Review Article
Sex Dev 2016;10:313–325
DOI: 10.1159/000452637
Mechanism of Sex Determination in
Humans: Insights from Disorders of Sex
Development
Anu Bashamboo Ken McElreavey
Human Developmental Genetics, Department of Development and Stem Cell Biology, CNRS, UMR 3738, Institut
Pasteur, Paris, France
Key Words
Ambiguous genitalia · Disorder of sex development ·
Gonadal dysgenesis · Mutation
Abstract
In this review we will consider the gene mutations respon-
sible for the non-syndromic forms of disorders of sex devel-
opment (DSD) and how recent genetic findings are provid-
ing insights into the mechanism of sex determination. High-
throughput sequencing technologies are having a major
impact on our understanding of the genetic basis of rare hu-
man disorders, including DSD. The study of human DSD is
progressively revealing subtle differences in the genetics of
the sex-determining system between the mouse and the hu-
man. This plasticity of the sex-determining pathway is appar-
ent in (a) the difference in phenotypes in human and mouse
associated with the same gene, (b) the different gene regula-
tory mechanisms between human and mouse, and finally (c)
the different and unexpected reproductive phenotypes
seen in association with mutations in well-studied sex-deter-
mining genes. © 2016 S. Karger AG, Basel
© 2016 S. Karger AG, Basel
1661–5425/16/0106–0313$39.50/0
E-Mail karger@karger.com
www.karger.com/sxd
Accepted: September 30, 2016
by M. Schmid
Published online: December 3, 2016
Disorders of sex development (DSD) are defined as
congenital conditions with discordant development of
chromosomal and gonadal/anatomical sex. Just over 10
years ago, at the Chicago Consensus conference, the term
DSD was coined to include previous descriptions such
as intersex, pseudohermaphroditism, hermaphroditism,
and sex reversal [Lee et al., 2006]. These terms were often
confusing, both to clinicians and patients as well as to
other family members. This umbrella definition of DSD
provides a rational basis for the classification of a range
of conditions, but more importantly, it avoids confusion
with terms such as transgender, gender dysphoria, or ho-
mosexuality.
46,XY DSD includes aberrant testis determination or
undermasculinization of an XY male due to errors in ei-
ther androgen synthesis or action. Errors in testis deter-
mination may manifest as either complete gonadal dys-
genesis (CGD) or partial gonadal dysgenesis (PGD).
46,XY CGD is characterized by completely female exter-
nal genitalia, well-developed Müllerian structures, and a
gonad composed of a streak of fibrous tissue, whereas
46,XY PGD is characterized by partial testis formation,
usually a mixture of Wolffian and Müllerian ducts, and
varying degrees of masculinization of the external genita-
lia. Embryonic testicular regression sequence can also be
Anu Bashamboo
Human Developmental Genetics, Institut Pasteur
25 Rue du Dr Roux
FR–75724 Paris cedex 15 (France)
E-Mail anu.bashamboo @ pasteur.fr
regarded as part of the clinical spectrum of 46,XY gonad-
al dysgenesis [Marcantonio et al., 1994]. Affected indi-
viduals have a 46,XY karyotype and usually present with
ambiguous external or internal genitalia. Gonad tissue is
absent on one or both sides. Patients with this condition
are considered to have incomplete testicular determina-
tion with the loss of gonad material early in gestation be-
fore testis differentiation is complete [Marcantonio et al.,
1994]. This also raises the concept that we are dealing
with a continuum of phenotypes rather than clearly dis-
tinct and unrelated categories of atypical testicular for-
mation. The genetic arguments also favor this, since in
some families with 46,XY DSD, the affected individuals
can present as girls with either CGD or PCG or as men
with isolated hypospadias and/or cryptorchidism [Le
Caignec et al., 2003]. Other forms of 46,XY DSD are dis-
orders in androgen synthesis or action. This includes an-
drogen biosynthesis defects such as 17-hydroxysteroid
dehydrogenase deficiency, 5α reductase deficiency, and
StAR mutations. Defects in androgen action include com-
plete or partial androgen insensitivity associated with
mutations in the androgen receptor. Recessive LH recep-
tor mutations result in Leydig cell hypoplasia or aplasia.
Central hypogonadotropic hypogonadism (CHH) occurs
when the physiologic function of the hypothalamic-pitu-
itary-gonadal axis is compromised. In 46,XY individuals
CHH is characterized by delayed or absent sexual devel-
opment and infertility associated with inappropriately
low gonadotropin (LH and FSH) and testosterone levels.
Male patients frequently show under-androgenisation
with micropenis and cryptorchidism observed at birth.
When anomalies of smell, hyposmia or anosmia, is asso-
ciated with hypogonadotropic hypogonadism, in 60% of
patients the disease is called Kallmann syndrome. This
combination of phenotypes is explained by the common
embryonic origins and developmental pathways of GnRH
and olfactory neurons. More than 20 genes are known to
cause CHH [Marino et al., 2014].
46,XX DSD includes overvirilization or masculiniza-
tion of an XX individual due to androgen excess, and the
vast majority of cases of 46,XX DSD are due to congen-
ital adrenal hyperplasia (CAH). The most common form
of CAH is due to deficiency of 21-hydroxylase, which
is caused by mutations in the 21-hydroxylase gene
(CYP21A2) and accounts for 90–95% of all cases [White
et al., 1984; Arlt and Krone, 2007]. The much rarer forms
are 46,XX testicular DSD (TDSD) and 46,XX ovotesticu-
lar DSD (OTDSD). Individuals with TDSD are males
with small and azoospermic testis [de la Chapelle, 1972]
and a normal male habitus. 46,XX OTDSD refers to indi-
314 Sex Dev 2016;10:313–325
DOI: 10.1159/000452637
viduals that have both ovarian and testicular tissue in the
gonads, usually ovotestes but less commonly a testis (or
ovotestis) on one side and an ovary on the other [Ergun-
Longmire et al., 2005]. The external genitalia are usually
ambiguous or feminine, with the degree of masculiniza-
tion broadly correlating with the amount of testicular tis-
sue present. In both TDSD and OTDSD the histological
examination of the gonads shows distinct tubule struc-
tures in the testicular-like tissue and the presence of fol-
licles in the ovarian-like tissue, in the case of OTDSD.
Sex chromosome DSD includes 47,XXY (Klinefelter
syndrome and variants), 45,X (Turner syndrome and
variants), 45,X/46,XY (mixed gonadal dysgenesis or
OTDSD), and 46,XX/46,XY (chimerism or OTDSD).
45,X/46,XY mosaicism is one of the most common causes
of DSD and also one of the most difficult for predicting
genotype-phenotype correlations. In several studies, no
correlation was found between the proportion of the
45,X/46,XY cell lines in the blood or the fibroblasts of the
patient and the phenotype. Furthermore, some of these
cases show mild intellectual disability or signs of autism.
A large number of syndrome associations of DSD have
been described including cloacal anomalies, Robinow,
Aarskog, Hand-Foot-Genital, popliteal pterygium and
Serkel syndrome, and Müllerian duct aplasia. This review
will focus on recent developments in our understanding
of the non-syndromic forms of DSD associated with er-
rors in sex determination.
Incidence of Disorders of Sex Development
There is very limited data available on the precise in-
cidence of DSD. This reflects both the rarity of some of
these conditions as well as the challenge of achieving a
definitive clinical diagnosis. In the newborn, truly am-
biguous genitalia that may pose a problem for binary gen-
der assignment has an estimated incidence of 1: 4,500–
5,500 births [Thyen et al., 2006; Sax, 2002]. Overall,
around 50% of all cases of DSD with truly ambiguous gen-
italia are due to either CAH or 46,XY mixed gonadal dys-
genesis caused by a 45,X/46,XY mosaicism [Thyen et al.,
2006]. The incidence of 46,XY DSD is estimated to be
1:20,000 births and of 46,XY gonadal dysgenesis around
1:100,000 births [Lee et al., 2016]. TDSD/OTDSD are es-
timated to occur in 1: 100,000 births [Sax, 2002]. More
commonly, developmental anomalies of the external gen-
italia may exist in 1 in 300 newborn infants [Nordenvall
et al., 2014]. These include undescended testis or anoma-
lies of the opening of the urethra on the penis (hypospa-
Bashamboo/McElreavey
dias). However, most of this published data on the inci-
dence of DSD is available only from Western countries;
therefore, the worldwide prevalence of DSD is unclear. A
German study indicated that the incidence of ambiguous
genitalia in infants of non-German background was 4×
higher compared to the general population [Thyen et al.,
2006], which they attributed to an increase in autosomal
recessive forms of DSD due to higher rates of consanguin-
ity in the migrant populations. There is some evidence to
support the hypothesis that there is a higher rate of DSD
in societies with a higher rate of consanguinity. The inci-
dence of ambiguous genitalia in Saudi Arabia has been
estimated at 1:2,500 live births, whilst in Egypt it has been
estimated at 1:3,000 live births [Abdullah et al., 1991; Ma-
zen et al., 2008], which is higher than the reported fre-
quency of 1:4,500–1:5,500 in European countries. Anoth-
er hindrance in defining the prevalence of DSD is the lack
of an accurate or even any diagnosis in many cases. In the
German study almost half of the children did not have a
definitive diagnosis by the age of 6 months [Thyen et al.,
2006]. Excluding cases where the biochemical profile in-
dicates a specific error in steroidogenesis, it has been es-
timated that a specific molecular diagnosis is obtained in
about 20% of cases of DSD and that only 50% of 46,XY
children with DSD will receive a definitive clinical diag-
nosis [Lee et al., 2006]. The detailed genetic analyses of
individuals with DSD have been a powerful tool in the
identification of genes involved in sex determination and
therefore DSD.
Gene Mutations and 46,XY Gonadal Dysgenesis
SRY and SOX9
Approximately 15% of 46,XY CGD patients carry mu-
tations in the testis-determining gene SRY, with the ma-
jority of these mutations localized within the functional
DNA-binding HMG-domain [McElreavey and Fellous,
1999] of the protein. Both CGD and PGD are also associ-
ated with small interstitial deletions 5′ and 3′ to the SRY
gene, as well as within the SRY promoter itself [McEl-
reavey et al., 1992, 1996; Assumpção et al., 2005]. In con-
trast to the mouse, the human SRY protein is expressed
in Sertoli cells and germ cells from the moment of testis
determination until adulthood. The role, if any, of SRY in
germ cells is unknown since SRY mutations are associ-
ated with a failure of testis development. SRY mutations
are usually de novo, but some are inherited from a fertile
father. Functional studies suggest that these inherited
mutant proteins retain partial biological activity, and the
Mechanism of Sex Determination in
Humans
incomplete penetrance could be caused by stochastic ef-
fects around a threshold level of biological activity re-
quired for testis formation [Phillips et al., 2011]. Although
SRY mutations are usually associated with gonadal dys-
genesis, a 46,XY woman with premature menopause was
reported to carry a de novo p.Gln2Ter mutation [Brown
et al., 1998].
The direct target of SRY is SOX9, another HMG-box
containing protein. Sox9 plays both an essential role in
the specification and differentiation of mesenchymal cells
toward the chondrogenic lineage through transcriptional
modulation of Col2a1, the major matrix protein of the
mature cartilage as well as establishing Sertoli cell iden-
tity in the developing testis immediately following the
expression of SRY. Many mutations have been reported
in the SOX9 gene and upstream and downstream flank-
ing regions associated with campomelic dysplasia (CD).
There is a variable severity of testicular dysgenesis in
about 75% of affected XY individuals [Foster et al., 1994].
However, recently 2 patients with male external genita-
lia, unpalpable testis, and either hypospadias or micro-
penis have been reported who carried p.Arg394Gly and
p.Arg437Cys heterozygous missense mutations [Katoh-
Fukui et al., 2015]. Neither of the boys had signs of CD,
although one boy carrying the p.Arg394Gly mutation was
reported as having spina bifida. Both of these mutations
are located in the C-terminal PQS-rich domain of SOX9
that is involved in protein-protein interactions with fac-
tors such as CREB-binding protein and p300, both of
which positively regulate gene expression. The boy carry-
ing the p.Arg437Cys mutation presented with testicular
regression sequence, reinforcing the hypothesis that both
gonadal dysgenesis and testicular regression sequence are
part of the same phenotypic continuum.
Besides the point mutations, rearrangements involv-
ing the SOX9 locus are also known to result in non-syn-
dromic 46,XY or 46,XX DSD. The developmental timing
and tissue-specific transcriptional regulation of SOX9 is
highly complex and involves multiple elements located in
flanking regions of at least 1 Mb upstream and 1.6 Mb
downstream. Upstream of SOX9, translocations and in-
version breakpoints associated with CD fall within 2 clus-
ters located about 400 kb apart [Leipoldt et al., 2007].
Large (>1 Mb) duplications 5′ to SOX9 are associated
with brachydactyly-anonychia (symmetric brachydactyly
of the hands/feet, hyponychia or anonychia) [Kurth et al.,
2009]. Pierre Robin sequence is a craniofacial disorder
characterized by micrognathia, cleft palate, and macro-
glossia that is associated with normal testis development
and is caused by either a 75-kb deletion located 1.38 Mb
Sex Dev 2016;10:313–325 315
DOI: 10.1159/000452637
upstream or a deletion located 1.56 Mb downstream of
SOX9 [Benko et al., 2009]. In mice, a testis-specific en-
hancer element has been mapped to a 1.4-kb region
termed Tesco that is located 13 kb upstream of Sox9 [Seki-
do and Lovell-Badge, 2008]. Both Sry and Nr5a1 bind to
the Tesco enhancer sequence in vivo, possibly through a
direct physical interaction to upregulate Sox9 gene ex-
pression. Once Sox9 protein levels reach a critical thresh-
old, several positive regulatory loops are initiated for its
maintenance, including autoregulation of its own expres-
sion and formation of feed-forward loops via Ffg9 or
Pgd2 signaling. Other cofactors are likely to be involved
in this process but have not yet been fully characterized.
Mutations involving the human TESCO element have not
been reported as a cause of DSD, however, rearrange-
ments involving another regulatory element, termed
RevSex, located 600 kb upstream of SOX9, are associated
with both XY and XX DSD. Five cases of 46,XX testicular
or ovotesticular DSD that carried duplications of this re-
gion and a familial case of 46,XY DSD that carried a dele-
tion of the element have been reported [Benko et al., 2011;
Cox et al., 2011; Vetro et al., 2011; Hyon et al., 2015]. The
minimal region associated with 46,XX-SRY negative DSD
has been narrowed down to a 40.7–41.9-kb element,
which contains 2 predicted enhancer motifs [Hyon et al.,
2015]. There is also data suggesting that deletions of an
immediately adjacent and non-overlapping region are as-
sociated with 46,XY gonadal dysgenesis [Kim et al., 2015].
In our experience, about 10% of cases of 46,XX TDSD/
OTDSD and 46,XY gonadal dysgenesis have rearrange-
ments involving the RevSex locus [unpubl. data].
NR5A1
Approximately 15% of all cases of 46,XY DSD are
caused by mutations involving the gene NR5A1. NR5A1
belongs to the subfamily of transcription factors known
as nuclear receptor subfamily 5 (group A, member 1),
which is highly conserved in vertebrates [Morohashi et
al., 1992]. The protein consists of a DNA-binding motif
composed of 2 zinc-chelating modules that coordinate
the interaction between the receptor and hormone re-
sponse element [El-Khairi and Achermann, 2012].
NR5A1 binds DNA as a monomer, with DNA binding
stabilized via a 30 amino acid extension termed the A-
box. The C-terminal ligand-binding domain (LBD) is re-
quired for maximal biological activity with co-activators
such as NCOA1. Posttranslational modification plays an
important role in modulating NR5A1 activation and re-
pressor functions. Phosphorylation of Ser203 within the
LBD enhances the positive interaction of regulatory co-
316 Sex Dev 2016;10:313–325
DOI: 10.1159/000452637
factors, whereas strong transcriptional repression re-
quires sumoylation of the lysines Lys119 and Lys194. Su-
moylation plays an important role in NR5A1 function. If
it is eliminated from the mouse Nr5a1 protein, the mu-
tant mice exhibit marked endocrine abnormalities and
changes in cell fate that reflect an inappropriate activation
of hedgehog signaling [Lee et al., 2011].
In humans, mutations involving NR5A1 are associated
with a wide range of reproductive anomalies including
46,XY gonadal dysgenesis with or without adrenal insuf-
ficiency, ambiguous genitalia, hypospadias, micropenis,
spermatogenic failure with normal genitalia, and primary
ovarian insufficiency [reviewed by El-Khairi and Acher-
mann, 2012]. Familial cases have been described where
both 46,XY gonadal dysgenesis and 46,XX ovarian insuf-
ficiency are present in the same family [Fabbri et al.,
2014]. In a study of 315 men with spermatogenic failure,
we identified heterozygous missense mutations in NR5A1
in 7 men with either azoospermia or severe oligozoosper-
mia [Bashamboo et al., 2010]. Testis histology in one man
with azoospermia was suggestive of a mild form of tes-
ticular dysgenesis rather than Sertoli-cell-only syndrome,
again reinforcing the idea that errors in testis determina-
tion can manifest as different human reproductive phe-
notypes. In all cases, the men carrying the NR5A1 muta-
tions had normal development of the external genitalia
[Bashamboo et al., 2010]. The observation that mutations
involving a key gene in human sex determination, NR5A1,
are associated with either male or female infertility es-
tablishes a link between human sex determination and
fertility.
The mechanism behind the phenotype variability as-
sociated with NR5A1 mutations is yet to be explained,
including variability associated with the same NR5A1
mutation. For example, male infertility, female infertility,
or 46,XY DSD are associated with the variants p.Gly123Ala
and p.Pro129Leu. It is possible that some patients with
NR5A1 mutations carry novel or rare variants in other
genes involved in sexual development that may influence
the severity of the phenotype. We reported 2 individuals
with the same missense mutation p.Arg313Cys in NR5A1
[Allali et al., 2011; Mazen et al., 2016]. In the first case it
was associated with mild hypospadias [Allali et al., 2011],
but in the second case it was associated with 46,XY go-
nadal dysgenesis [Mazen et al., 2016], and in both cases
the mutation was de novo. The more severe phenotype
may be explained by digenic inheritance since the patient
carried a missense mutation in the MAP3K1 gene (see
below). In other cases of DSD carrying mutations in
NR5A1 the severity of the phenotype may be a conse-
Bashamboo/McElreavey
quence of the specific amino acid involved (see NR5A1
p.R92W below). A homozygous p.R103Q NR5A1 muta-
tion was reported in a child with severe 46,XY DSD and
absent spleen. The mutation decreased the ability of the
mutated NR5A1 protein to transactivate TLX1, a tran-
scription factor that is essential for spleen development
[Zangen et al., 2014]. The R103Q mutation impaired ac-
tivation of steroidogenic genes without affecting the syn-
ergistic NR5A1/SRY co-activation of SOX9.
GATA4 and the Cofactor FOG2
GATA4 belong to a class of evolutionarily conserved
lineage-limited zinc finger transcription factors charac-
terized by the presence of 2 conserved type IV zinc finger
domains that participate in cell fate determination, pro-
liferation, and maturation [Zaytouni et al., 2011]. Both
GATA4 and GATA6 are expressed in the somatic tissues
of the embryonic testis [Ketola et al., 1999]. GATA4 co-
operatively interacts with NR5A1 to regulate the expres-
sion of genes critical for testis determination and differ-
entiation [Viger et al., 2008]. Male mice lacking Gata4
show partially descended small testis with irregular
cords, are infertile, and lack expression of Dmrt1 during
embryogenesis [Manuylov et al., 2011]. Gata4ki mice,
which carry a p.Val217Gly mutation in the N-terminal
zinc finger domain of the protein that abrogates the
physical interaction of Gata4 with the cofactor Fog2,
present with severe testicular dysgenesis [Molkentin et
al., 1997; Crispino et al., 2001; Bouma et al., 2007]. Gata4
also plays a pivotal role in Leydig cell function, for ex-
ample a Gata4/Mef2 complex regulates Star gene expres-
sion in mouse Leydig cells [Bergeron et al., 2015; Daems
et al., 2015; Schrade et al., 2015]. Although mutations in
human GATA4 are associated with congenital heart
anomalies, a proportion of XY males carrying deletions
of 8p23.1 that includes the GATA4 gene have hypospa-
dias and bilateral cryptorchidism [Wat et al., 2009]. We
identified a familial case of 46,XY DSD and congenital
heart disease that affected both 46,XX and 46,XY indi-
viduals [Lourenço et al., 2011]. This family carried a het-
erozygous missense mutation (p.Gly221Arg) located im-
mediately adjacent to the mouse p.Val217Gly Gata4ki
mutation in the N-terminal zinc finger domain [Louren-
ço et al., 2011]. In functional studies the p.Gly221Arg
variant failed to bind to DNA, did not to transactivate the
AMH promoter in a transient gene activation assay, and
lacked the ability to bind to its protein partner FOG2.
Although the incidence of GATA4 mutations in associa-
tion with DSD has yet to be established, we have identi-
fied other heterozygous missense mutations in GATA4
Mechanism of Sex Determination in
Humans
in 46,XY DSD patients who have no evidence of heart
disease [unpubl. data].
FOG2 (also known as ZFPM2) is a zinc finger cofactor
that modulates the activity of GATA4 by binding to the
N-terminal zinc finger [Zaytouni et al., 2011]. XY Fog2–/–
mice fail to develop testis, and since the expression of key
genes involved in testis determination such as Sry and
Sox9 is dramatically reduced, it suggests that Fog2 is in-
volved in the early stages of testis determination. The
evidence that FOG2 may be involved specifically in hu-
man testis determination was suggested by 2 cases of
46,XY gonadal dysgenesis in association with apparent-
ly balanced translocations that included the FOG2 lo-
cus on chromosome 8 (t(8;10)(q23.1;q21.1) and t(8;18)
(q22;q21)) [Finelli et al., 2007; Tan et al., 2012]. In both
cases other complex somatic anomalies were reported.
Using exome sequencing, we identified 2 independent
cases, of 46,XY gonadal dysgenesis each with missense
mutations in the FOG2 gene [Bashamboo et al., 2014].
There was no history of cardiac anomalies in either the
patients or their families. Functional studies indicated
that the failure of testis development in these cases could
be explained by the impaired ability of the mutant FOG2
proteins to interact with GATA4. These studies estab-
lished GATA4 and FOG2 mutations as causes of 46,XY
DSD.
CBX2
CBX2 encodes for chromobox homolog 2, a compo-
nent of the polycomb group (PcG) complex of regulatory
proteins. In mammals, PcG proteins are associated with
2 main families of complexes, referred to as polycomb re-
pressive complex 1 (PRC1) and PRC2. These complexes
catalyze mono-ubiquitination of histone H2A on lysine
119 and tri-methylation of histone H3 on lysine 27, re-
spectively. Human CBX2 exists in 2 isoforms, a 532 ami-
no acid isoform termed CBX2.1 and a second shorter 211
amino acid isoform termed CBX2.2. Mice lacking Cbx2
display posterior transformation of the vertebral columns
and sternal ribs, failure of T cell expansion, and XY mice
show male-to-female sex reversal whereas XX animals
have either absent or smaller ovaries [Katoh-Fukui et al.,
1998].
A single patient with 46,XY gonadal dysgenesis and
mutations in the human CBX2 gene has been reported.
This was a 46,XY girl who carried 2 independent muta-
tions in CBX2 – a paternally inherited p.Pro98Leu muta-
tion and a maternally inherited p.Arg443Pro mutation
[Biason-Lauber et al., 2009]. Histology of the gonads at
4.5 years revealed apparently normal ovaries. Although
Sex Dev 2016;10:313–325 317
DOI: 10.1159/000452637
polycomb group proteins are traditionally regarded as
transcriptional repressors, there is evidence that at least
in some cellular or promoter contexts CBX2 acts as a
transcriptional activator of NR5A1 and SRY expression
[Biason-Lauber et al., 2009]. In the patient described
above, the presence of apparently normal ovaries suggests
that CBX2 actively represses fetal ovarian development in
an XY individual. Recently, several potential downstream
targets of CBX2 that are relevant to testis determination
have been reported using a DamID-NGS approach [Eid
et al., 2015]. The gene targets of CBX2 include many fac-
tors that are known or suspected to be involved in sex
development including SOX9, MAMLD1, SOX3, FGFR2,
ATRX, TEX10, EXO1, TBX2, TSPYL4, WTAP, and MTM1
[Eid et al., 2015]. The exact role of CBX2 (and by implica-
tion the PcG complex) in sex determination is unknown,
although both the human and mouse phenotypes suggest
that it is acting at an early stage of gonad formation.
MAP3K1
The mitogen-activated protein kinases (MAPKs) are
activated through an evolutionarily conserved three-
component signal transduction cascade composed of a
mitogen-activated protein kinase kinase kinase 1
(MAP3K1), a MAP2K and a MAPK. Historically, these
were considered to be cellular housekeeping factors, and
it was a surprise to find that mutations in at least some of
these factors could generate gonad-specific phenotypes.
In mice, XY embryos lacking functional Map3k4 on a
predominantly C57BL/6J background exhibit embryonic
gonadal XY sex reversal associated with a failure to tran-
scriptionally upregulate Sry [Bogani et al., 2009]. Mice
lacking Gadd45g, which encodes a protein that interacts
with Map3k4, also show a lack of testis determination that
is associated with a delay in Sry expression [Gierl et al.,
2012]. Furthermore, the absence of both the p38a and
p38b MAPK isoforms results in XY sex reversal associ-
ated with reduced Sry expression [Warr et al., 2012]. Re-
cent data also suggest that Map2k6 is required for the nor-
mal spatiotemporal expression profile of Sry [Warr et al.,
2016]. Therefore, at least in mice, available data are con-
sistent with a GADD45γ/MAP3K4/p38 pathway which is
required for the appropriate timing of Sry expression in
sex determination.
Nineteen MAP3Ks are present in mammals, though
their precise biological roles are not fully understood.
MAP3K1 (also known as MEKK1) plays a role in lympho-
cyte differentiation and function [Suddason and Galla-
gher, 2016], vasculature remodeling [Li et al., 2005], car-
diogenesis [Minamino et al., 2002], as well as injury repair
318 Sex Dev 2016;10:313–325
DOI: 10.1159/000452637
[Deng et al., 2006]. The MAP3K1 protein contains an
amino terminal plant homeodomain (PHD) motif that
has E3 Ub ligase activity, a caspase-3 cleavage site, and a
conserved kinase domain. MAP3K1 is the only MAP3K
that has a PHD motif. MAP3K1 is activated in response
to a number of different stimuli. These include growth
factors, hyperosmolarity, microtubule disruption, cell
shape disturbance, pro-inflammatory cytokines, and
many other physiological stresses [Yujiri et al., 1998]. In
humans, mutations in MAP3K1 have been identified in
cases of 46,XY DSD [Pearlman et al., 2010; Loke et al.,
2014]. The mechanism whereby MAP3K1 mutations
cause a failure of testis determination is unclear. The mu-
tations are heterozygous and are either missense, splice
site, or in-frame deletions. A clearly disruptive mutation,
such as nonsense or frameshift mutation, has not been
identified, and considering the fact that MAP3K1 has a
widespread expression pattern these more severe muta-
tions may be embryonic lethal. Available data suggest that
mutations observed in DSD cases may be subtle gain-of-
function variants that result in the increased phosphory-
lation of the downstream MAPK proteins p38 MAPK and
ERK1/2 [Pearlman et al., 2010; Loke et al., 2014]. Patients
carrying these mutations show no other apparent pheno-
typic anomalies other than 46,XY gonadal dysgenesis [Le
Caignec et al., 2003; unpubl. data].
Mice lacking Map3K1 are viable and fertile but with an
increased embryonic gonadal length [Warr et al., 2011].
XY Map3k1mPHD/+ mice that are heterozygous for an in-
active PHD motif have a significantly enlarged testes but
with a reduced number of Leydig cells [Charlaftis et al.,
2014]. Another Map3k1 mouse model, goya, exhibits a
severe hearing loss [Parker et al., 2015]. This data suggest
that in testis determination either the MAP kinase signal-
ing pathways in human or mouse have diverged or the
difference in phenotype is caused by an intrinsic differ-
ence in the type of mutation. In our experience about 10%
of 46,XY DSD cases with gonadal dysgenesis carry rare or
novel variants in the MAP3K1 gene that could potential-
ly contribute to the phenotype. Although the MAP kinase
signaling pathway involved in mouse testis determina-
tion is becoming clearer, the MAP3K1 pathway or the
targets of MAP Kinase signaling in human testis deter-
mination are unknown. The phenotype associated with
MAP3K1 mutations is 46,XY CGD, a phenotype that is
also associated with mutations in the SRY gene. This sug-
gests that MAP3K1 signaling, like the equivalent pathway
in the mouse, is required for the early stages of testis de-
termination in humans, too.
Bashamboo/McElreavey
 DMRT1, an Evolutionary Conserved Sex-Determining
Gene
Deletions of terminal 9p are associated with monoso-
my 9p syndrome, which is characterized by intellectual
disability together with a distinctive series of somatic
anomalies, and in approximately 70% of 46,XY individu-
als anomalies of testis development are seen that range
from a completely female phenotype to a male phenotype
with hypospadias and/or cryptorchidism [Ottolenghi
and McElreavey, 2000]. Two DMRT genes, DMRT1 and
DMRT3 (DMRTA3), which are orthologues of the dou-
blesex (dsx) of Drosophila and mab-3 of Caenorhabditis
elegans, are located within the minimal recurrently de-
leted region [Raymond et al., 1998]. Dsx controls the ter-
minal switch of the pathway leading to sex fate choice in
Drosophila, and mab-3 is necessary to confer male traits
in C. elegans. In mice, Dmrt1 is not required for testis de-
termination, however, its continuous expression in the
adult testis is required to maintain organ identity, because
forced attenuation of Dmrt1 expression in adult testis re-
sults in transdifferentiation of the testis to an ovary [Mat-
son et al., 2011]. Although deletions of 9p24 suggest that
DMRT1 hemizygosity is sufficient in some individuals to
lead to a failure of testicular development, these deletions
usually remove other genes, including the evolutionary
related DMRT2 and DMRT3 genes [Ottolenghi and
McElreavey, 2000]. Formally, the phenotype could be due
to haploinsufficiency of 1, 2, or all 3 of these DMRT genes.
Evidence to indicate that the key player in human testis
determination is DMRT1 came through the identification
of a de novo missense mutation in the functionally im-
portant DM-DNA-binding domain in a patient with
46,XY CGD [Murphy et al., 2015]. There were no other
somatic anomalies in this healthy girl. The histology of
the gonad was similar to that of an SRY mutation and
showed no evidence of testicular material, suggesting that
the mutation was indeed impacting on primary testis de-
termination. In vitro studies indicated that the mutant
protein had reduced DNA affinity, altered sequence spec-
ificity and when mixed with wild-type protein, it altered
the stoichiometry of the wild-type protein [Murphy et al.,
2015]. This suggests that the lack of testis determination
seen in this patient is due to a combination of haploinsuf-
ficiency and dominant negative activity. This observation
may also explain, at least in part, the absence of mutations
that have been identified thus far in cases of 46,XY go-
nadal dysgenesis. In a screen of over 100 cases of XY go-
nadal dysgenesis, we have only identified a single muta-
tion associated with the phenotype. The residual varia-
tion intolerance score (RVIS) ranks human genes by their
Mechanism of Sex Determination in
Humans
deviation from the genome-wide average number of non-
synonymous mutations found in genes with a similar
amount of global mutational burden (http://genic-intol-
erance.org/). Mendelian disease causing genes are less
tolerant to coding variations than other genes. Put sim-
ply, genes known to carry few common functional vari-
ants in healthy individuals may be considered to be more
likely to cause certain diseases, such as rare forms of DSD,
than genes known to carry many functional variants. The
intolerance score is based upon allele frequency obtained
from whole exome sequence data within the NHLBI-
ESP6500 data set. A good example is the gene SOX9,
which has an RVIS score of 14.4% and a deficit in loss-of-
function variants (%ExAC_RVIS) among the 9.81% low-
est of the human genome. Equivalent %ExAC_RVIS fig-
ures for NR5A1, MAP3K1 and CBX2 are 15.76, 6.58 and
11.63%, respectively. However, DMRT1 has an RVIS
score of 25.56% and a deficit in loss-of-function variants
among the 40% lowest of the human genome. This indi-
cates that DMRT1 is more tolerant to genetic variation in
healthy individuals than the other genes mentioned
above. This suggests that for a DMRT1 mutation to be
pathogenic (or penetrant) resulting in a failure of testis
determination, it may be required to show either domi-
nant negative activity on the wild-type allele or alter the
normal interactions of the protein.
Gonadal anomalies may not be the only phenotype as-
sociated with mutations involving DMRT1. Recently, ge-
nome-wide association studies indicated a variant in the
putative promoter of DMRT1 for sex-specific asthma
[Schieck et al., 2016]. The role, if any, of DMRT1 in the
regulation of allergic immune responses is unclear, but
the expression of DMRT1 was found to be higher in lung
macrophages from men with various lung diseases
[Schieck et al., 2016].
46,XX Testicular and Ovotesticular DSD
SOX Gene Mutations
In recent years it has become evident that the ectopic
expression of HMG-box containing proteins in the uro-
genital ridge at the moment of sex determination may
result in testicular development in a chromosomal XX
female. Although most cases of 46,XX TDSD/OTDSD are
caused by the presence of the SRY gene, usually on the X
chromosome, the remaining cases stay unexplained. As
compared to 46,XY DSD, there are relatively fewer known
causes of 46,XX DSD. In human, SOX3 loss-of-function
mutations are associated with mental retardation and
Sex Dev 2016;10:313–325 319
DOI: 10.1159/000452637
growth hormone deficiency [Laumonnier et al., 2002].
However, three 46,XX SRY-negative testicular DSD pa-
tients have been reported who carry rearrangements at
the SOX3 locus on the X chromosome. One patient car-
ried 2 microduplications, one of ∼123 kb that spanned
the entire SOX3 gene and another of 85 kb that was lo-
cated 350 kb proximal to SOX3. A second patient carried
a single 343-kb microdeletion immediately upstream of
SOX3, and a third XX male with multiple congenital
anomalies carried a 6-Mb duplication including SOX3
and at least 18 other genes [Sutton et al., 2011]. 46,XX
testicular DSD has also been reported in association with
a 774-kb insertion translocation from chromosome 1 into
a palindromic sequence 82 kb distal to SOX3 [Haines et
al., 2015]. Three further duplications of the SOX3 gene
have been reported in XX males [Moalem et al., 2012;
Vetro et al., 2015; Grinspon et al., 2016].
Complete or partial duplications of chromosome 22 in
46,XX-SRY negative individuals are associated with vari-
ous degrees of masculinization [Nicholl et al., 1994; Aleck
et al., 1999; Seeherunvong et al., 2004]. Further delimita-
tion of the minimal region was demonstrated by a de
novo duplication of 22q11.2q13 in a 46,XX SRY-negative
male with mild hypospadias, dysmorphic features, and
hypotonia [Polanco et al., 2010]. Human SOX10 maps to
22q13.1 and may be responsible for the phenotype. Loss-
of-function heterozygous mutations in SOX10 are as-
sociated with Waardenburg-Shah and Waardenburg-
Hirschsprung disease [Pingault et al., 1998; Touraine et
al., 2000], however, in the mouse, transgenic expression
of Sox10 in the gonads of XX mice results in testis forma-
tion [Polanco et al., 2010].
RSPO1/WNT4/β-Catenin Signaling
Little is known about the genetic pathway(s) involved
in human ovary development. In XX individuals, activa-
tion of the β-catenin signaling pathway by the proteins
RSPO1 and WNT4 is necessary for granulosa cell differ-
entiation leading to ovarian development. Stabilization of
β-catenin by the RSPO1/WNT4 pathway results in tran-
scription of its target genes. The mechanism by which
RSPO1 stimulates WNT4 in the developing ovary is un-
known. In general, RSPOs stimulate WNT signaling by
binding to the leucine-rich repeat-containing G protein-
coupled receptors LGR4, LGR5, and LGR6 [Wang et al.,
2013]. RSPOs can also bind to 2 negative feedback regula-
tors of the WNT signaling pathway, the RING-type E3
ubiquitin ligases ZNRF3 or RNF43, leading to their clear-
ance and resulting in enhanced WNT signaling [Jiang et
al., 2015]. Mutations involving RSPO1 and WNT4 are as-
320 Sex Dev 2016;10:313–325
DOI: 10.1159/000452637
sociated with exceptionally rare syndromic forms of
46,XX testicular/ovotesticular DSD. Human homozy-
gous RSPO1 mutations are associated with a rare reces-
sive syndrome, which is characterized by XX testicular
DSD, palmoplantar hyperkeratosis, and predisposition to
squamous cell carcinoma of the skin [Parma et al., 2006].
Mutations involving RSPO1 have not been reported in
non-syndromic cases of testicular and ovotesticular DSD
[unpubl. data].
The absence of Wnt4 in XX mice results in a partial
masculinization of the gonad including the differentia-
tion of some Leydig-like cells. In human, 4 dominant
heterozygous missense mutations in WNT4 have been
reported in 46,XX women with various degrees of viril-
ization including androgen excess and abnormal devel-
opment of Müllerian ducts [Baison-Lauber et al., 2004,
2007; Philibert et al., 2008, 2011]. A single homozygous
WNT4 mutation was reported in a consanguineous fam-
ily with an embryonic lethal syndrome of 46,XX testicular
DSD and dysgenesis of kidneys, adrenals, and lungs
(SERKAL syndrome; SEx Reversion, Kidneys, Adrenal
and Lung dysgenesis) [Mandel et al., 2008].
NR5A1 p.Arg92Trp and 46,XX DSD
Primary ovarian insufficiency, also termed premature
ovarian failure, is defined by the arrest of normal ovarian
function before the age of 40 years and includes prema-
ture menopause, primary and secondary amenorrhea as
well as ovarian dysgenesis. In 2007, when we were ana-
lyzing cases of 46,XX primary ovarian insufficiency
for mutations in the NR5A1 gene, a patient (sporadic case
2; Lourenco et al. [2009]) was clinically investigated at 4
months of age because of a hypertrophy of the clitoris.
The girl had high levels of FSH indicating ovarian insuf-
ficiency and carried an NR5A1 mutation. The clitoral hy-
pertrophy suggested that the girl had been exposed to an-
drogens in utero. Therefore, we decided to screen idio-
pathic cases of 46,XX TDSD/OTDSD for mutations in the
NR5A1 gene. We rapidly discovered a small family with
2 virilized sibs who carried a p.Arg92Trp mutation. The
unaffected mother also carried this mutation. At the time,
this observation was interesting but could simply have
been explained as a chance finding – a rare genetic variant
unrelated to the phenotype. This view was supported by
our screen of further 40 cases of 46,XX DSD that did not
reveal any other NR5A1 mutations. In 2015, we were con-
tacted by different groups who had also identified the
same NR5A1 p.Arg92Trp mutation in association with
46,XX testicular DSD [Bashamboo et al., 2016]. Two fam-
ilies had a single affected child who carried a de novo
Bashamboo/McElreavey
Fig. 1. NR5A1 may regulate anti-testis gene expression in the ova-
ry. In a 46,XY individual, NR5A1 synergizes with SRY to upregu-
late male-specific gene expression (e.g., SOX9) leading to testis for-
mation. In 46,XX individuals, NR5A1 synergizes with β-catenin to
upregulate the expression of anti-testis genes (e.g., DAX-1/NR0B1)
and possibly pro-ovarian genes. In the 46,XY DSD case, the
p.Arg92Trp mutant shows a reduced ability to upregulate SOX9
NR5A1 p.Arg92Trp mutation, and this formally excluded
a founder mutation. A fourth family was identified with
2 affected sibs, one with 46,XY gonadal dysgenesis and
raised as a girl and the other a boy with 46,XX testicular
DSD. They both carried the mutation. A number of mu-
tations have been described in NR5A1 in 46,XX indi-
viduals in association with ovarian insufficiency, but we
are unaware of any other amino acid changes in NR5A1 ex-
cept p.Arg92Trp that is associated with virilization in
46,XX individuals. Interestingly, a homozygous p.Arg92Gln
change has been observed in a 46,XX girl with no evi-
dence of virilization [Guran et al., 2015]. This suggests
that the p.Arg92Trp mutation specifically results in testis
formation in a chromosomal female background (fig. 1).
The mutation involves a highly conserved amino acid res-
idue located in the A-box motif of the protein. The A-box
consists of a 30 amino acid basic region carboxyl-terminal
to the DNA binding domain. NR5A1 binds to DNA as a
monomer with the A-box recognizing DNA sequences 5′
to the NR5A1 consensus motif in the minor groove. The
p.Arg92Trp mutation is predicted to disrupt DNA bind-
ing and this is what we observed using the consensus
binding motif CCAAGGTCA as a target. In contrast the
p.Arg92Gln mutant has essentially wild-type binding ac-
tivity. However, this difference in DNA binding activity
cannot explain why testis determination is occurring due
to the 92Trp substitution. The p.Arg92Trp mutation
could be associated either with inappropriate activation
of testis-specific pathways in the ovary or with disruption
Mechanism of Sex Determination in
Humans
gene expression leading to a lack of testis formation. In a 46,XX
child with TDSD/OTDSD (shown on the right), the same mutant
shows reduced ability to synergize with β-catenin to upregulate the
expression of anti-testis genes. As a consequence of this lack of re-
pression, the expression of pro-testis genes (e.g., SOX9) leads to
testis formation.
of pathways that oppose testis development and maintain
ovarian integrity. Transient transfection assays demon-
strated that the p.Arg92Trp NR5A1 mutant had reduced
activation of several minimal promoters involved in testis
development as well as the Sox9 Tesco enhancer. This is
consistent with a 46,XY PGD phenotype, but it does not
explain testis formation in an XX background. We then
assayed the ability of the 92Trp and 92Gln mutations to
influence the pro-ovary canonical WNT signaling path-
way. β-Catenin can interact functionally with NR5A1 to
modulate target gene expression [Gummow et al., 2003;
Hossain and Saunders, 2003; Jordan et al., 2003; Kennell
et al., 2003; Mizusaki et al., 2003; Parakh et al., 2006; Salis-
bury et al., 2007; Ehrlund et al., 2012]. Specifically, the
Nr5a1 and β-catenin proteins physically interact to up-
regulate the expression of the Nr0b1 (Dax-1) gene on the
X chromosome [Mizusaki et al., 2003]. In 46,XY individ-
uals, 2 copies of NR0B1 result in a failure of testis deter-
mination and are associated with 46,XY gonadal dysgen-
esis, indicating that NR0B1 is an anti-testis gene [Mus-
catelli et al., 1994]. In contrast to the p.Arg92Gln mutant,
we found that the p.Arg92Trp NR5A1 mutant showed
loss of synergy with β-catenin to activate target gene ex-
pression. The phenotype in the 46,XX children with the
p.Arg92Trp variant may be due to altered regulation of
the expression of pro-ovarian genes that normally sup-
press testis development (fig.  2). In humans, NR5A1 is
expressed in the granulosa cells of the early developing
ovary, whereas in the mouse the expression of Nr5a1 in
Sex Dev 2016;10:313–325 321
DOI: 10.1159/000452637
 Schematic diagram of mammalian somatic sex determination

MAP kinases
Genital ridge
GATA4/FOG2/WT1 ?
SRY
FGF9, DMRT1 NR5A1 DHH
PTGDS
?
WNT4 -catenin Foetal
SOX9 Sox8/SOX9 Leydig Cell
RSPO1
NR5A1,GATA4
? NR5A1
WT1, SOX8
NR5A1
AMH steroidogenesis

NR5A1

steroidogenesis DMRT1
DMRT1?
Adult
FOXL2 SOX9 FOXL2 SOX9

TIME
OVARY TESTIS

Fig. 2. The molecular and genetic events in mammalian sex deter-
mination and differentiation. In the XY gonad the activation of
SRY expression, possibly initiated by CBX2/WT1/GATA4/FOG2/
NR5A1, leads to the upregulation of Sox9 expression via a synergy
with Nr5a1 at a Sox9 enhancer such as Tesco [Sekido and Lovell-
Badge, 2008]. In the XX gonad the supporting cell precursors ac-
cumulate β-catenin in response to RSPO1/WNT4 signaling, which
either directly or indirectly represses SOX9 expression, perhaps at
least in human, by interacting with NR5A1 [Bashamboo et al.,
2016]. Once Sox9 levels reach a critical threshold, several positive
regulatory loops are initiated, including autoregulation of its own
expression and formation of feed-forward loops via FGF9 or PGD2
signaling. At later stages, Foxl2 may repress Sox9 expression to
maintain ovarian identity [Uhlenhaut et al., 2009]. In the testis,
Sox9, together with other Sox proteins including Sox8, promotes
the testis pathway by for example stimulating Amh expression, and
it also probably represses the ovarian genes Wnt4 and Foxl2
[Uhlenhaut et al., 2009]. DMRT1 controls sex determination in
some species of fish and may be the master sex-determining switch
in birds. In mice it is not involved in male primary sex determina-
tion, but there is evidence to indicate that it is important in human
testis determination [Murphy et al., 2015].

the embryonic gonad is sexually dimorphic. It is continu- Conclusions

ously expressed in the mouse embryonic testis, but Nr5a1
transcripts are absent during the period of ovarian forma-
tion between E13.5–E16.5 [Ikeda et al., 1994]. Therefore
in this case, the mouse model may not be informative for
elucidation of the mechanism. This further reiterates the
differences between the molecular mechanisms involved
in gonad determination in mouse and human and em-
phasizes the need to analyze human cases of DSD to es-
tablish novel causes of 46,XX and 46,XY DSD.
The last few years have seen a considerable number of
new genes involved in human sex determination that
when mutated cause non-syndromic DSD. A failure of
testis determination, 46,XY gonadal dysgenesis is mainly
associated with point mutations in coding sequences (e.g.
NR5A1, MAP3K1, GATA4, FOG2), whereas gene muta-
tions leading to testis formation in a chromosomal XX
female background are mainly associated with dysregula-
tion of the expression of SOX genes (e.g. SOX9, SOX3,

322 Sex Dev 2016;10:313–325 Bashamboo/McElreavey

DOI: 10.1159/000452637
SOX10). In the latter, point mutations in coding genes are
usually associated with syndromic forms of DSD involv-
ing the RSPO/WNT signaling pathway. Overall, the ge-
netic etiology in the majority of cases of these extreme
forms of DSD is unknown and reflects our poor knowl-
edge of the genetic pathways that are involved. High
throughput sequencing will reveal rare genetic mutations
that are responsible for these phenotypes; however, if we
are to fully establish the causality of these mutations and
understand the mechanisms, a series of complimentary
combinatorial studies using different model systems are
required.
Acknowledgements
A.B. is funded in part by the program Actions Concertees Inter-
pasteuriennes (ACIP). A.B. and K.McE. are funded by a research
grant from the EuroDSD in the European Community’s Seventh
Framework Programme FP7/2007-2013 under grant agreement No.
201444 as well as grant No. 295097 entitled GM_NCD_in_Co – Re-
inforcing IPT capacities in Genomic Medicine, Non Communicable
Diseases Investigation and international cooperation as part of the
EU call FP7-INCO-2011-6. The work is also funded by a Franco-
Egyptian AIRD-STDF grant and the Agence Nationale de la Recher-
che (Laboratoire d’Excellence Revive).
Disclosure Statement

The authors have no conflicts of interest to declare.

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