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Identification of a new nonsense mutation (Tyr129Stop) of the SRY gene in a newborn infant with XY sex-reversal.

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American Journal of Medical Genetics 128A:46 –47 (2004)
Identification of a New Nonsense Mutation (Tyr129Stop) of
the SRY Gene in a Newborn Infant With XY Sex-Reversal
Mario Giuffrè,1* Piero Sammarco,2 Carmelo Fabiano,2 Fabio Giardina,2 Fabio Lunetta,2 and Giovanni Corsello1
Dipartimento Materno Infantile, Universitá degli Studi di Palermo, Italy
Neonatologia e Terapia Intensiva Neonatale, Azienda Ospedaliera ‘‘Cervello,’’ Palermo, Italy
Point mutations and deletions of SRY gene have
been described in several cases of XY gonadal
dysgenesis. To date, most of these mutations affect
the HMG domain of SRY which plays a central role
in DNA binding activity of SRY. We report on a
non-mosaic XY sex-reversed newborn girl (completely female external genitalia). The direct
sequencing of SRY showed a new nonsense mutation in a codon of SRY gene flanking the 30 end of
the HMG domain: a thymine is replaced by a
guanine at position þ387 in codon 129, resulting in
the replacement of the amino acid tyrosine (TAT)
by a stop codon (TAG). The new mutation of this
patient provides further evidence to support the
functional importance of the putative DNA binding activity of the HMG-box domain.
ß 2004 Wiley-Liss, Inc.
gonadal dysgenesis; nonsense
mutation; sex determining region
Y; sex reversal
In males, the sex determining region Y (SRY) gene encodes a
protein (TDF, testis determining factor) that causes the
undifferentiated gonads to develop into testes during embryogenesis. The SRY gene is located on the short arm of the
Y chromosome and consists of an open reading frame (ORF)
single exon within a region of 35 kb adjacent to the
pseudoautosomal boundary. Analysis of the ORF has shown
that the central third encodes a high-mobility-group (HMG)
DNA binding domain. This protein binds to target DNA
sequences and bends it. From this protein–DNA interaction
and DNA flexing arises the main regulatory activity of the
SRY gene.
Point mutations and deletions of SRY gene have been
described in several cases of XY gonadal dysgenesis. To date,
most of these mutations affect the HMG domain of SRY. Here
we report on a non-mosaic XY sex-reversed girl (completely
female external genitalia) caused by a new nonsense mutation in a codon of SRY gene flanking the 30 end of the HMG
The present patient was the third child born to nonconsanguineous healthy parents; mother and father were,
respectively, 40- and 39-years old. Family and pregnancy
history were unremarkable. Because of the mother’s age,
amniocentesis was performed and the karyotype was found to
be 46,XY. Birth weight was 3,600 g, Apgar score was 10 at 10
and 50 , physical examination showed an apparently normal
female with external female genitalia normal in shape and
pigmentation. There were no palpable masses along the inguinal duct; all physical findings were normal.
Cytogenetic analysis performed on T lymphocyte cultures
from baby’s peripheral blood sample confirmed a normal 46,XY
karyotype in all 50 cells examined. Abdominal ultrasound
investigation documented the presence of a uterus; gonads
were not visible. The levels of gonadotropins and gonadal
steroids were normal with a slight increase of 17-OHprogesterone (Table I).
Direct sequencing of the polymerase chain reaction (PCR)
product of SRY was performed. A salting-out method [Miller
et al., 1988] was used to prepare genomic DNA from peripheralblood leukocytes. The SRY gene was amplified using the
upstream primer extF. 50 -ATGCAATCATATGCTTCTGCTA30 complementary to nucleotides þ1 bp to þ 22 bp and reverse
primer extR. 50 -CTACAGCTTTGTCCAGTGGCTG-30 corresponding to nucleotides þ593 bp to þ615 bp relative to the
ATG start codon. Using these primers, a 615 bp PCR product,
encompassing the entire SRY gene, was amplified from
genomic DNA. PCR product was purified, and both strands
were sequenced directly using the ABI Prism 310 Capillary
Sequencer and the following internal primers: intF. 50 ATCATATGCTTCTGCTATGTTA-30 and intR. 50 -GCTTTGTCCAGTGGCTGTAGCG-30 . A new nonsense mutation within
the HMG-box of the SRY gene was discovered in the patient
(Fig. 1): a thymine is replaced by a guanine at position þ387 in
codon 129, resulting in the replacement of the amino acid
tyrosine (TAT) by a stop codon (TAG). This point mutation has
not been described previously.
TABLE I. Serum Hormonal Levels Compared With Normal
Control Females of the Same Age
*Correspondence to: Dr. Mario Giuffrè, Dipartimento Materno
Infantile, Universitá degli Studi di Palermo, Via Cardinale
Rampolla, 1, 90142 Palermo, Italy. E-mail:
Received 8 July 2003; Accepted 13 October 2003
DOI 10.1002/ajmg.a.30075
ß 2004 Wiley-Liss, Inc.
Total testosterone (ng/dl)
Free testosterone (pg/ml)
17-OH-progesterone (ng/ml)
D4-Androstenedione (ng/ml)
Cortisol (mg/dl)
FSH (mU/ml)
LH (mU/ml)
ACTH (pg/ml)
New Mutation (Tyr129Stop) in the SRY Gene
between species and the site of almost all mutations causing
XY gonadal dysgenesis [Harley, 2002].
Affara NA, Chalmers IJ, Ferguson-Smith MA. 1993. Analysis of the SRY
gene in 22 sex-reversed XY females identifies four new point mutations
in the conserved DNA binding domain. Hum Mol Genet 2:785–789.
Bilbao JR, Loridan L, Castaño L. 1996. A novel postzygotic nonsense
mutation in SRY in familiar XY gonadal dysgenesis. Hum Genet 97:
Fig. 1. Sequence analysis of SRY DNA from the patient showing a novel
nonsense point mutation (thymine to guanine) at position 387 determining a
stop signal instead of incorporation of tyrosine at position 129 of the protein
Analysis of the SRY ORF demonstrated a T to G transversion
at the third position of codon 129 (TAT, Tyr) giving rise to a stop
codon (TAG). This point mutation may result in a truncated
non-functional protein leading to complete 46,XY gonadal
dysgenesis. The present patient underwent surgical removal of
the gonadal streaks in the first year of life because of the high
risk of developing a gonadoblastoma.
Mutations in the SRY gene are among the known causes of
XY sex reversal, and an increasing variety of mutations within
this gene have been reported in XY sex reversal patients with
gonadal dysgenesis [Hawkins et al., 1992; McElreavey et al.,
1992a,b; Müller et al., 1992; Affara et al., 1993; Iida et al., 1994;
Bilbao et al., 1996; Veitia et al., 1997; Brown et al., 1998].
Literature data suggest that there is an SRY mutation
incidence ratio of about 10–15% in 46,XY females [Cameron
and Sinclair, 1997], including point mutations, frame-shifts,
and deletions. The novel point mutation, identified in this
patient, creates a stop codon at the 30 end of the HMG-box and is
very likely to produce an impairment of its DNA binding
activity. The ability of the SRY protein to bind and bend the
DNA helix appears to be critical to its function. Mutations in
the SRY HMG box region decrease DNA binding of the mutant
SRY protein and often result in 46,XY unambiguous females
with no testicular differentiation [Hawkins, 1994]. The HMG
domain plays a central role, being the only region conserved
Brown S, Yu CC, Lanzano P, Heller D, Thomas L, Warburton D, Kitajewski
J, Stadtmauer L. 1998. A de novo mutation (Gln2Stop) at 50 end of the
SRY gene leads to sex reversal with partial ovarian function. Am J Hum
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determining genes. Hum Mutat 9:388–395.
Harley VR. 2002. The molecular action of testis-determining factors SRY
and SOX9. Novartis Found Symp 244:57–66.
Hawkins JR. 1994. Sex determination. Hum Mol Genet 3:1463–1467.
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PN. 1992. Mutational analysis of SRY: Nonsense and missense
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McElreavey KD, Vilain E, Abbas N, Costa JM, Souleyreau N, Kucheria K,
Boucekkine C, Thibaud E, Brauner R, Flamant F, Fellous M. 1992a. XY
sex reversal associated with a deletion 50 to the SRY ‘‘HMG box’’ in the
testis-determining region. Proc Natl Acad Sci USA 89:11016–11020.
McElreavey KD, Vilain E, Boucekkine C, Vidaud M, Jaubert F, Richaud F,
Fellous M. 1992b. XY sex reversal associated with a nonsense mutation
in SRY. Genomics 13:838–840.
Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure for
extracting DNA from human nucleated cells. Nucleic Acids Res 16:12–
Müller J, Schwartz M, Skakkebæk NE. 1992. Analysis of the sexdetermining region of the Y chromosome (SRY) in sex reversed patient:
Point-mutation in SRY causing sex-reversion in a 46,XY female. J Clin
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sex, infant, nonsense, mutation, reversal, tyr129stop, identification, genes, sry, newborn, new
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