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Annals of RSCB
Vol. XVII, Issue 1/2012
FISH TECHNIQUE USEFULNESS FOR THE EVALUATION OF
TURNER SYNDROME IN PATIENTS WITH SHORT STATURE
Diana Miclea, I. V. Pop
MOLECULAR SCIENCE DEPARTMENT, UNIVERSITY OF MEDICINE AND
PHARMACY « IULIU HAŢIEGANU », CLUJ, ROMANIA
Summary
Turner syndrome is very heterogeneous, short stature often being the only clinical sign.
In this pathology, classical karyotype is the gold standard in genetic investigation for the
positive diagnosis, and it is recommended to be performed in every girl with unexplained
short stature. We investigated by standard and molecular cytogenetics a girl who
presented for short stature not explained by common medical reasons. Standard
karyotype showed homogeneous monosomy, 45,X, FISH technique revealing a
mosaicism undetected before, 45,X/46,XX, the level of mosaicism for the second cell line
being of 22%. This difference could be explained by an augmented number of cells
analyzed by FISH, thus improving the cytogenetic diagnosis. FISH technique is useful in
medical practice, complementary to the classical techniques due to a greater resolution
and for the detection of the mosaicism.
Keywords: Turner syndrome, short stature, X chromosome, FISH technique, karyotype
[email protected]
2001); the ovarian function is related to the
genes situated on the long arm of X, Xq26
(POF1) and Xq13-21 (POF2) (Cabrol, 2007);
the congenital lymphedema is associated with
Xp11.4 region, cardiac malformations and
aortic coarctation being presumed to be caused
by the haploinsufficiency of the lymphogenic
gene (Cabrol, 2007).
The change of the vision concerning
Turner syndrome has been remarked in the last
years due to the knowledge of the great
variability of its clinical expression. On the
other hand, this entity should not be
considered a devalorized handicap, due to the
therapeutic progress. Actually, in Turner
syndrome there is the possibility to treat the
growth retard by growth hormones, there is
also a better approach to pharmacological
feminization and the possibility of maternity
by egg cell donation from wealthy women and
in vitro fertilization. Therefore, we may say
that this pathology is not what it was a while
ago and it benefitted from a therapeutic
revolution. By a precocious diagnosis and
therapy, the quality of life of these patients
should be improved. Beyond all the aspects, a
correct and precocious diagnosis also permits
Introduction
Turner syndrome is observed in 1:2500 female
newborn, the characteristic phenotype being
characterized by dysmorphy, short stature,
hypogonadism and internal malformations
(Bondy, 2006). Turner syndrome is very
heterogeneous, the clinical spectrum ranging
from a complete form to an almost
asymptomatic picture, short stature being
present in the majority of cases. The
phenotype of the patients with Turner
syndrome is explained by the aneuploidy
himself or by the haploinsufficiency of the
genes which escape from the mechanism of X
inactivation (Cabrol, 2007; Ranke and
Saenger, 2001; Zinn and Ross, 1998). The
study of structural abnormalities permitted us
to determine the role of certain genes which
intervene to explain some phenotype features
of Turner syndrome, thus: the loss of the distal
extremity on Xp leads to short stature and to
typical skeleton abnormalities, due to the
haploinsufficiency of the SHOX gene, situated
on the pseudoautosomal region of X
chromosome (Xp11-22) and of Y
chromosome (Yp11) (Cabrol, 2007; Kosho et
al., 1999; Pienkowsk, 2004; Ogata et al.,
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Vol. XVII, Issue 1/2012
gestational age. Statural growth rate was
always poor. It isn’t mentioned a family
history of short stature, midparental height
being of 171 cm (+1,3DS). Clinical exam
showed: height of 112 cm (-2,5 DS), weight of
24 kg (+3,2 DS), bilateral short 4th metacarpal.
Routine laboratory investigations performed
for the short stature reveals no changes. Thus,
genetic tests were indicated to characterize sex
chromosomes.
to diagnose and treat the associated
pathologies. A better informing is therefore
necessary to know this progress.
As a consequence, in the developed
countries it has been observed in the last 10-20
years, a progressive growth of the number of
cases yearly diagnosed, by more careful
investigation of the short stature and by better
prenatal and postnatal genetic diagnosis, using
a complex cytogenetic techniques (Fluorescent
in situ Hybridization - FISH)( Pienkowski,
2004; Ogata et al., 2001; Stochholm et al.,
2006). The tendency of lower age at the
diagnosis has also been observed, the majority
of cases being diagnosed in antenatal and
postnatal period until the age of 10-12 years
(in Belgium the average age at diagnosis was
in 1991 of 11,2 years, and in 2005 of 6,6 years,
the average height at diagnosis being -2,9 DS
in 1991, and -2,4DS in 2005) (Massa et al.,
2005; Carel et al., 2004).
However, in Romania, there are many
undiagnosed cases of Turner syndrome, partly
because of an inadequate clinical evaluation
and on the other hand due to the lack of
advanced cytogenetic techniques. They are
diagnosed tardily, often at the age of puberty
(14-16 years) or later, thus delaying the
institution of a treatment which, otherwise,
could ameliorate significantly the disease
(Grigorescu-Sido, 2000).
There is still no consensus on warning
clinical sign that should lead to an indication
of karyotype. However, for an early diagnosis
it is generally recommended to perform a
karyotype in every girl with unexplained short
stature, because it can often be the only sign in
Turner syndrome (Savendahl and Davenport,
2000). In literature it is described that, among
the girls with short stature, up to 19% have
Turner syndrome (Lam et al., 2002; Gicquel et
al., 1998; Moreno-Garcia et al., 2005).
The purpose of this paper was to
highlight the role of FISH technique in
cytogenetic diagnosis of Turner syndrome.
Case. An 8-yr-old girl presented with short
stature. Auxological parameters at birth
showed a weight of 2900g (-0,6DS) and a
length of 47 cm (-1,5DS) at 38 weeks of
Material and methods
Cytogenetic techniques were done on the
lymphocytes of the peripheral blood. 10 cells
were examined, and for 4, karyotype was
performed. In FISH techniques, specific
probes for X and Y centromeres were used
(Vysis®, Abbott, Abbott Park). The threshold for
the diagnosis of a mosaic was 2%. 200
interphase cells and 50 metaphases were
examined by FISH.
The protocol for standard karyotype
included:
culture
peripheral
blood
lymphocytes (culture environment with
phytohemaglutinine), use of colchicines to
block the cells in metaphase, hypotonic shock
with a solution of KCl, fixation with acetic
acid:methanol (1:3), spreading on the slide.
FISH technique consisted in:
pretreatement of the slide by plunging in
successive baths of PBS 1X (5’) – PFA (10’) –
PBS 1X (5‘), then treatment with HCl 0,01 N
and pepsin at 37°C (8’); suspension in
successive baths of PBS 1X (2’) – PFA (10’) –
PBS 1X (2‘); dehydration in 70% (2’), 85%
(2’), 100% (2’) alcohol bath; application of the
XY centromeric probe – 75°C (5’);
maintaining the slide overnight in a
hybridizing room at 37°C; slides lavage in a
solution of SSC (3M de NaCl + 0,3 M sodium
citrate) 0,4X, than at 70°C (2’); slides
suspension in SSC 2X solution; DAPI
application and examination in microscopy.
Results and discussion
Standard cytogenetic evaluation revealed a
45,X, homogeneous monosomy. FISH
technique
evidenced
the
karyotype
45,X/46,XX, the second normal cell line being
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important in prenatal diagnosis, its rapidity
indicating that as a technique of first choice.
Actually, the karyotype is the golden standard
in aneuploidies diagnosis, having the
maximum of sensibility and specificity of
detecting them. One advantage of performing
a complete karyotype is that it allows the
global study of the chromosomes, revealing
numeric or structural abnormalities, larger than
5Mb.
Standard
karyotype
has
also
disadvantages. One would be the time required
for cells culture (2-3 days in lymphocytes, 2-3
weeks in amniocytes), and for chromosome
analysis. Therefore, it has been tried to obtain
such molecular cytogenetic techniques, which
can be used in the absence of cell culture and
thus allowing faster results. The main
disadvantage of these molecular techniques, as
FISH, is that they do not allow the detection of
other chromosome abnormalities outside the
target anomalies.
By
conventional
cytogenetic
techniques, the karyotypes observed in patients
with Turner syndrome are in half of the cases
45, X, in approximately 15% - numeric mosaic
of X (45, X/46, XX; 45, X/46, XX/47, XXX;
45, X/47, XXX); in 30% - structural
abnormalities of X, homogeneous or in mosaic
(i(Xq); i(Xp); del(Xp), del(Xq), r(X)) and in
5% anomalies implying Y chromosome,
normal or reshuffled, homogenous or in
mosaic (Pienkowski, 2004).
By these classical techniques it cannot
always be established a correlation karyotypephenotype, cases being evidenced with typical
phenotype of Turner syndrome, but with
normal karyotype, in these situations the
karyotype with FISH being indicated. It was
observed, by using this technique, that the
frequencies of the mosaics is greater (some
authors suggest that more than 98% of cases
are mosaics, and that only the mosaics fetus
are viable), the great variability of Turner
phenotype, being connected with the
proportion of the number of cells 45, X (Ranke
and Saenger, 2001; Kosho et al., 1999). The
severity of the phenotype depends on the
complexity of the mosaic, the patients which
observed in 22% cells. Regarding Hook’s
tables, we observe that by the analysis of 10
cells, as it has been done in standard
cytogenetic analysis, we can detect a level of
mosaicism superior to 25% with 95%
confidence and a inferior level of mosaicism
cannot be excluded (Hook, 1977). 22% level
of mosaicism was identified only by FISH and
not by standard cytogenetics, because it was
based on the analysis of a greater number of
cells, 200 metaphases and 50 interphases,
which allowed the detection of a mosaicism
level superior to 2% (Hook, 1977).
Diagnostic guidelines in Turner
syndrome recommend to perform a
cytogenetic analysis in every girl with short
stature, after the exclusion of common causes
(Bondy, 2006; Savendah and Davenport,
2000). Chromosomal analysis in this case is
recommended to be done on 10 cells and in 4
cells a karyotype should be done. In the
laboratories which have FISH techniques
available, it is indicated to do a supplementary
analysis of a least 100 interphase cells and 10
metaphases for the mosaicism detection,
complementary to standard cytogenetic
techniques.
FISH technique is based on
hybridization by complementarities of a single
stranded DNA-probes, fluorescently marked,
with a specific region of a chromosome. It can
mark chromosomes in metaphase, interphase
nuclei, elongated chromosomes, even
chromatin fiber. Metaphase cells most often
used in FISH are cultured lymphocytes, also
the cells obtained from bone marrow and
cultured fibroblasts. Interphase cells which are
most often uses in FISH technique are the
amniocytes, lymphocytes, buccal swab cells or
histopathological fragments. The analysis of
the results is done using a microscope,
analyzing fluorescent signals number per cell.
To exclude 10% mosaicism a minimum of 100
cells should be examined.
An advantage of FISH technique
would be that it can be applied in interphase
cells, thus ameliorating the technical steps in
interpreting the results. This is particularly
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chromosome is detected (Gravholt et al., 2000;
McDonough and Tho, 2006).
Among the clinical uses of FISH
techniques, it can be mentioned, in the cases of
prenatal diagnosis, the detection of 13, 18, 21
or sex chromosomes aneuploidy, or in the
postnatal period, in genetic diagnosis of
microdeletions syndrome, sexual development
anomalies, short stature, mental retardation
and also in oncology. The main limitation of
FISH technique is that it is an expensive
technique, not suitable for automation, as
molecular biology techniques are, which could
allow costs minimizing.
It can be affirmed that FISH technique
presents similar specificity and sensibility with
standard karyotype when we refer to targeted
diagnosis of different aneuploidies (Grimshaw
et al., 2003), and in mosaicism detection it
ameliorates the technical steps for the
diagnosis.
present a 45,X/46,XX karyotype having a
better prognostic then those with homogenous
45, X karyotype (Abulhasan et al., 1999). The
patients with more attenuated clinical traits,
such as the patients who have only short
stature, have a greater frequency of mosaics,
compared to those diagnosed in the first years
of life, with a more severe phenotype, in which
a greater frequency of homogeneous X
monosomy has been observed (Ogata et al.,
2001, Gicquel et al., 1998). The dissociation
between phenotype and karyotype is probably
caused by the mosaics that are not diagnosed.
The exact evaluation of the mosaic could
demonstrate a better correlation between
karyotype and phenotype.
Thus, the new techniques of
cytogenetic using in situ hybridization are
indispensable for the detection of some
mosaics or structure abnormalities (Abulhasan
et al., 1999, Van Dyke and Wiktor, 2006).
Even if the karyotype effectuated on the
lymphocytes of the peripheral blood is usually
adequate, if there is a powerful clinical
suspicion of Turner syndrome, in contrast with
a normal karyotype on lymphocyte, the second
tissue should be analyzed, such as skin tissue
(Saenger et al., 2001; Battin et al., 2003).
Also, the cytogenetic exam by FISH may
better reveal the abnormalities of structure of
sex chromosome. By FISH technique the
origin of the marker chromosomes can be
established: X or Y. It can also be observed
the deletion of the SHOX gene (Rappold et al.,
2002).
FISH technique is also useful in Y
chromosome detection. The research activities
from the last years, indicate the fact that the
presence of Y chromosome fragments in the
patients with Turner syndrome imply up to
30% risk of developing gonadoblastoma (in
situ cancer of germinal cells, associated with
dysgerminoma in 50-60% of cases) (Gravholt
et al., 2000; Cantoa et al., 2004). The
investigation of Y chromosome is necessary to
be made in all the patients with Turner
syndrome, even more in the presence of
virilization signs or when a marker
Conclusions
Genetic evaluation is required in any patient
with short stature, FISH technique being very
useful. These techniques are complementary to
the classical cytogenetics, improving the
diagnosis of chromosome abnormalities,
particularly the mosaicism and structural
abnormalities. These techniques are useful for
a precise genetic diagnosis, which is important
for the evolution and prognosis, in terms of
final height, sexual development and fertility
of Turner patients, these data being
particularly important in the genetic advice, in
antenatal and postnatal period.
References
Abulhasan, S.J.; Tayel, S.M.; Al-Awadi, S.A.:
Mosaic Turner syndrome: cytogenetics versus
FISH. Ann. Hum. Genet., 63, 199-206, 1999.
Battin, J.; Lasfargues, G.; Jaffiol, C.; Rethore, M.;
Canlorbe, P.: Turner's syndrome and mosaicism.
Discussion. Bull. Acad. Natl. Med., 187, 2, 359370, 2003.
Bondy, C.A.: Turner's Syndrome and X
Chromosome-Based Differences in Disease
Susceptibility. Gend. Med., 3, 18-30, 2006.
Cabrol, S.: Syndrome de Turner. Encyclopedie
Orphanet [online]. [cited 2007 oct]. Available
99
Annals of RSCB
Vol. XVII, Issue 1/2012
from:
URL:
www.orpha.net/data/patho/Pro/fr/TurnerFRfrPro44v02.pdf, 2007.
Cantoa, P.; Kofman-Alfarob, S.; Jimenez, A.L.;
Soderlunda, D.; Barronc, C.; Reyesd, E.; et al.:
Gonadoblastoma in Turner syndrome patients
with nonmosaic 45,X karyotype and Y
chromosome sequences. Cancer Genet.
Cytogenet., 150, 70–72, 2004.
Carel, J.C.; Bastie-Sigeac, I.; Ecosse, E.; Coste, J.:
La santé des jeunes adultes atteintes de syndrome
de Turner en France. Arch. Pediatr., 11, 559-561,
2004.
Gicquel, C.; Gaston, V.; Cabrol, S.; Le Bouc, Y.:
Assessment of Turner’s syndrome by molecular
analysis of the X chromosome in growthretarded girls. J. Clin. Endocrinol. Metab., 83,
1472–1476, 1998.
Gravholt, C.H.; Fedder, J.; Naeraa, R.W.; Muller,
J.: Occurrence of Gonadoblastoma in Females
with Turner Syndrome and Y Chromosome
Material: A Population Study. J. Clin.
Endocrinol. Metab., 85, 9, 3199-3202, 2000.
Grigorescu-Sido, P.: Sindromul Turner. In: Tratat
elementar de pediatrie, vol.4, chap.1, pp. 23-32,
2000. Edited by Casa Cartii de Stiinta, ClujNapoca.
Grimshaw, G.M.; Szczepura, A.; Hulten, M.;
MacDonald, F.; Nevin, N.C.; Sutton, F.; Dhanjal,
S.: Evaluation of molecular tests for prenatal
diagnosis of chromosome abnormalities. Health
Technol. Assess., 7, 10, 1–146, 2003.
Hook EB: Exclusion of chromosomal mosaicism:
tables of 90%, 95% and 99% confidence limits
and comments on use. Am. J. Hum. Genet., 29,
94–97, 1977.
Kosho, T.; Muroya, K.; Nagai, T.; Fujimoto, M.;
Yokoya, S.; Sakamoto, H.; Hirano, T.; Terasaki,
H.; Ohashi, H.; Nishimura, G.; Sato, S.; Matsuo,
N.; Ogata, T.: Skeletal Features and Growth
Patterns in 14 Patients with Haploinsufficiency
of SHOX: Implications for the Development of
Turner Syndrome. J. Clin. Endocrinol. Metab.,
84, 12, 4613-4621, 1999.
Lam, W.F.; Hau, W.L.; Lam, T.S.: An evaluation
of referrals for genetics investigation of short
stature in Hong Kong. Chin. Med. J., 115, 607–
611, 2002.
Massa, G.; Verlinde, F.; De Schepper, J.; Thomas,
M.; Bourguignon, J.; Craen, M.; De Zegher, F.;
François, I.; Du Caju, M.; Maes, M.; Heinrichs,
C.: Trends in age at diagnosis of Turner
syndrome. Arch. Dis. Child., 90, 267-268, 2005.
McDonough, P.G., Tho, S.P.T.: Clinical
implications of overt and cryptic Y mosaicism in
individuals with dysgenetic gonads. Int. Congr.
Ser., 1298, 13–20, 2006.
Moreno-Garcia, M.; Fernandez-Martinez, F.J.;
Barreiro Miranda, E.: Chromosomal anomalies
in patients with short stature. Pediatr. Int., 47,
546-549, 2005.
Ogata, T.; Muroya, K., Matsuo, N., Shinohara, N.,
Yorifuji, T.; Nishi, Y.; Hasegawa, Y.; Horikawa,
R.; Tachibana, K.: Turner Syndrome and Xp
Deletions: Clinical and Molecular Studies in 47
Patients. J. Clin. Endocrinol. Metab., 86, 11,
5498-5508, 2001.
Pienkowski, C.: Syndrome de Turner [online].
Available
from:
URL:
https://w3med.univlille2.fr/pedagogie/contenu/di
scipl/gyneco-medic/module1/08-1204/turner1.pdf, 2004.
Ranke, M.B.; Saenger P.: Turner’s syndrome.
Lancet, 358, 309–314, 2001.
Rappold, G.A.; Fukami, M.; Niesler, B.; Schiller,
S.; Zumkeller, W.; Bettendorf, M.; Heinrich, U.;
Vlachopapadoupoulou, E.; Reinehr, T.; Onigata,
K.; Ogata, T.: Deletions of the homeobox gene
SHOX (short stature homeobox) are an
important cause of growth failure in children
with short stature. J. Clin. Endocrinol. Metab.,
87, 1402–1406, 2002.
Saenger, K.; Albertsson, W.; Conway, G.S.;
Davenport, M.; Gravholt, C.H.; Hintz, R.; et al.:
Recommendations for the Diagnosis and
Management of Turner Syndrome. J. Clin.
Endocrinol. Metab., 86, 7, 3061-3069, 2001.
Savendahl, L.; Davenport, M.L.: Delayed
diagnoses of Turner’s syndrome: proposed
guidelines for change. J. Pediatr., 137, 455-459,
2000.
Stochholm, K.; Juul, S.; Juel, K.; Naeraa, R.W.;
Gravholt,
C.H.:
Prevalence,
Incidence,
Diagnostic Delay, and Mortality in Turner
Syndrome. J. Clin. Endocrinol. Metab., 91, 10,
3897-3902, 2006.
Van Dyke, D.L., Wiktor A.E.: Testing for sex
chromosome mosaicism in Turner syndrome.
Int. Congr. Ser., 1298, 9–12, 2006.
Zinn, A.R.; Ross, J.L.: Turner syndrome and
haploinsufficiency. Curr. Opin. Genet. Dev, 8,
322-327, 1998.
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