Clin Genet 1999: 55: 356–361

Printed in Ireland. All rights reser6ed

Short Report

FISH and PCR analysis of the presence of

Y-chromosome sequences in a patient with
Xq-isochromosome and testicular tissue
Álvarez-Nava F, Martı́nez MC, González S, Soto M, Borjas L, Rojas Francisco Álvarez-Nava, Marı́a
A. FISH and PCR analysis of the presence of Y-chromosome se- C Martı́nez, Sandra González,
quences in a patient with Xq-isochromosome and testicular tissue. Marisol Soto, Lisbeth Borjas
Clin Genet 1999: 55: 356 – 361. © Munksgaard, 1999 and Alicia Rojas
Unidad de Genética Médica, Facultad de
Mixed gonadal dysgenesis includes a heterogeneous group of different Medicina, Universidad del Zulia, Maracaibo,
chromosomal, gonadal, and phenotypic abnormalities, characterized by Venezuela
the presence of a testis on one side and streak or an absent gonad on
the other, persistence of müllerian duct structures and/or wolffian Key words: FISH – PCR – SRY –
derivatives, and a variable degree of genital ambiguity. Here, we de- testicular development –
scribe a patient with virilized external genitalia and phenotypic features Xq-isochromosome
of Turner syndrome, whose blood karyotype was 45,X/46,X,i(Xq). The
presence of a unilateral dysgenetic testis was confirmed by histopathol- Corresponding author: Francisco Álvarez-
ogy. Using fluorescence in situ hybridization (FISH) and polymerase Nava, Unidad de Genética Médica, Univer-
chain reaction (PCR)-based analysis to detect Y-specific sequences, Y- sidad del Zulia, Hospital Universitario de
chromosome material was not detected. To date, this is the first case Maracaibo, Ave. 23, ZIP 4001, Maracaibo,
Estado Zulia, PO Box 15374, Venezuela.
reported of Xq-isochromosome associated with the presence of testicu-
Fax: +58-61-519496; e-mail:
lar tissue.
f.marfan@iamnet.com

Received 27 November 1998, revised and

accepted for publication 14 January 1999

The term ‘46,XY gonadal dysgenesis’ has been
used to describe various conditions of abnormal
gonadal differentiation. ‘46,XY complete gonadal
dysgenesis’ is defined by the absence of the testes,
female external genitalia, normal müllerian struc-
tures, and streak gonads. Subjects with 46,XY
partial gonadal dysgenesis have incomplete testicu-
lar development, with the gonads presenting with a
broad range in the degree of testicular develop-
ment, and subjects usually have ambiguous geni-
talia (1). It is important to emphasize that,
although this condition is common in 45,X/46,XY
mosaicism, the gonadal findings are not specific for
this karyotype (2). In the group of patients with
the 45,X/46,XY karyotype, the phenotypic spec-
trum varies from normal women, women with clas-
sic signs of Turner syndrome with bilateral streak
gonads, to normal phenotypic men with bilateral
atrophic scrotal testes, through different degrees of
genital ambiguity, with unilateral abdominal streak
gonad and contralateral abdominal or inguino-
scrotal testis (3, 4). There is evidence that partial
(mixed) gonadal dysgenesis is a heterogeneous syn-
356
drome, with varying phenotypic, gonadal, and cy-
togenetic abnormalities, and it has been associated
with a 45,X karyotype (5), a 46,X,+ marker kary-
otype (6), among others (7–9).
In this context, here we report the clinical pre-
sentation, histopathologic findings, and molecular
analysis of 1 patient with mixed gonadal dysgenesis
and a 45,X/46,X,i(Xq) karyotype.
Clinical report
The patient was first seen at 13 years of age be-
cause of short stature, absence of menses, and
signs of external virilization (Fig. 1). At birth, the
patient had clitoromegaly and a tumor in the right
inguinal region. The proband was the first child of
healthy, unrelated parents and has two older sisters
and one brother. The pregnancy was uneventful.
She was born at full term after an uncomplicated
pregnancy. The family history was otherwise non-
contributory. She had features of Turner syn-
drome, including low posterior hairline, low-set
ears with posterior rotation, increased internipple

Fig. 1. External genitalia of the patient.
distance, cubitus valgus, short fourth metacarpals,
and short stature (136 cm, less than the third
percentile). In addition, clitoromegaly (phallus was
4 cm), generalized hyperpigmentation of the geni-
tal skin, no labial fusion, and normal female ure-
Fig. 2. Histopathology of the right gonad. Different testicular structures were present.
Xq-isochromosome and testicular tissue
thra and vagina were found. A pea-sized gonad
was palpable on the right side. She exhibited hir-
sutism, with increase of hair on the arms and legs,
facial and periareolar regions, and pubic hair along
the linea alba and perianal region. She had a
deepened voice and scanty breast development
(Tanner II). Intellectual development was normal.
Genitogram was performed by retrograde injection
of contrast dye, which showed a normal vagina
and uterine cervix. The hemogram profile and bio-
chemistry, intravenous pyelogram, echocardio-
gram, and electrocardiogram were normal.
The presence of functional testicular tissue was
suspected by the elevated serum testosterone re-
sponse to stimulation by human chorionic go-
nadotropin (hCG). The plasma 17-hydroxyproge-
sterone concentration was normal. Exploratory la-
parotomy was performed. The left side contained
an intraabdominal streak gonad macroscopically
with Fallopian tubes. The right inguinal hernia
contained a dysgenetic testis with Fallopian tubes.
In the midline, an immature uterus was present.
The streak gonad consisted of a thin cortex com-
posed of an elongated wavy stroma and a
medullary region composed of loose stroma with
numerous vessels. No primordial follicles were
present. The dysgenetic testis was predominantly
testicular tissue (Fig. 2). The majority of the testic-
ular parenchyma consisted of closely apposed sem-
iniferous tubules with intertubular diffuse Leydig
cell hyperplasia. The most tubules contained only
mature Sertoli cells. The dysgenetic testis was also
composed of a region of wavy ovarian-like stroma,
but without primordial follicles.
357
Álvarez-Nava et al.

Cytogenetic and DNA analyses 70 of 122 cells analyzed. The abnormal X chromo-
some consisted of a metacentric chromosome, with
Peripheral blood metaphases were obtained from
two arms of equal length, whose banding pattern
routine phytohemagglutinin-stimulated 72-h cul-
was similar to that of the long arm of the normal
tures and from methotrexate-synchronized cul-
X chromosome (Xq-isochromosome). A 45,X cell
tures. Chromosomes were examined using GTG
line was found in 52 (42%) of the 122 cells. Periph-
banding. The X-inactivation pattern was studied
eral blood chromosome analysis revealed a 45,X/
by means of acridine-orange reverse banding after
46,X,i(Xq) (42%/58%) karyotype (Fig. 3).
5-bromodeoxyuridine (BrdU) incorporation during
Chromosome replication analysis after 5-BrdU
the last 4–6.5 h prior to terminating peripheral
treatment showed early replication of the normal
blood cultures.
X chromosome and late replication of the Xq-
Fluorescence in situ hybridization (FISH) of the
isochromosome in all metaphases of the total 50
patient’s chromosomes, using the biotin-labeled Y
cells scored. Thus, X-inactivation studies showed
centromeric probe DYZ3 and Y-chromosome
that the Xq-isochromosome was inactived.
painting probe, was carried out, according to the
The Y centromeric probe DYZ3 and the Y-chro-
published method (10).
mosome painting probe were not found to hy-
DNA samples from various sources, such as
bridize to either normal X chromosome, the
blood lymphocytes, skin, and gonad tissues, were
Xq-isochromosome, or autosomal chromosomes in
obtained. DNA was prepared from peripheral
the patient.
blood lymphocytes by the sodium dodecyl sulfate
Using eight different Y-chromosome-specific
(SDS)–proteinase K method, according to stan-
primers for PCR, there was no evidence for Y-spe-
dard procedures (11). The gonadal tissues were
cific sequences in DNA from peripheral blood
dissected into testicular and ovarian-like stroma
lymphocytes, skin fibroblasts, and gonads. The
components. Then, DNAs of the testicular and
molecular investigations disclose the absence of
ovarian-like stroma tissues from the right and left
SRY. Hidden mosaicism with a Y-bearing cell line
gonads were obtained, as described previously (12).
in all tissues was ruled out, due to the absence of Y
DNA was isolated from genital skin fibroblasts of
centromeric and other Y-specific sequences. In ad-
the patient, according to Eisentein (13).
dition, negative results were observed for all go-
Nine different sets of oligonucleotide primers
nadal tissues, irrespective of testicular and
were used for PCR: 1) PABY, a pseudoautosomal
ovarian-like stroma nature.
boundary region; 2) human SRY (the sex-deter-
mining gene of the Y chromosome), which pro-
duces a fragment 607 bp in length; 3) ZFY, a gene Discussion
located at the Yp; 4) DYS19, clustered GATA
Several lines of evidence suggest that this patient
repeats that mapped to the short arm of the Y
has partial (mixed) gonadal dysgenesis. First, the
chromosome; 5) DYZ3, which flanks a 170-bp
patient had ambiguous genitalia, indicating that
fragment of the alphoid repeats from the cen-
the solitary dysgenetic testis must have had dimin-
tromere human Y chromosome; 6) amelogenin,
ished Leydig cell production of testosterone during
which amplifies X- and Y-specific products with a
embryogenesis, which was insufficient to promote,
size difference; 7) Kal-Y, Kallman gene located on
via 5-a-dyhidrotestosterone, complete virilization
the proximal Yq; 8) DYZ1, a repetitive segment
of external genitalia. Secondly, the absence of wol-
that hybridizes to the distal long arm of the Y
ffian duct derivatives also suggests that the testis
chromosome; 9) CFTR gene from human chromo-
must have had diminished Leydig cell function.
some 7, an autosomal control that was co-am-
The presence of müllerian derivatives on the testic-
plified with the SRY primer set. Control DNA
ular side demonstrates that Sertoli cell function
from a normal male and a normal female were
was also abnormal. Finally, the histopathological
included in molecular studies. Oligonucleotide se-
appearance of the gonads was abnormal. The
quences and the PCR protocol have been previ-
histopathologic findings found in this patient are
ously described (14 – 22). All experiments were
diagnostic of mixed gonadal dysgenesis, according
carried out by the same female (46,XX karyotype).
to Robboy et al. (8).
The presence of Xq-isochromosome and testicu-
lar development does not correspond with any of
Results
the previously described forms of mixed gonadal
Peripheral blood chromosome analysis of G- dysgenesis. A review of the literature revealed no
banded metaphases revealed a normal autosomal reports of cases with the Turner phenotype, 45,X/
complement and an abnormal X chromosome in 46,X,i(Xq) karyotype, and testicular development.
358

Fig. 3. Details of metaphase spreads (GTG-staining), showing the patient’s Xq-isochromosome.
Isochromosome of the long arm of the X chromo-
some [i(Xq)] is a frequent structural chromosomal
abnormality. Some reported patients with this
chromosomal aberration have features of Turner
syndrome, and others have pure gonadal dysgene-
sis. Patients with i(Xq) are invariably short of
stature and have streak gonads, although some
menstruate spontaneously. In our patient, the Xq-
isochromosome was observed to be late replicating
in all cells examined ( \ 50), and by inference, was
the inactive X chromosome in each cell. The Xq-
isochromosome in our patient must have been in-
active in embryonic cells, and the features of
Turner syndrome must have been produced in
either 45,X cell lines or 46,X,i(Xq) cell lines. The
equal size of Barr bodies noted in our patient
(much larger than normal) supports the hypothesis
of selective inactivation (our patient had only 11%
Barr bodies). It is difficult to explain the presence
of testicular development with two X chromo-
somes, one morphologically normal and another
presumably inert, in the patient we described.
Male sex determination depends on the Y chro-
mosome gene known as SRY (23). It is generally
agreed that it involves a pathway of gene regula-
tion. The observation that SRY protein can bind
to DNA with sequence specificity indicates that
Xq-isochromosome and testicular tissue
SRY exerts its function through transcriptional
regulation of ‘downstream’ genes (24). Activation
of ‘downstream’ genes regulated by SRY may ex-
plain cases of 46,XX sex reversal with no iden-
tifiable SRY sequence. Such components of the
testicular development pathway have not yet been
identified. Mechanisms that trigger testicular dif-
ferentiation on the right gonadal ridge must have
been present to explain the partial development of
the testis in this patient, rather than the presence of
an ovary or streak gonad. It is possible that incom-
plete testicular development in this patient could
be related to a defect in one of the genes involved
in the early stages of testicular determination or
differentiation. Presumably, somatic mutation of a
gene downstream from SRY in the right gonadal
ridge could explain this patient’s phenotype.
In spite of the molecular similarity of this pa-
tient to Y-negative-46,XX males and -46,XX-true
hermaphrodites, our patient obviously does not
have such conditions. Several hypotheses have
been proposed to explain the etiology of 46,XX
males and 46,XX true hermaphrodites (6, 25). The
same possible etiologies that can be invoked to
explain the complete or incomplete testicular devel-
opment in 46,XX males or 46,XX true
hermaphrodites can explain the partial gonadal
dysgenesis in this patient.
359
Álvarez-Nava et al.
Ogata et al. (26) have proposed that a testis-
determining factor (TDF) subject to X-inactiva-
tion is present on Xp (TDF-X) and that two
active copies of TDF-X hinder testis formation in
the presence of SRY. In a further study, they
refined the chromosomal location of TDF-X to
Xp21.2-p21.3 and speculated that TDF-X may
function as a suppressor of genes involved in the
formation of the testis, and SRY may permit the
operation of the testis formation by repressing
the function of TDF-X (27). This hypothesis is
primarily based on the evidence that SRY can
function as a transcriptional repressor (28). These
investigators have suggested that the interaction
between SRY and TDF-X may constitute the
switch system for testis determination. On the ba-
sis of this hypothesis, a loss of function mutation
of TDF-X may lead to male sex reversal in a
female. It may be possible that this patient has a
mutation on the TDF-X gene, permitting partial
development of the testis during the embryonary
period. Complete testicular development did not
occur in this patient, probably because the 45,X
cell lines or 46,X,i(Xq) cell line (both altered)
could have disrupted the expression other testis
formation genes. 46,XX males may display a loss
of function of TDF-X similar to our patient, but
in the presence of two normal X chromosomes,
complete testicular development is reached in
46,XX males. Presumably, the DAX1 gene is
TDF-X. The DAX1 is a gene that has been
cloned from this region, and it may be responsi-
ble for the sex-reversal syndrome. The results of
Swain et al. (29) show that this gene is responsi-
ble for dosage-sensitive sex reversal and provide
a model for early events in mammalian sex deter-
mination, when precise levels and timing of gene
expression are critical. The study of the DAX1 in
46,XX males and our patient could explain these
phenotypes.
In conclusion, in this report, we present the
results of our studies of a 13-year-old girl with
several features of Turner syndrome, 45,X/
46,X,i(Xq) karyotype, and testicular development.
The findings in this patient illustrate the complex
nature of sexual determination and emphasize the
need for a multidisciplinary approach in this type
of investigation. Data from the case herein de-
scribed suggest that testicular development may
occur in the absence of SRY.
Acknowledgements
This study was supported by a CONDES grant (0543-97)
from the University of Zulia.
360
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