blood
samples from patients 1 and 2 and on fibroblast cultures from
skin and gonadal biopsies of patient 1 and from gonadal
biopsies of patient 2. Prometaphase cells were obtained by
the ethidium bromide method of Ikeuchi (10). The chromo-
somes were stained by G-banding, R-banding, C-banding,
DNA was prepared from blood samples and fibroblast
cultures of both patients, their parents, and normal control
male and female individuals. The DNAs were digested to
completion with the restriction endonuclease Taq I (for probe
pDP34) or EcoRI (for probes 50f2, 52d, 47b, 118, pl2f2, and
pl2f3). The DNA fragments were separated by agarose gel
(0.7%) electrophoresis and transferred to membrane filters
(Nytran or GeneScreen) for Southern blot hybridization. The
probes were labeled with 32Pby nick-translation. Hybridiza-
tion and washing of the blots were as described (11). The
following probes were used. Probe pDP34 (DXYSI) detects
homologous sequences on the short arm of the Y chromo-
some and on the long arm of the X chromosome (12). Probe
50f2 (DYS7) hybridizes with DNA sequences located on the
Y chromosome (long and short arm) and on an autosome.
Probe 52d (DXYSJJ) detects sequences on the long arm ofthe
X chromosome and on the short and long arm of the Y
chromosome (2, 4, 13). Probe 47b (DXYS5) detects Y (short
arm), X, and autosomal sequences, whereas probe 118
(DYS8) is Y-specific, detecting sequences on the long and
Abbreviation: kb, kilobase(s).
§Present address: Clinical Genetics and Child Development Center,
Department of Maternal and Child Health, Dartmouth Medical
School, Hanover, NH 03756.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 83 (1986)
short arm (2, 4, 13). Two additional probes, p12f2 (DYSII)
and p12f3, detect sequences on the long arm of the Y
chromosome (13). When hybridized to Southern blots, the
probes listed above detect restriction fragments that have
been mapped to specific chromosomal locations (2, 4, 13).
The blots were washed in 75 mM NaCl/7.5 mM sodium
citrate/1% NaDodSO4 at room temperature, 50'C, and 650C
and autoradiographed for 7 days with an enhancing screen.
Two female patients with a 46,XY karyotype were reevalu-
ated by cytogenetics using prometaphase banding and by
molecular analysis using Y chromosome-specific DNA
probes. A small deletion of the short arm of the Y chromo-
some was identified in both patients, as shown in Fig. 1, in
which the Y chromosomes of patient 1 and patient 2 are
compared to a normal Y chromosome after G- and Q-
bandings. The deletions were barely detectable on metaphase
chromosomes. Although it appeared that the deletions might
be interstitial rather than terminal, it was not possible to
determine their nature with certainty orwhether they differed
in the two patients. The long arm of the Y chromosome of
both patients appeared of normal size, including a Q-bright
heterochromatic region of average size. The Y chromosome
ofthe father ofeach patient was normal. No other karyotypic
abnormalities were identified in either patient and no evi-
dence of mosaicism was found in the tissues analyzed: in
patient 1, 105 cells, 50 cells, 50 cells, and 51 cells were
examined in the blood, skin, and right gonadal and left
gonadal samples, respectively, whereas in patient 2, 120
cells, 54 cells, and 58 cells were examined in the blood and
bilateral gonadal samples, respectively.
To characterize the deletions seen in the 46,XY female
patients, seven 32P-labeled DNA probes that detect Y chro-
mosome-specific restriction fragments were hybridized to
patient and control genomic DNA blots.
Fig. 2 shows the results of the hybridization of probe
pDP34 to patient and control DNAs digested with Taq I.
Lanes 1 and 4 contain normal male and female control DNAs,
respectively. A 15-kb band characteristic ofthe male (Fig. 2,
lane 1) is the Y chromosome-specific band, whereas a
polymorphic band at 11 or 12 kb is X chromosome-specific
(14). Patient 1 (Fig. 2, lane 2) showed a hybridization pattern
characteristic of a normal male with bands at 11 and 15 kb,
indicating that theY chromosome-linked locus ofDXYSI was
not deleted in this case. Patient 2, however (Fig. 2, lane 3),
was missing the band at 15 kb, indicating that the Y
chromosome-specific DNA homologous to DXYSI had been
deleted in that patient. The father ofthe latter patient showed
patient 2 (no. 2), and from a normal male control (no. 3). The lower
row shows Y chromosomes stained by Q-banding; staining in the
upper row was by G-banding.
Examples ofY chromosomes from patient 1 (no. 1), from
DNA from a normal male (lane 1), a normal female (lane 4), patient
1 (lane 2), and patient 2 (lane 3). Five micrograms ofDNA was loaded
on each lane ofa 0.7% agarose gel. After transfer to GeneScreenPlus
filters, 32P-labeled probe pDP34 was hybridized to the blots at 47°C.
Autoradiograph of a Southern blot with Taq I digests of
normal Y and X chromosome-specific bands at 15 and 12 kb,
respectively (data not shown).
Fig. 3 shows the hybridization of 32P-labeled probes 118
(Fig. 3a), 52d (Fig. 3b), 50f2 (Fig. 3c), and 47b (Fig. 3d) to
EcoRI-digested DNAs from the patients and their parents.
With all four probes, patient 1 (lane 3) showed deletions ofa
total of seven of the male-specific bands (7-, 2.6-, and 3.2-kb
bands in Fig. 3a, 1.8-kb band in Fig. 3b, 10- and 8-kb bands
in Fig. 3c, 7.8-kb band in Fig. 3d), whereas her father (lane
2) had a normal male pattern and her mother (lane 1) had a
normal female pattern. The data summarized in Table 1
confirmed the cytogenetic data indicating that patient 1 had
a deletion ofa portion ofthe short arm ofthe Y chromosome.
Probes 118, 52d, and 50f2 showed some male-specific bands
that were not deleted in patient 1 (lane 3, 7.8- and 6-kb bands
in Fig. 3a, 10- and 0.8-kb bands in Fig. 3b, and 6-, 4.5-, and
1.7-kb bands in Fig. 3c) as compared to her father (lane 2),
indicating that these three probes recognized sequences
located in regions of the Y chromosome other than that
deleted in patient 1. These bands have been mapped to the
long arm or the pericentromeric region ofthe Y chromosome
Patient 2 did not appear to be missing Y chromosome-
specific bands with probes 118 and 52d. However, with probe
50f2, the father ofpatient 2 (Fig. 3c, lane 5) and patient 2 (Fig.
3c, lane 6) were missing the 6-kb, Y chromosome-specific
band, which represents a variant band. With probe 47b,
patient 2 (Fig. 3d, lane 6) showed a missing Y chromosome-
specific band at 7.8 kb identical to the one missing in patient
1 (Fig. 3d, lane 3) but present in normal males, including the
father of patient 2 (Fig. 3d, lane 5). Studies with two
additional probes, pl2f2 and p12f3, which are located on the
long arm of the Y chromosome (13), showed normal male
hybridization patterns on patients 1 and 2 (data not shown).
Table 1 summarizes the hybridization data, which indicate
that patients 1 and 2 have different deletions. The only DNA
fragment these patients are both missing in common is
homologous to probe 47b.
We have described the molecular analysis of two female
individuals with a Y chromosome. A common region of the
Y chromosome was found to be missing in both individuals.
This region was detected by probe 47b, which is adjacent to
probe 47c (subclone of the same cosmid). Interestingly, the
latter probe has been found to be most often present in
patients who are 46,XX males (2-4). One or both of the
deletions described here are likely to be interstitial, extending
on either side ofthe location of47b. The overlapping portion
of the deletions is likely to be essential for male determina-
tion. The Y chromosome rearrangements in our 46,XY
female patients could be more complex than simple deletions.
They could also involve inversions or translocations.
Genetics: Disteche et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
(lane 2), patient 1 (lane 3), the mother of patient 2 (lane 4), the father of patient 2 (lane 5), and patient 2 (lane 6). Ten micrograms ofDNA was
loaded on each lane of 0.7% agarose gels. After transfer to a Nytran membrane, 32P-labeled probes 118 (a), 52d (b), 50f2 (c), and 47b (d) were
hybridized to the blots. The male-specific bands are indicated by their size in kb.
Autoradiographs of Southern blots containing EcoRI-digested DNAs from the mother of patient 1 (lane 1), the father of patient 1
The hypothesis of exchange between the X and Y chro-
mosomes, put forward by Ferguson-Smith (5), predicted that
some individuals would be 46,XX males carrying Y chromo-
some material on one X chromosome, and others would be
46,XY females missing Y chromosome material. The descrip-
tion of46,XX males with a small translocation ofY chromo-
some-specific material (1-4) and the present report agree
with this prediction. The recent finding of pseudoautosomal
sequences near the telomere of the X and Y chromosomes
provides a way to explain exchanges between the two
nonhomologous sex chromosomes (15-17). Cytologically, it
appears that the region of pairing at meiosis extends into the
nonhomologous region of the sex chromosomes and it was
suggested that exchanges might occur due to abnormal delay
in the segregation of the chromosomes (18).
The two patients reported here have different but overlap-
ping deletions, which could explain that although both
patients have features of Turner syndrome, they differ in
some respects. Patient 2, but not patient 1, developed
gonadoblastoma; however, the gonads were removed at an
early age in patient 1.
Two previous cases of Yp deletions have been reported.
Rosenfeld et al. (6) described a patient who had typical
Turner phenotype with lymphedema at birth and short
stature. Her gonads were fibrous with no primordial follicles
or seminiferous tubules. Magenis et al. (7) recently reported
a Yp deletion in another patient with features of Turner
syndrome but ofnormal height, like our patients 1 and 2. That
patient developed gonadoblastoma, like our patient 2. By in
situ hybridization, deletion of a Y chromosome-specific
Hybridization data indicating that patients 1 and 2 have different deletions
For each probe, a + indicates the presence of the Y chromosome-specific band and a -indicates
the absence of that band. The bands were generated by Taq I digestion ofDNA for pDP34 and EcoRI
digestions of DNA for the other probes. The bands scored correspond to sequences detected on the
Y chromosome only and their location is indicated as Yp (short arm), Yq (long arm), or Yc
(pericentromeric region) (see Figs. 2 and 3). Some of the probes also detect sequences located on
autosomes or the X chromosome.
*Phenotype, Turner stigmata; sex, female; gonads, streak.
tPhenotype, Turner stigmata; sex, female; gonads, streak and gonadoblastoma.
tThis fragment was also absent from the father of patient 2.
Genetics: Disteche et al.
Proc. Natl. Acad. Sci. USA 83 (1986) Download full-text
sequence was demonstrated in this patient (19). Patients with
isochromosomes of the long arm of the Y chromosome or
with isodicentric Y chromosomes missing portions of the
short arm may also have stigmata of Turner syndrome and
develop gonadoblastoma (8, 9). However, interpretation of
the phenotype-genotype relationships in these patients is
more difficult due to the presence of 45,X mosaicism and of
two Yq regions in addition to the absence of Yp.
It is likely that some 46,XY female patients with no
apparent chromosomal deletion have Y chromosome dele-
tions that are smaller than can be detected on a cytogenetic
level. The study of these 46,XY female patients with addi-
tional Y chromosome-specific probes should aid our under-
standing of the role of the different Y chromosomal regions
and Y chromosome-specific genes in male differentiation.
We thank Dr. J. Weissenbach for some of the Y chromosome
probes. We thank Shawna Gandy, Laura Brown, and Janice Garrfor
their help. The study was partially supported by National Institutes
of Health Grants GM15253, GM30476, and HD20059 and by March
of Dimes Grant 5-353.
de la Chapelle, A., Tippett, P. A., Wetterstrand, G. & Page, D.
(1984) Nature (London) 307, 170-171.
Guellaen, G., Casanova, M., Bishop, C., Geldwerft, D.,
Andre, G., Fellous, M. & Weissenbach, J. (1984) Nature
(London) 307, 172-173.
Page, D. C., de la Chapelle, A. & Weissenbach, J. (1985)
Nature (London) 315, 224-226.
Vergnaud, G., Page, D. C., Simmler, M.-C., Brown, L.,
Rouyer, F., Noel, B., Botstein, D., de la Chapelle, A. &
Weissenbach, J. (1986) Am. J. Hum. Genet. 38, 109-124.
Ferguson-Smith, M. A. (1966) Lancet u", 475-476.
Rosenfeld, R. G., Luzzatti, L., Hintz, R. L., Miller, 0. J.,
Koo, G. C. & Wachtel, S. S. (1979) Am. J. Hum. Genet. 31,
Magenis, R. E., Tochen, M. L., Holahan, K. P., Carey, T.,
Allen, L. & Brown, M. G. (1984) J. Ped. 105, 916-919.
Buhler, E. M. (1980) Hum. Genet. 55, 145-175.
Munke, M., de Martinville, B. & Francke, U. (1985) Am. J.
Med. Genet. 22, 361-374.
Ikeuchi, T. (1984) Cytogenet. Cell Genet. 38, 56-61.
Disteche, C. M., Tantravahi, U., Gandy, S., Eisenhard, M.,
Adler, D. & Kunkel, L. M. (1985) Cytogenet. Cell Genet. 39,
Page, D. C., Harper, M. E., Love, J. & Botstein, D. (1984)
Nature (London) 311, 119-123.
Bishop, C., Guellaen, G., Geldwerth, D., Fellous, M. &
Weissenbach, J. (1984) J. Mol. Biol. 173, 403-417.
Page, D., de Martinville, B., Barker, D., Wyman, A., White,
R., Francke, U. & Botstein, D. (1982) Proc. Natl. Acad. Sci.
USA 79, 5352-5356.
Cooke, H. J., Brown, W. R. A. & Rappold, G. A. (1985)
Nature (London) 317, 687-692.
Simmler, M.-C., Rouyer, F., Vergnaud, G., Nystrom-Lahti,
M., Ngo, K. Y., de la Chapelle, A. & Weissenbach, J. (1985)
Nature (London) 317, 692-697.
Buckle, V., Mondello, C., Darling,
Goodfellow, P. N. (1985) Nature (London) 317, 739-741.
Chandley, A. C., Goetz, P., Hargreave, T. B., Joseph, A. M.
& Speed, R. M. (1984) Cytogenet. Cell Genet. 38, 241-247.
Magenis, R. E., Brown, M. G., Donlon, T., Olson, S. B.,
Sheehy, R. &Tomar, D. (1985) in The YChromosome, PartA:
Basic Characteristics of the Y Chromosome, ed. Sandberg,
A. A. (Liss, New York), pp. 537-574.
I. W. &
Genetics: Disteche et al.