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The Journal of Clinical Endocrinology & Metabolism 90(4):2429 –2435
Copyright © 2005 by The Endocrine Society
doi: 10.1210/jc.2004-1110
CLINICAL CASE SEMINAR
Three New Novel Point Mutations Localized within and
Downstream of High-Mobility Group-box Region in SRY
Gene in Three Indian Females with Turner Syndrome
Mohammed Shahid, Varinderpal S. Dhillon, Mohammed Aslam, and S. A. Husain
Human Genetics Lab (M.S., M.A., S.A.H.), Department of Biosciences, Jamia Millia Islamia, New Delhi 110 025, India; and
Commonwealth Scientific and Industrial Research Organization Health Sciences and Nutrition (V.S.D.), Adelaide, Adelaide
5000, Australia
Point mutations and deletions in the SRY gene result in XY sex
reversal in pure gonadal dysgenesis. To date, a majority of
these affect the high-mobility group (HMG) domain of SRY,
which plays a key role in its DNA binding activity. We carried
out molecular genetics studies in three Turner syndrome patients all presenting with 45,X/46,XY mosaic karyotype. Case
1 demonstrated an insertion of T (thymine) within helix I of
HMG box leading to frame shift mutation (N82X). In case 2,
insertion of A (adenine) downstream of HMG box resulted in
a nonsense frame shift mutation (L159fsX167). These mutations resulted in truncated and altered proteins. In case 3,
D
URING MAMMALIAN EMBRYOGENESIS, the presence of the SRY gene determines whether the gonads
develop as testes, which in turn determines whether the
embryo will develop as a male; in its absence, the fetal gonads
will develop as ovaries (1). XY gonadal dysgenesis results
from an embryogenic testicular regression sequence and can
occur in a pure or partial form. Gonadal histology presents
with hypoplastic testicular tubules intermixed with ovarian
stroma (2). These individuals may have unilateral or bilateral
dysgenetic gonads and/or streak gonads. Several genetic loci
may play important roles in testis-determining pathways.
Male-to-female reversal in 46,XY patients results in failure to
develop testis. This could be attributed to the mutations in
the SRY gene (3). Sex determination in humans depends on
the action of a testis-determining factor encoded by the SRY
gene consisting of a single exon. It is located on the short arm
of Y chromosome and encodes a protein of 204 amino acids.
The human Y chromosome is reported to have about 76
protein-coding genes, although only 27 distinct proteins
have been identified thus far. Earlier thought to be a genetic
wasteland, the Y chromosome is now found to have expanded its repertoire during the course of evolution by seFirst Published Online February 1, 2005
Abbreviations: ASO, Allele-specific oligonucleotide; F, forward;
HMG, high-mobility group; R, reverse; SSCP, single-stranded conformational polymorphism.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
G>C missense mutation is found at codon 74 within helix I of
HMG box (Q74H). No other mutations were found in the SRY
gene of these patients. An allele-specific oligonucleotide study
further confirmed that these variants are not common polymorphisms. To our knowledge, this is the first time these mutations are described at these codons resulting in mutated
SRY proteins. Lack of a second sex chromosome in a majority
of cells [mosaic karyotype and mutation(s) in the SRY gene] in
these patients may have triggered the short stature. (J Clin
Endocrinol Metab 90: 2429 –2435, 2005)
quence amplification and selectively importing genes from
autosomes and X chromosomes. This presents evolutionary
conserved DNA-binding domain [the high mobility group
(HMG) box], suggesting this protein regulates gene expression. SRY gene is essential for initiating testis development
and differentiation of indifferent and bipotential gonads into
testicular pathway (3, 4). In SRY presence, supporting cells
of indifferent gonads become testicular Sertoli cells, whereas
in its absence, these become ovarian follicular cells (5). SRY
protein has been shown to possess sequence-specific DNAbinding activity and is assumed to regulate other genes involved in male determination pathways (6). Normal SRY
changes the architecture of DNA, thus allowing access of
other factors needed for its expression (7). This protein binds
to target DNA sequences and facilitates DNA bending and
hence acts as the main regulatory activity of the SRY gene (8).
Mutations in the SRY gene have been found to account for
approximately 15% of cases with gonadal dysgenesis (9 –12).
However, a majority of these patients may have mutations in
other genes involved in sex-differentiation pathway or in the
regulatory elements of the SRY gene. To date 44 mutations
have been identified within the open reading frame of the
SRY gene, and most are located within the HMG box, thus
highlighting the critical role of this domain (11, 13, 14). Of
these, only 10 mutations outside the HMG box [eight are
located in the 5⬘ region upstream of the HMG box, and the
remaining two lie downstream, 3⬘ of the HMG box] have
been reported so far (14, 15). There is one interesting report
describing the mutations in the SRY gene occurring in two
of three subjects with the 45,X/46,XY karyotype, one with
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J Clin Endocrinol Metab, April 2005, 90(4):2429 –2435
cytogenetically normal Y chromosome and the other with a
Y-derived marker chromosome. An identical missense mutation (G2128A, Ser18Asn) upstream of the HMG box of the
gene has been found in two patients (16).
Turner syndrome is characterized by short stature, gonadal dysgenesis, and dysmorphic features, including neck
webbing (17). Almost half of all cases have typical Turner
syndrome karyotype (45,X), whereas the remaining cases
have either a derivative sex chromosome in the investigated
cells or a mosaic karyotype, with the second cell line having
a normal or structurally rearranged sex chromosome (18).
Various studies have revealed either a normal or derivative
Y chromosome in a high proportion of cases with clinical
phenotype of Turner syndrome (19, 20). Turner syndrome
with a mosaic 45,X/46,XY karyotype comprises a phenotypic
spectrum of females (10 –15%) having SRY mutations.
Here we describe three novel point mutations in the SRY
gene in three Turner syndrome patients. To our knowledge,
no mutations at these positions have been reported previously in the literature.
Subjects and Methods
Informed consent was obtained from all patients and control individuals. This study was approved by the ethics committee of the hospital
and university.
Subjects
Cases 1 and 2. Patients 1 and 2, 24- and 20-yr-old females, respectively,
born to healthy but unrelated parents, were referred because of primary
amenorrhea. Physical examination revealed a height of 135 and 146.5 cm,
respectively (below the fifth percentile). External genitalia were unambiguous female type. Histological investigations revealed white fibrous
stromal tissue (with some similarities to testicular histopathology) in
streak gonads. Both patients have multiple Turner stigmata (i.e. webbed
neck, high arched palate, cubitus valgus, broad shield-like chest with
widely spaced nipples, low hairline, disproportionately short legs, presence of müllerian structures, sexual infantilism at puberty, hypertension,
and glucose intolerance). Scoliosis, nail dysplasia, and ear anomalies
were also found in these patients. Endocrinological studies demonstrated hypergonadotropic hypogonadism (estradiol ⬍ 13.0 pg/ml; LH
32.3 and 26.4 mIU/ml; FSH 56.7 and 48.8 mIU/ml, respectively) as well
as normal female concentrations of testosterone and androstenedione.
Both suffer from psychological problems and primary amenorrhea. Axillary and pubic hairs were scanty. Case 1 has a blood karyotype as
45,X(80%)/46,XY (20%), whereas case 2 has 45,X (86%)/46,XY (14%)
karyotype.
Case 3. The proposita presented at 22 yr of age with short stature,
ambiguous external genitalia [a birth defect in which the outer genitals
do not have typical appearance of either sex] and absence of pubertal
development. Clinical examination revealed a height of 151 cm (below
the fifth percentile) and multiple Turner stigmata (i.e. micrognathia,
low-set ears, high-arched palate, short and webbed neck, bilateral cubitus valgus, and multiple nevi). Sparse axillary and pubic hairs (Tanner
stage II) were observed, and there was no clitoromegaly. On arousal,
phallus length increased from 20 to 30 mm. The position of urinary
meatus lies just beneath the phallus. Sonographic examination revealed
a normal-sized uterus with a thin endometrium, bilateral müllerian
derivatives, and right intraabdominal testis. Endocrinological studies
demonstrated hypergonadotropic hypogonadism (estradiol ⬍ 13.0 pg/
ml; LH 32.5 mIU/ml; FSH 74.2 mIU/ml) as well as normal female
concentrations of testosterone and androstenedione. Ovaries were not
visible, but thickened structures resembling streak gonads were found
on both sides of the uterus. Histological investigations revealed fibrous
stromal tissue. No gonadoblastoma and dysgerminomas were found in
the streak gonads. She had no psychological problems. Blood karyotype
was 45,X (89%)/46,XY (11%).
Shahid et al. • SRY Mutations and Turner Syndrome
Extraction of DNA
Genomic DNA from the blood samples was extracted by digestion
with proteinase K (Boehringer, Mannheim, Germany) followed by routine phenol chloroform isolation and precipitation with ethanol or isopropanol with chilled 3 m sodium acetate (pH 5.2). Vacuum-dried DNA
samples were dissolved in Tris-EDTA, and DNA concentration was
measured by gel electrophoresis method. All reagents used in the DNA
isolation were procured from E. Merck India Ltd. (Mumbai, India).
PCR
Complete SRY exon was studied by overlapping primers; however,
only the relevant primers (which helped to identify these mutations)
were discussed. The following primer sets were used to amplify fragments of 254 and 351 bp, respectively, from the open reading frame of
the SRY gene (17): forward (F)1, 5⬘-CATGAACGCATTCATCGTGTGGTC-3⬘; reverse (R)1, 5⬘-CTGCGGGAAGCAAACTGCAATTCTT-3⬘; F2,
5⬘-CAGTGTGAAACGGGAGAAAACAGT-3⬘; and R2, 5⬘-GTTGTCCAGTTGCACTTCG CTGCA-3⬘. Exon 5 of p53 gene was amplified as
internal control using oligonucleotide primers 5⬘-TACTCCCCTGCCCTAACAA-3⬘ (sense) and R, 5⬘-CATCGCTATC TGAGCAGCGC-3⬘
(antisense) to amplify the 184-bp PCR product. PCR amplification was
performed in 25 ␮l reaction volume containing 10 mm Tris HCl (pH 8.4),
50 mm KCl, 1.5 mm MgCl2, 200 ␮m each of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), 5 pmol oligonucleotides primers,
100 ␮g DNA and 0.5 U Taq DNA polymerase (Perkin-Elmer Cetus,
Norwalk, CT). PCR was performed as follows: 4 min at 95 C followed
by 35 cycles at 95 C for 30 sec; 55 C for 30 sec; 72 C for 30 sec, and a final
extension step of 72 C for 7 min. Every PCR included negative (normal
XX female) and positive (normal XY male) controls. PCR products (7 ␮l)
were run on 2% agarose gels, and bands were visualized by ethidium
bromide staining on an UV transilluminator. For all agarose gels, a
100-bp ladder was used as size standard.
Allele-specific oligonucleotide (ASO) hybridization
With primers F2 and R2 as described above, a 351-bp segment of SRY
(including the mutation sites) was amplified from the DNA of the
patients and 44 randomly selected normal XY males. The 351-bp SRY
PCR products were then arrayed on two identical nylon membranes.
Oligonucleotides were designed and synthesized for use as probes in
this study: one specific for wild-type SRY (5⬘-tctcgcgatcagaggcgcaagatggc-3⬘) and the other specific for Q74H SRY (5⬘-tctcgcgatcacaggcgcaagatggc-3⬘), differing only at the 12th nucleotide: wild-type (5⬘-atggctctagagaatcccagaatgcg-3⬘) and N82X SRY (5⬘-atggctctagagtaatcccagaatgc-3⬘)
differing in having additional “t” at the 13th nucleotide and wild-type
SRY (5⬘-agcgaagtgcaactggacaacagg-3⬘) and L159fsX167 SRY (5⬘-agcgaagtgcaaactggacaacagg-3⬘) differing in an additional “a” at the 13th
nucleotide position. Twenty-five nanograms of these oligonucleotides
were end labeled with ␥-32P by T4 polynucleotide kinase (Fermentas Inc.,
Hanover, MD) and used separately to probe the duplicate membranes
for 16 h at 65 C. The membranes were subsequently washed and exposed
to film at ⫺80 C. Hybridizations were done separately with each category of wild-type and mutant oligonucleotides.
Single-stranded conformational polymorphism (SSCP)
PCR products were labeled with (␣-P32) dCTP by performing 15
additional cycles of PCR and loaded on 6% nondenaturing polyacrylamide gel containing 5% glycerol. The electrophoresis was carried out
overnight at 200 V at 17 ⫾ 1 C. The dried gel was exposed to x-ray film
for 48 h at ⫺70 C (21).
Automated DNA sequencing
PCR products were purified using QIAquick PCR purification kit
(QIAGEN, Santa Clarita, CA) before being sequenced using an ABI
Prism 310 automated sequencer (Applied Biosystems, Foster City, CA).
The cycle sequencing of the purified PCR products was performed using
BIG Dye terminator sequencing ready reaction mix with AmpliTaq DNA
polymerase FS on GeneAmp PCR 9700 (Applied Biosystems). PCR conditions were set as: 96 C ⫻ 10 sec, 56 C ⫻ 5 sec and 60 C ⫻ 4 min for
25 cycles. After cycle sequencing, extension products were purified to
Shahid et al. • SRY Mutations and Turner Syndrome
remove any unincorporated dye-labeled terminators using ethanol/
sodium acetate precipitation method. Template suppressor reagent was
added, and samples were heat denatured, chilled on ice, and loaded on
the Prism 310 sequencer. The sequences were analyzed using sequencing
analysis software 3.4.1 on a Mac OS 9.1 (Applied Biosystems).
Results
Chromosome analysis from cultured peripheral blood
lymphocytes showed mosaic karyotype (45,X/46,XY). Biochemical parameters showed altered levels of estradiol, LH,
and FSH, but normal female concentrations of testosterone
and androstenedione. These patients showed altered migration of PCR products in SSCP assay (Fig. 1). In reference to
SRY mutations, patient 1 has an insertion of T (thymine)
leading to frame shift within the open reading frame inside
highly conserved DNA-binding motif-HMG box (N82X;
Figs. 2, A and B, and 3, A and B), and the mutated protein
lacks the last 123 amino acid residues. The father and other
male members of the family did not consent to DNA analysis,
but some are already parents of healthy children. Patient 2
showed insertion of A (adenine) leading to frame shift mutation downstream of the HMG box, resulting in an altered
protein at the C terminus (L159fsX167; final 46 amino acids),
lacking the last 37 residues (Figs. 2, C and D, and 3, A and
C). In patient 3, G⬎C transversion was observed leading to
Q74H in the HMG box (Figs. 2, E and F, and 3A). We also
performed an ASO hybridization study on fragments of SRY
amplified from patients’ DNA and 44 randomly selected
normal males. Figure 4 shows that these variants were
found only in patients and not among any of 44 normal
SRY alleles sampled in the present study. The data suggest
that these variants are indeed mutations and not common
polymorphisms.
Discussion
Normal male sex determination in mammals is targeted by
the SRY gene on the Y chromosome. Timing and expression
of this gene are exquisitely regulated and must probably
reach the required threshold for testis formation in the developing embryo (9). These patients have mosaic 45,X/46,XY
karyotype with multiple Turner stigmata. We identified two
new SRY mutations in two phenotypic females with 45,X/
46,XY mosaic karyotype. Both mutations result in altered
nonfunctional protein, which may explain the female phe-
FIG. 1. PCR-SSCP analysis of single exon of SRY gene in Turner
syndrome patients. Lane 1, SRY from normal male control DNA; lane
2, SRY with shifted band from patient 1; lane 3, SRY with missing
band from patient 2; and lane 4, SRY with shifted band from patient
3 indicates that these patients carry mutations in SRY gene.
J Clin Endocrinol Metab, April 2005, 90(4):2429 –2435
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notypic characteristics and presence of streak gonads. Sexual
differentiation during early embryonic stages is characterized by complex interaction of various genetic and nongenetic factors (22). In cascade of gonosomal and autosomal
genes, the SRY gene located on the short arm of the Y chromosome is one of the first activated genes. There is evidence
that SRY is essential for sex determination (8). However,
there are many examples of SRY-negative individuals who
differentiate to males (20% XX males).
These observations strengthen the notion that there are
few other (so far unidentified) genes that could influence the
male differentiation. There are few reports of patients representing clinical phenotype resembling Turner syndrome
with either normal or derivative Y chromosome in a high
proportion of cells. Despite the presence of a structurally
abnormal SRY gene, there was a dysfunction at the hormonal
level during male sexual differentiation. Apparently, a relatively small proportion of the 45,X cell line is sufficient
enough to suppress the male-determining function of SRY
gene. This suggests that patients did not result from either
abnormal sex chromosomes pairing or deletion of SRY region
of the Y chromosome. Loss of the Y chromosome by nondisjunction/mitotic loss after normal disomic fertilization
could lead to mosaic karyotype in these patients. It is not
known, whether there is a predisposition toward the loss of
the Y chromosome or it is merely a random event caused by
the inherent instability/inability of the XY chromosome pairing. It is possible that even if the Y chromosome appears to
be normal, mutations in other genes that reside on this chromosome, necessary for chromosome integrity, may lead to its
eventual loss. This may, therefore, explain the emergence of
the abundant 45,X cell line.
The presence of streak gonads may be attributed to the
invasion of primary genital ridge by the 45,X cell line during
early developmental stages. Mutations in the SRY gene and
presence of the dominant 45,X cell line in these patients may
have acted in a cumulative manner to induce a cause-effect
relationship. This may account for nonmasculinization in an
otherwise originally XY embryo. Unequal distribution of two
cell lines as seen in these patients may have originated at the
time of the implantation as per their distribution into fetal
and placental poles. Presence of the 45,X cell line may have
determined the gonadal development into abdominal streak
gonads, whereas the 46,XY cell line with the SRY mutation
might have contributed toward the development of intraabdominal testis as in patient 3.
The assignment of the SRY as the testis-determining
factor is further supported by many studies of human
intersex abnormalities (1, 23–27). These reports indicate
the association of SRY mutations with gonadal dysgenesis.
SRY protein belongs to the SOX family of transcription
factors characterized by the HMG domain having DNA
binding and bending properties. It has the ability to mediate protein-protein interactions and contains signals for
nuclear import (28, 29). The rapid degradation of the truncated proteins in both these patients cannot be verified in
vivo because their embryonic expression may be time and
tissue specific. However, these truncated proteins found in
these patients may be stable, although they are missing
C-ter region, which is considered to be necessary for the
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J Clin Endocrinol Metab, April 2005, 90(4):2429 –2435
Shahid et al. • SRY Mutations and Turner Syndrome
FIG. 2. Partial electropherograms of the SRY gene in patients. Patient 1 shows mutation N82fsX82 (A) and normal or wild-type sequence (B).
Patient 2 shows a mutation L159fsX167 (C) and wild-type sequence (D). Patient 3 shows mutation Q74H (E) and wild-type sequence (F).
nuclear import of these proteins. This change may form an
electrostatic and hydrophobic interaction with phosphate
and sugars, respectively, of DNA. This can alter the specific orientation and binding usually to DNA bases in the
major groove. This change totally or at least partially may
inhibit the interactions of SRY to interact with DNA (30).
Insertion of adenine at codon 159 resulted in a frame shift
mutation (L159fsX167) downstream of the HMG box, resulting in an altered protein at the C terminus (final 46
amino acids) lacking the last 37 residues. Only two other
alterations in the C-ter region (L163X, resulting truncated
protein identified in two sisters with 46,XY karyotype, and
Q158fsX180 in another 46,XY patient with primary amen-
orrhea, resulting in a truncated protein both at 3⬘ end) have
been reported in the SRY region (15, 31). Mutation detected in the present case was not found in her paternal or
sibling normal males’ DNA. It was therefore, considered
to be a de novo mutation, although a paternal germ cell
mosaicism cannot be ruled out. To our knowledge this is
the first time that a truncated SRY protein has been found
in Turner syndrome with mosaic 45,X/46,XY karyotype.
These three mutations further strengthen the functional
significance of the 3⬘ downstream region of the HMG box.
Transversion mutation in patient 3 at codon 74 lies in the 5⬘
region of the HMG box, which contains N-terminal nuclear
localization signals, highly conserved in mammals and is
Shahid et al. • SRY Mutations and Turner Syndrome
J Clin Endocrinol Metab, April 2005, 90(4):2429 –2435
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FIG. 3. A, Partial upstream and downstream sequence of HMG box showing
the position of mutations (circled amino
acids, affected residues). B and C, Partial sequence of SRY resulting from mutation indicating altered protein at C
terminus, respectively. *, Stop or termination codon.
believed to be required for complete nuclear localization (28).
There is just one report showing a mutation at codon 74 in
which glutamine is changed to a stop codon in a pure gonadal dysgenesis patient (24). The HMG domain is composed of three ␣-helices and adopts an L shape (32). Change
of glutamine (polar, hydrophilic, and neutral amino acid) at
codon 74 to histidine (polar, hydrophilic, aromatic, and
charged amino acid) within helix 1 may have a direct impact
on the nuclear localization of the protein that itself may
influence the helix structure and/or impair its DNA binding
activity. Takagi et al. (33) reported in a phenotypical female
with mosaic 45,X/47,XYY karyotype a frame-shift mutation
at position 422 of SRY gene. They have shown that mutant
SRY may be assumed to induce a nonfunctional SRY-coded
protein that lacks a DNA-binding motif. These results also
explain the phenotypic female and the gonadal dysgenesis in
the 45,X/47,XYY sex-reversed affected individual. Similarly,
Canto et al. (16) reported in two patients presented with
mosaic karyotype as 45,X/46,XY with a missense mutation
as S18N in the 5⬘ non-HMG box region in DNA from both
blood and streak gonads. However, Yorifuji et al. (34) could
not find any mutations in the SRY gene in 11 patients with
Turner syndrome. Previous studies demonstrated the presence of whole Y chromosome or Y-derived material in varying frequencies, and the presence of Y chromosome material
has increased the chances of the gonadoblastoma development in these patients. The presence of intraabdominal testis
in patient 3 has further strengthened the critical role of SRY
sequences in this patient to develop the testis. But the predominance of the 45,X cell line, besides the presence of mutated SRY, might have impaired the total development of
testicular tissue.
The majority of the mutations detected so far in the SRY
gene lie within the conserved motif, causing alterations in
DNA binding/bending activity and therefore the origin of
46, XY females. To date only 12 mutations that lie outside the
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Shahid et al. • SRY Mutations and Turner Syndrome
Acknowledgments
We thank the patients involved in this study and the clinicians from
the Department of Obstetrics and Gynecology (Maulana Azad Medical
College and Lok Nayak Hospital, New Delhi, India).
Received June 17, 2004. Accepted January 21, 2005.
Address all correspondence and requests for reprints to: Dr.
Varinderpal S. Dhillon, Genome Stability Laboratory, Commonwealth
Scientific and Industrial Research Organization Health Sciences and
Nutrition, Gate No. 13, Kintore Avenue, P.O. Box 10041, Adelaide BC,
Adelaide 5000, Australia. E-mail: [email protected].
This work was supported by Grant 3/1/2/18/2001-RHN from the
Indian Council of Medical Research, New Delhi, India.
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FIG. 4. ASO hybridization of wild-type (A) or mutant (B) SRY probes
to SRY alleles amplified from the patients and 44 randomly chosen
normal males.
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