biology

DSDs: genetics, underlying pathologies and psychosexual differentiation

Valerie A. Arboleda, Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90095-7088, USA

David E. Sandberg, and Department of Pediatrics, Division of Child Behavioral Health and Child Health Evaluation & Research (CHEAR) Unit, University of Michigan, 300 North Ingalls Street, Ann Arbor, MI 48109-5456, USA

Eric Vilain Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90095-7088, USA

Abstract

Mammalian sex determination is the unique process whereby a single organ, the bipotential gonad,

undergoes a developmental switch that promotes its differentiation into either a testis or an ovary.

Disruptions of this complex genetic process during human development can manifest as disorders

of sex development (DSDs). Sex development can be divided into two distinct processes: sex

determination, in which the bipotential gonads form either testes or ovaries, and sex

differentiation, in which the fully formed testes or ovaries secrete local and hormonal factors to

drive differentiation of internal and external genitals, as well as extragonadal tissues such as the

brain. DSDs can arise from a number of genetic lesions, which manifest as a spectrum of gonadal

(gonadal dysgenesis to ovotestis) and genital (mild hypospadias or clitoromegaly to ambiguous

genitalia) phenotypes. The physical attributes and medical implications associated with DSDs

confront families of affected newborns with decisions, such as gender of rearing or genital

surgery, and additional concerns, such as uncertainty over the child’s psychosexual development

and personal wishes later in life. In this Review, we discuss the underlying genetics of human sex

determination and focus on emerging data, genetic classification of DSDs and other considerations

that surround gender development and identity in individuals with DSDs.

© 2014 Macmillan Publishers Limited. All rights reserved

Correspondence to: E.V. [email protected].

Competing interests The authors declare no competing interests.

Author contributions The authors contributed equally to all aspects of the article.

HHS Public Access Author manuscript Nat Rev Endocrinol. Author manuscript; available in PMC 2015 October 01.

Published in final edited form as: Nat Rev Endocrinol. 2014 October ; 10(10): 603–615. doi:10.1038/nrendo.2014.130.

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Introduction

Sex development is a critical component of mammalian development that provides a robust

mechanism for continued generation of genetic diversity within a species. In mammals, sex

development occurs in two distinct and sequential stages: sex determination and sex

differentiation.1 Mammalian sex determination is dictated by the complement of sex

chromosomes within an organism and refers to the mechanisms that lead to specification of

either a male or female gonad from a single undifferentiated bipotential gonad.2 The

presence of a Y chromosome drives the bipotential gonad toward testis-specific

differentiation and formation of a male-specific gonad, whereas the absence of a Y

chromosome results in development of an ovary, which is the female-specific gonad. In the

process of male sex determination, expression of SRY, which is located on the Y

chromosome, initiates a cascade of gene expression within Sertoli cells that ultimately drives

the morphological differentiation of the testis.3,4 In sex differentiation, secretion of

endocrine and local factors, such as testosterone, dihydrotestosterone and anti-Müllerian

hormone, by the testis results in the development of male internal and external genitalia

(prostate, vas deferens, penis and scrotum) and a reciprocal regression of the Müllerian

ducts, which are the precursors of female internal genital structures (Fallopian tubes, uterus

and vagina).5,6 Disorders of sex development (DSDs) arise when this tightly regulated

process is disrupted, which occurs primarily as a result of genetic mutations that interfere

with either the development of the testes or ovaries or the actions of endocrine and local

factors in extragonadal tissues.7,8

This Review covers the current classifications and emerging knowledge of genetic

mechanisms that contribute to sex and gender development among individuals with errors in

sex determination that culminate in the development of DSDs. Current concepts of human

sex determination and data from mouse models that have contributed to understanding of

mammalian gonadal development are discussed. Aberrations in the process of sex

differentiation (for example, defects in androgen biosynthesis) are also known to result in

DSDs; however, these mechanisms have been reviewed elsewhere.9,10

Classification of DSDs

In 2006, the Consensus Statement on Management of Intersex Disorders was drawn up by

international experts and patient advocates under the auspices of the Pediatric Endocrine

Society and the European Society for Paediatric Endocrinology. The consensus conference

was convened to review clinical management practices in DSDs given new advances in

diagnosis, introduction of modified and new surgical techniques, studies of psychosocial

factors pertinent to outcomes and challenges to clinical practices voiced by patient advocacy

organizations. A key consensus recommendation involved the introduction of new

terminology to replace confusing and potentially stigmatizing terms, including intersex,

pseudohermaphroditism, hermaphroditism and sex reversal.7,8 Instead, the expert panel

recommended that all variations of sex development should be incorporated under the

superordinate term DSDs, which they defined as “congenital conditions in which

development of chromosomal, gonadal, or anatomic sex is atypical”. Within this definition,

DSDs can have a wide range of gonadal phenotypes, such as partial or complete gonadal

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dysgenesis and ovotestis, and external genital phenotypes, such as hypospadias,

clitoromegaly and ambiguous genitalia or fully masculinized or feminized genitalia that are

discordant with karyotype or gonadal phenotypes. Using this inclusive definition, DSDs are

estimated to occur in approximately one in 100 live births.8,11 However, the incidence of

46,XY gonadal dysgenesis, in which genetic mutations result in disruption of testis

development, is estimated at less than one in 10,000 live births. Given the rarity of this

condition, neither the distribution of genetic mutations within these patients nor the long-

term consequences of the diagnostic and management challenges associated with 46,XY

gonadal dysgenesis are well understood. To address this issue and other gaps in our

understanding, large multicentre studies in both the USA12,13 and Europe11,14 are ongoing.

Despite rapid and broad acceptance of the consensus definition of DSDs by clinical and

research communities, controversy remains regarding whether particular conditions (such as

Turner and Klinefelter syndromes) and genital phenotypes (such as hypospadias) constitute

DSDs and, therefore, require a multidisciplinary approach to clinical management. The

continuing uncertainty regarding definitions of DSDs carries with it a number of potential

negative consequences. Most important among these drawbacks is the reduced likelihood

that a patient with a DSD will receive comprehensive and integrated care if seen outside of a

multidisciplinary DSD clinic.7,8 For example, endocrine abnormalities in a patient who has

descended testes but has a history of mild or severe hypospadias might remain undetected

until puberty.15,16 Additionally, despite positive and objectively-assessed cosmetic results

following surgery to repair hypospadias, patients are frequently less satisfied with sexual

function and less likely to experience intimate relationships than unaffected individuals,

highlighting the importance of behavioural and/or sexual health involvement in the model of

care.17 Incorporation of a multidisciplinary healthcare team that includes psychological

counselling at the time of diagnosis could contribute to identification and prevention of

adverse effects consequent to the DSD. One barrier to providing multidisciplinary care to a

large population of patients with DSDs is the perception of a lack of resources. Contrary to

this misperception, increased numbers of patients can make the logistics of multidisciplinary

clinics practical. With regard to behavioural health services only a small proportion of

patients with a DSD and their families would be expected to require services beyond

anticipatory guidance and even fewer patients and families would be expected to need

additional targeted interventions, such as addressing pre-existing psychosocial stressors that

include financial problems or family conflict. Finally, clinical treatment would be reserved

for patients and/or their families who exhibit multiple risk factors for maintaining distress,

such as chronic and marked anxiety, depressive symptoms or other individual or family-

based psychosocial problems.18 Triaging of resources can be effectively accomplished using

psychometrically robust psychosocial screening tools, such as the Psychosocial Assessment

Tool and Child Behavior Checklist.19

The distinction and clarification of the terms DSD and intersex is important and necessary.

The term DSD was introduced to emphasize underlying genetic and hormonal factors

responsible for atypical somatic sex development. However, there are individuals with

DSDs who have assumed the term intersex as an identity and reject the notion that the

human body must be dichotomous. These individuals view the term DSD as a negative label

that implies that atypical sex anatomy must be corrected with surgical or hormonal

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interventions.20,21 Supporters of this position recognize that some interventions may be

necessary in order to maintain physical health, such as removal of dysgenetic gonads in

patients at high risk for malignancy, but call for a clear distinction between what is

medically necessary versus what is elective or cosmetic.22–24

Genetic pathways of sex determination

Testis

The identification of genes involved in sex determination was spurred on by the discovery

and characterization of the testis-determining gene SRY, which is a Y-chromosome-linked

gene that encodes a transcription factor. In humans, levels of SRY mRNA are upregulated in

the urogenital ridge at 7 weeks after conception and drive the bipotential gonad towards

testis formation in 46,XY individuals.25,26 After translation, the SRY protein translocates to

the nucleus and binds to the enhancer regions of SOX9, to drive the differentiation and

proliferation of Sertoli cells and testis tubule organization.27–29 Definitive evidence that SRY

is the initiating factor in human male sex determination was provided by the discovery of

missense mutations that disrupt the DNA-binding region of SRY, nonsense mutations, and

deletions in the 5′ or 3′ regulatory regions of SRY that alter the timing or levels of SRY

expression and lead to 46,XY gonadal dysgenesis.3,30–33

The second major gene involved in male sex determination is the gene that encodes the

SRY-related transcription factor SOX9. In humans, autosomal dominant mutations in SOX9

cause campomelic dysplasia with a 46,XY DSD and external genitalia ranging from

ambiguous genitals to a female phenotype.34,35 SOX9 protein expression is required for

testis determination and, in conjunction with SRY and the transcription factor NR5A1, the

SOX9 protein binds to its own promoter to perpetuate a positive feedback loop that

maintains high levels of SOX9 expression. In mouse models, Sox9 mRNA expression is also

maintained by activation of the Fgf9–Fgfr236–38 and prostaglandin D2 (PGD2) signalling

pathways.39 Clinical syndromes associated with skeletal dysplasias have been described for

mutations in FGFR2.40 However, mutations that result in gonadal dysgenesis and DSDs in

humans have not yet been identified in components of the FGF9–FGFR2 or PGD2

signalling pathways, which suggests that phenotypes associated with such mutations might

result in embryonic lethality or have redundant functions in the genetic networks that drive

sex determination.

A number of mutations in the genes encoding additional transcription factors that are

involved in testis determination have been identified in human DSDs. NR5A1 (also known

as steroidogenic factor 1, or SF-1), encodes an orphan nuclear receptor, which is a major

contributor to the development of the hypothalamic–pituitary–gonadal–adrenal axis.41–43

High expression of Nr5a1 in the mouse bipotential gonad suggested a role for this gene in

cell proliferation prior to the onset of testis determination and in the upregulation of both Sry

and Sox9 gene expression.44 Initial reports highlighted the involvement of NR5A1 mutations

in gonadal and adrenal dysgenesis in 46,XY individuals with a female phenotype.43 In pre-

Sertoli cells, NR5A1 synergizes with the transcription factor GATA4 at the onset of testis

determination and binds to the SRY promoter to upregulate SRY expression.45 Further

analysis of NR5A1 in individuals with DSDs or with infertility issues has shown that

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mutations in this gene are associated with a variable range of phenotypes from mild

hypospadias to ambiguous genitalia46,47 as well as infertility in adulthood.48,49 Emerging

studies have also demonstrated that 46,XY individuals with mutations in NR5A1 who are

phenotypically female may present with clitoromegaly that is secondary to elevated

testosterone levels at the onset of puberty despite their dysgenetic gonads.50

NR0B1 (also known as DAX1) is located on the X chromosome (at p21.3) and like NR5A1

encodes an orphan nuclear receptor that has a function in mammalian sex determination.51

Duplications encompassing NR0B1 in humans52 or transgenic overexpression of Nr0b1 in

mice53,54 lead to dose-dependent XY gonadal dysgenesis and a female phenotype. As XY

individuals have only one copy of this gene, a duplication that results in NR0B1

overexpression is sufficient to block testis determination. One of the molecular mechanisms

identified in XY mice transgenic for Nr0b1 is through direct inhibition of Nr5a1-mediated

transcription of Sox9.54 In 46,XX female individuals, the two functioning copies of the

NR0B1 gene are crucial to preventing testis formation. Loss-of-function mutations or

deletions of NR0B1 lead to congenital adrenal hypoplasia and life-threatening adrenal failure

that is associated with abnormalities in male genital development owing to decreased

steroidogenesis.55

The GATA4 and ZFPM2 (also known as FOG2) genes encode transcription factors that are

critical for testis development. The discovery of a familial heterozygous missense mutation

in GATA4 that resulted in a Gly221Arg mutation and development of a 46,XY DSD, which

was also associated with congenital heart disease, underscores the fundamental role of

GATA4 in both gonad and cardiac development.56 Genetic associations between two

unrelated patients with rare mutations in ZFPM2 who also have 46,XY gonadal dysgenesis

have recently been described, which further underscores the important role of GATA4–

ZFPM2 interactions in testis determination.57 In mouse models, mutations that disrupt

associations between Gata4 and Zfpm2 give rise to abnormal testis development.58 In a

porcine model, GATA4 directly activated the SRY promoter; however, in humans and mice

direct activation of SRY expression has only been observed when the WT1 protein is

coexpressed.59 The studies in mice support the findings in patients with mutations in these

genes and offer additional mechanistic insights into sex determination. Mutations in Gata4

or Zfpm2 resulted in decreased interactions between Gata4 and Zfpm2 proteins, which led to

decreased ability of either gene (independently or when coexpressed) to activate

transcription of target genes such as Amh, Sry, and Sox9.56,57,59

Deletions that encompass the region surrounding the DMRT1 and DMRT2 genes, which are

located on chromosome 9p, have been identified in multiple cases of 46,XY gonadal

dysgenesis with ambiguous genitalia.60,61 Fine mapping of this region has narrowed the

minimal region associated with 46,XY gonadal dysgenesis to a small 260 kb region

upstream of the DMRT1 and DRMT2 genes.60 In addition to its role in somatic cell

determination within the gonads, a highly significant locus on chromosome 9p near DMRT1

was identified in genome-wide association studies performed in patients with gonadal germ-

cell tumours.62 The finding that DMRT1 is associated with a propensity towards germ-cell

tumours is consistent with the phenotype of Dmrt1-knockout mice, which have a profound

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failure in postnatal maintenance of the germline63,64 and, on specific genetic backgrounds,

also have increased susceptibility to gonadal germ-cell tumour formation.65

Within testis differentiation, DMRT1 is critical for the maintenance of Sertoli cell fate. Once

the testis fate has been established, the phenotypes of the heterogeneous cells within the

testis must be actively maintained. In mice, postnatal expression of Dmrt1 simultaneously

promotes testis-specific gene expression in Sertoli cells, through maintenance of high levels

of Sox9 expression, and represses ovary-specific granulosa cell differentiation. Loss of

Dmrt1 from Sertoli cells in the postnatal mouse testis results in transdifferentiation of these

cells into granulosa cells.66

An individual with multiexonic deletions in the WWOX gene on chromosome 16 presented

with 46,XY gonadal dysgenesis (see Table 1),67 which suggested that this gene was also

involved in testis development. Pathological analysis of the gonads of this patient revealed

immature testis and the presence of premalignant gonadal germ cells.67 Given this

phenotype it is possible that WWOX may function in the same developmental pathway as

DMRT1 to promote somatic cell differentiation of Sertoli cells and maintain germ cells.

Additional studies in mice support this hypothesis, as XY mice homozygous for a

hypomorphic Wwox allele have testicular atrophy and decreased fertility.68

Ovary

Before the emergence of molecular and genomic tools in the past decade, ovarian sex

determination was considered a passive default pathway that occurred in the absence of SRY

expression and testis development. An equivalent gene to SRY has not yet been identified for

ovarian differentiation. Furthermore, few morphological changes indicative of ovarian

development have been identified that occur at the equivalent developmental time point as

when an XY bipotential gonad begins to develop the organizational structure of the testis.

Despite the lack of visible morphological changes, gene expression patterns within XX

somatic cells in the bipotential gonad have been shown to drive differentiation of granulosa

cells and steroid-producing theca cells.69 The primary signals for initiation of granulosa cell

differentiation are unclear. Nevertheless, high mRNA levels of the signalling factors WNT4

and RSPO1 upregulate expression of and stabilize the transcription factor CTNNB1 (also

known as β-catenin), which suppresses male-specific SOX9 expression, maintains WNT4

gene expression and promotes germ-cell proliferation (Figure 1).70–73

Morphological differentiation of human ovaries occurs at week 7 of gestation, which is the

developmental stage in which female germ cells enter the first steps of meiosis. Much of the

knowledge we have of this developmental process has been gleaned from studies in animal

models. In mice, meiosis in female germ cells is triggered by cell-extrinsic factors, such as

retinoic acid and expression of Stra8.74,75 At this point, high levels of Wnt4 signalling

through either Frizzled or Lrp5–Lrp6 receptors drive both germ cell meiosis and

differentiation of theca cells.76 Similar to the developing testis, after the point of fetal

ovarian determination, the ovarian phenotype and granulosa cell differentiation are actively

maintained by expression of the FoxL2 protein and the estrogen receptors α and β.77–79 Loss

of Foxl2 expression in mouse adult granulosa cells results in upregulation of Sox9 and

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transdifferentiation towards a Sertoli cell phenotype. Thus, like male sex determination,

initial ovarian determination is dependent on Wnt4 and Rspo1 signalling, while maintenance

of germ cells and the ovarian phenotype requires other proteins, such as FoxL2 and the

estrogen receptors.

Failure of correct ovarian development in individuals with the 46,XX karyotype can result in

one of two distinct states: gonadal dysgenesis or the presence of some testicular tissue

within a 46,XX gonad. Gonadal dysgenesis has been suggested to have a distinct aetiology

from that of a DSD; however, we believe that failure of ovarian development, particularly

during the early stages, is similar to 46,XY gonadal dysgenesis. The primary difference

between 46,XX and 46,XY gonadal dysgenesis is that, in the former case, the phenotype of

the internal and external genitalia is congruent with the complement of sex chromosomes.

By contrast, in individuals with the 46,XY karyotype, there is some degree of phenotypic

variability of the external genitals that can range from hypospadias to a female phenotype

and can include ambiguous genitalia.7

Gonadal dysgenesis associated with the 46,XX karyotype manifests clinically as primary

ovarian insufficiency, which is defined as premature depletion of ovarian follicles and onset

of menopause before 40 years of age.80 This condition is represented by a range of

phenotypes, from individuals who have not entered puberty by 15 years of age81 to those

who experience early cessation of ovulation known as secondary amenorrhoea.80 Although

primary ovarian insufficiency can occur as a result of a variety of nongenetic aetiologies (for

example, chemotherapy, auto-immunity and environmental factors), a genetic basis is

thought to be the primary cause in 10–15% of all cases.82,83

Female sex determination is intricately linked with the initiation of meiosis in germ cells.84

The most frequent genetic cause of gonadal dysgenesis in a phenotypic female is 45,X

Turner syndrome, which is estimated to occur in one in 2,000 live female births.85 The

follicle depletion observed among patients with 45,X Turner syndrome can result both from

haploinsufficiency of critical genes on the X chromosome that escape X-inactivation and

from incorrect pairing of the X chromosomes during meiosis.

Mutations or deletions that affect the expression of the autosomal gene FOXL2 can lead to

46,XX gonadal dysgenesis with blepharophimosis, ptosis and epicanthus inversus syndrome

(BPES) in humans and goats.86,87 BPES in humans is divided into two types. BPES type I is

a sex-limited, autosomal dominant form with the full spectrum of disease. This severe

phenotype results from truncating mutations that lead to haploinsufficiency of FOXL2

protein.86 BPES type II is limited to the blepharophimosis phenotype, is present in both

male and female individuals and has no gonadal phenotype. The subset of patients who have

this limited form of the disease have small duplications within the FOXL2 gene.88 BPES

types I and II can rarely occur together within a single family.88 Autosomal dominant

mutations in the nuclear orphan receptor NR5A1, described in detail above, can also result in

premature ovarian failure in 46,XX individuals and account for <3% of all genetic cases of

this condition.48,89,90

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In addition to FOXL2, ovarian determination is dependent on the presence of either

functional WNT4 or functional RSPO1.91 Mutations in WNT4 and RSPO1 (Tables 1 and 2,

Figure 1) are discussed in detail in the section of this Review focused on signal transduction

pathways. Genetic mutations in the WNT4 gene can have a wide range of functional effects,

including impaired lipid modification, defects in receptor signalling, and aggregate

formation.92,93

Testicular or ovotesticular DSDs that are associated with the 46,XX genotype are rare and

arise primarily as a result of ectopic expression of SOX-family genes that are related to the

major testis determining gene, SRY, within the fetal bipotential gonad. Up to 90% of isolated

46,XX testicular DSDs involve a translocation of SRY to the X chromosome or to an

autosome.94 A portion of the remaining cases can be explained by rare duplications or

deletions in the promoter and enhancer regions of SOX genes, including SOX9,95–98

SOX399,100 and SOX10101,102 (Table 1). In such cases, testicular tissue develops and causes

some degree of masculinization of both the internal and external genital structures.

Individuals with rare syndromic 46,XX testicular or ovotesticular DSDs have been identified

who harbour mutations in RSPO1.71

Pathways of sex determination

As discussed above, transcription factors have a key role in male sex determination;

however, other factors and pathways have recently been recognized to play an important

part in this process.

Signal transduction pathways

A number of studies have addressed the role of cellular proliferation in the bipotential gonad

and in the period before the onset of sexually dimorphic changes in sex determination. At

the initiation of male sex determination, the WNT4 and RSPO1 signalling pathways become

sexually dimorphic, with downregulation of WNT4 and RSPO1 expression and upregulation

of SOX9 expression in the developing testis.70,71,73

Mitogen-activated protein kinase (MAPK) signalling has is the dominant pathway in testis

determination (Figure 1). Heterozygous missense mutations in MAP3K1 have been

described in six published cases of 46,XY DSDs.103,104 Additionally, functional studies in

human cell lines suggest that gain-of-function mutations in MAP3K1 shift the balance of sex

determination from testis development (driven by SOX9–FGF9 signalling) towards ovarian

development (mediated by WNT4 and β-catenin signalling).105 Furthermore, studies in mice

have demonstrated that phosphorylation of the transcription factor Gata4 is regulated

through MAP3K4 signalling. This pathway might also regulate Sry expression during fetal

gonadal development via modulation of chromatin structures.106

Mutations in another signalling pathway, the desert hedgehog (DHH) pathway, have also

been described in a small subset of patients with 46,XY DSDs with complete gonadal

dysgenesis, as well as in a subset of patients with minifascicular neuropathy.107,108

Additionally, autosomal recessive mutations in the hedgehog acyltransferase gene, HHAT,

have been identified in a patient with a 46,XY DSD with gonadal dysgenesis and

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chondroplasia.109 Gonad-specific deletion of Hhat in mice resulted in abnormal testis tubule

formation, decreased gonadal size, and testicular dysgenesis. High expression of Sox9 and

Cyp11a1 (which encodes a steroidogenic enzyme marker of Leydig cells) in these mice

showed that differentiation of both Sertoli and Leydig cells was affected; however, Sry

expression remained normal, which indicated that these disruptions occur downstream of

Sry expression.109

In female sex determination, WNT4 and RSPO1 signalling via β-catenin is crucial for

normal ovarian development. In humans, dominant missense mutations in WNT4 have been

associated with Müllerian aplasia and hyperandrogenism,92,93 a finding that is supported by

the phenotypes of female Wnt4-deficient mice.70 Rare recessive mutations in WNT4 cause

SERKAL syndrome, which is associated with multiple developmental anomalies in the

kidneys, adrenal gland and lungs, as well as testicular DSDs with ambiguous genitalia in

individuals with the 46,XX karyotype.110 Findings in Wnt4-knockout mice predated the

human discoveries and demonstrated early loss of germ cells and increased expression male

steroidogenesis enzymes, which leads to elevated androgen levels driving Wolffian duct

formation.70 Further studies have shown that Wnt4 also functions to upregulate retinoic acid

signals that both initiate the onset of meiosis in germs cells and maintain germ cells

throughout adult life.74,75,111

In conjunction with WNT4, RSPO1 is upregulated in the developing human ovary, and

missense mutations in this gene result in syndromic forms of 46,XX testicular and

ovotesticular DSDs that are associated with palmoplantar hyperkeratosis and a

predisposition for development of squamous cell carcinoma in the skin.71,73 Findings in

mouse models suggest that Rspo1 is required for ovarian determination in both normal XX

individuals and in individuals with XY sex reversal.37,71 Thus, the interplay between

signalling pathways and transcription factors in ovary determination is without clear

hierarchy, unlike that which exists with SRY signalling in testis determination. What is clear

in both ovary and testis determination is that multiple pathways are required to program the

heterogeneous group of cells within the differentiating gonads.

Epigenetic pathways

The emergence of sequencing technologies has enabled dissection of epigenomes in vivo on

a large scale and has brought heightened recognition of the importance of crosstalk between

epigenetic and transcriptional factors in developmental processes. Epigenetic factors that

influence the expression of SRY have a substantial role in sex determination.

Chromobox2 (CBX2) has been shown to modulate DNA histone marks, which alter the

expression of downstream genes in other developmental processes. To date, a single case of

an individual with compound heterozygote mutations in CBX2 that result in Pro98Leu and

Arg443Pro amino acid substitutions has been reported.112 A follow-up study of 47 patients

with 46,XY or 46,XX DSDs did not identify any pathogenic CBX2 mutations, which

indicates that CBX2 is probably not a frequent cause of these disorders.113 The two CBX2

mutations were placed independently into the gene that encodes the long-CBX2 isoform and

transfected into a variety of human cell lines. Introduction of each mutation independently

disrupted DNA transactivation of downstream sex determination genes, such as NR5A1, and

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transfection of the same cell with a gene that carried both mutations had a synergistic effect

and further decreased activation of the sex determination genes.112

Mechanistic studies using a human epithelial cell line grown in culture suggest that CBX2

functions as a component of the Polycomb repressive complex, which silences transcription

through binding to H3K27me3 histone tags (Figure 1)114 In addition to CBX2’s known role

as part of a polycomb repressive complex, emerging evidence in both Drosophila

melanogaster and mouse models suggests that the long isoform of the protein may influence

the transactivation of sex determination genes through its DNA-binding domain.115,116

Findings from mouse models of sex determination suggest that Cbx2 acts upstream of Sry to

either directly or indirectly upregulate gene expression and suppress the ovarian

determination pathway in XY mice.117 However, whether one or both of the known roles of

CBX2 are important in human gonadal development has yet to be determined.

Copy number variations

The emergence of high-density single nucleotide polymorphism analyses to identify small

and large copy number variations (CNVs) on a genome-wide scale has highlighted the

importance of noncoding regions of the genomic DNA in the development of DSDs. A

summary of duplications and deletions that have been implicated in DSDs is shown in Table

1.

Large and rare chromosomal duplications and deletions are associated with DSDs and these

genomic regions are likely to harbour genes involved in sex determination. The most

prominent chromosomal anomalies include 9p deletion60 (the region that contains the

known sex determination genes DMRT1 and DMRT2), Xp duplication52,101 (the location of

NR0B1), 10q deletion118,60 (encompassing FGFR2) and 22q duplication60,102 (the location

of SOX10).

There remain a number of rare DSD-associated CNVs in which a causative gene in sex

determination has not yet been identified or in which the putative gene has not been well

studied in the context of sex determination. Heterozygous deletions in the 13q33.2

chromosomal region result in a 46,XY male phenotype with ambiguous genitalia and growth

restriction and, depending on the size of the deletion, multiple organ malformations. The

critical region encompasses a 9.5 Mb region that contains over 20 genes, of which EFNB2 is

a potential candidate given that the phenotype of mice with ephrin-b2 knocked out show

defects in urorectal development.119,120

Deletions on 19q12q13 have been described in several patients with 46,XY DSDs

characterized by cryptorchidism, ambiguous genitalia, intrauterine growth restriction and

other developmental anomalies;60,121 however, no genes have been associated with DSDs

located in this region. The smallest region of overlap in these patients that is associated with

a DSD phenotype has been difficult to pinpoint, as deletions in this region have a wide range

of variable phenotypes with approximately 75% of affected individuals demonstrating a

DSD phenotype.121

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Small chromosomal deletions or duplications that result in either haploinsufficiency or

overexpression of a single sex determination gene can also lead to development of a DSD.

For example, deletions in WWOX67 and duplications in NR0B152 can each lead to

development of 46,XY DSDs. In addition, CNVs within noncoding regulatory regions of sex

determining genes are associated with development of DSDs in individuals with 46,XY and

46,XX karyotypes.31,100,122 The exact mechanisms by which these noncoding CNVs

function is incompletely understood; however, it has been postulated that they disrupt long-

range repressor or activator elements or alter the balance between these elements, which are

critical for correct spatiotemporal expression of nearby sex determination genes.100 Several

noncoding CNVs have been described for SOX9.96,98,101 Nevertheless, it is important to

note that while 98% of the human genome is non-protein-coding, emerging evidence

suggests that a large proportion of the non-protein-coding DNA is transcribed into

functional RNA molecules, such as long non-coding RNA and microRNA species. These

regions may harbour causative mutations in non-protein-coding genome that might function

to regulate developmental gene expression.123

Multigenic inheritance

The availability of next-generation sequencing technologies has provided the potential to

identify concurrent mutations in genes that are associated with DSDs, which might

contribute to pathogenesis in a cumulative fashion. One such example is the discovery of

mutations in AKR1C4 and AKR1C2, which are involved in regulating fetal testosterone

synthesis. In two families with multiple affected members who presented with 46,XY DSDs

and varying degrees of undervirilization and cryptorchidism, mutations in AKR1C4 and

AKR1C2 were identified using Sanger sequencing of coding regions of genes within an

overlapping linkage peak. Variable inheritance of splicing and missense mutations in

AKR1C4 and AKR1C2, respectively, were found to result in a dose-dependent 46,XY DSD

with ambiguous genitalia.124 The variable expression that is often observed in large families

with multiple members affected by DSDs might be influenced by co-inheritance of other

mutations in sex determination genes or modifier genes.

The shift from traditional viewpoints of Mendelian genetic inheritance as relates to diseases

has been cultivated as the complexities of the human genome continue to be unravelled.

Mutations within the same developmental pathways may be additive or multiplicative and

can result in an increased mutational load and increased severity of the DSD phenotype. An

example of a DSD with complex inheritance is Bardet–Biedl syndrome. This syndrome is a

ciliopathy characterized by dysfunction in multiple systems and is associated with obesity,

developmental delays, renal malformations and genital anomalies. The pattern of inheritance

of Bardet–Biedl syndrome displays substantial locus heterogeneity and has been shown to

map to at least 15 loci with rare instances of triallelic inheritance.125,126 Advances in next-

generation sequencing will enable rapid genetic diagnosis in patients with DSDs that arise

from multigenic inheritance, such as Bardet–Biedl syndrome or in cases with multiple

defects within the same steroidogenic pathway.

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Environmental factors

In addition to the genetic factors addressed in this Review, the contribution of environmental

factors to gonadal and genital development, which range from intra-uterine environment and

placental insufficiency127,128 to low doses of endocrine disruptors, such as industrial and

agricultural chemicals,129 cannot be discounted. Emerging evidence suggests that an

association might exist between environmental factors and development of DSD-associated

conditions such as cryptorchidism and hypospadias.129–131 However, precise understanding

of the contributions of genetic, hormonal and environmental factors to genital development

and their interactions with genes remains limited.

Genetic diagnosis

The genetic and clinical diagnosis of DSDs has become increasingly complex as a

consequence of the rapid expansion of knowledge about the genetic mechanisms that cause

DSDs over the past 20 years. Mutations in the same gene can result in a wide range of

genital phenotypes among individuals with DSDs; conversely, similar genital and gonadal

phenotypes in different individuals can be caused by mutations in any one of 20 or more

genes.12 When an individual presents with a suspected DSD, a minimal initial

endocrinological and genetic evaluation is required to rule out life-threatening adrenal

disorders, such as congenital adrenal hyperplasia.132 However, technological advances have

provided the tantalizing possibility that next-generation sequencing might be used for the

primary identification of genetic mutations associated with DSDs, either using a targeted

sequencing panel or whole-exome sequencing with a focus on genes known to be associated

with DSDs.132,133

Given the limitations of current understanding of the human genome, it is imperative to

evaluate the parental DNA alongside that of the affected child in order to narrow down the

pathogenic genetic variant, particularly in the case of previously unidentified mutations.

Many types of genetic mutations (missense, nonsense, small deletions, small insertions,

deletions and duplications) and inheritance patterns can be ascertained using whole-exome

sequencing, with the exception of deletions, duplications, or chromosomal inversions that

are limited to the non-coding portion of the human genome.

Next-generation sequencing provides a rapid and unbiased approach to detection of genetic

mutations compared with the approach of sequentially sequencing individual genes, which is

both expensive and can delay diagnosis and initiation of appropriate treatments for

individuals with DSDs. Next-generation sequencing can identify a genetic diagnosis in

almost 40% of cases of DSDs, a number which is likely to be an underestimate, as only

cases that are refractory to other diagnostic approaches, such as a thorough endocrine work-

up and single-gene sequencing are currently sent for this type of analysis. Given the

limitations of whole-exome sequencing mentioned above, the cases in which no causative

mutation is identified should be further analysed by microarray or complete genomic

hybridization for CNVs.

Using a next-generation-sequencing diagnostic approach, after one or more genetic

mutations are identified an astute clinician may pursue functional validation of the predicted

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effects of the identified variant(s) in vivo using endocrinological and/or imaging approaches.

For example, in cases in which a mutation in NR5A1 is identified as causative, one should

perform adrenal function tests such as measurement of serum cortisol and

adrenocorticotropic hormone levels. A mutation in GATA4 would prompt imaging of the

heart to rule out the possibility of an associated congenital heart defect.

From the perspective of both clinicians and researchers, correct genetic classification is

essential to best assess the pathophysiology and treatment outcomes in patients with DSDs.

Use of terminology and classifications should be consistent and careful and a genetic

diagnosis should be obtained for all patients with DSDs. Currently, only a small fraction of

individuals with a DSD receive a genetic diagnosis,132 but patients and their families

embrace such a diagnosis for the purposes of fully understanding and planning for the

potential health-related and psychological issues that might be associated with an

individual’s mutation(s). The particular genetic mutation(s) that underlie a specific DSD

diagnosis can influence health-related considerations include management of complications

such as cancer within the gonad and in other organ systems (for example Wilms tumours,

skin tumours and adrenal tumours), whose risk might be increased; the potential need for

hormone replacement therapy; and family planning considerations, both for the affected

individual and for their parents.

Psychosexual differentiation

The terms sex and gender are often used interchangeably in studies with human patients;

however, there are important distinctions between them. According to recommendations of

an Institute of Medicine report, the term sex should be used to refer to classifications of male

or female individuals according to physical attributes that are determined by an individual’s

karyotype, specifically the reproductive organs and their functions. By contrast, the term

gender is defined by the individual’s self-representation and identity as either a male or a

female person, as well as by society-specific expectations regarding the appropriateness of

attributes, activities, or behaviours for boys and men or girls and women.134

Psychosexual differentiation involves the developmental unfolding of gender identity,

gender role and sexual orientation. Gender identity refers to a person’s identification of self

as a girl (or woman), a boy (or man) or a mixture of both. Gender role refers to behaviours

that differ in frequency or level between male individuals and female individuals in culture

and time (such as toy play or maternal interest). Sexual orientation refers to sexual arousal to

persons of the same sex (homosexual), opposite sex (heterosexual) or both sexes (bisexual),

and is expressed in behaviour, fantasies and attractions.135 The consensus statement advises

that gender identity, gender-typical behaviour, and sexual orientation should all be viewed

as separate components. As such, if an individual with a DSD exhibits atypical gender role

behaviour this is not an indication of having been reared in the wrong gender.7

The issue of gender identity in DSDs has been reviewed elsewhere.136–138 Gender identity

has been suggested to generally follow gender of rearing,136,137 with the exception of

syndromes that result from errors in biosynthesis of androgens, such as deficiencies of 5-α-

reductase-2 and 17β-hydroxysteroid dehydrogenase-3.138–140 In these conditions, the

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masculinizing puberty that results from endogenous testosterone production is frequently

associated with a shift in gender identity among individuals reared as girls.138

Unfortunately, the details provided in published case series are inadequate to test competing

explanations for the observed change in gender identity in these individuals, such as the role

of atypical genital appearance, masculinized gender behaviour, a contra-sexual puberty and

cultural pressure that favours living as a man.141

Other challenging considerations from the perspective of gender assignment are those

associated with non-hormonal DSDs, such as cloacal exstrophy and penile agenesis. A

report of self-initiated gender change in a cohort of patients with 46,XY cloacal exstrophy

who were reared as girls142 has been associated with a shift in gender assignment

recommendations by paediatric urologists, from recommendations of rearing as a girl to

recommendations of rearing as a boy for these patients.143,144 In a six-year follow-up survey

of US paediatric urologists, 79% recommended gender assignment as a boy for patients with

cloacal exstrophy, with 97% identifying ‘brain imprinting’ by prenatal androgens as an

important factor in their decision. Nonetheless, a review of literature on gender identity

stability in female-reared individuals with a 46,XY karyotype who have nonhormonal

genital defects concluded that evidence is lacking to infer that gender identity is fully

determined by prenatal androgen exposure.145

DSDs have been studied to test hormonal hypotheses of gender development that extend

beyond gender identity. In contrast to the broadly accepted notions that gender is determined

by both biological and social environmental factors,146 associations between DSDs and

gender-atypical behavioural development have often been interpreted as directly resulting

from prenatal androgen exposure.147,148

Prenatal androgens have been shown to exert a masculinizing effect on the development of

behaviours that exhibit gender-related variability (for example, childrens’ toy and play

preferences) and sex differences in neurocognitive function.149,150 As noted, less certainty

exists regarding the role of early androgens in shaping gender identity and this is also the

case for sexual orientation. The possibility that early androgen exposure shapes sexual

orientation has been investigated, in particular among women with classic congenital

adrenal hyperplasia, a population in which increased rates of non-heterosexual orientation

have been reported.149,151,152 The reason for this observed correlation remains to be

determined but could be attributed to a number of factors, such as a direct influence of

androgens on sexual differentiation of the brain, or influences of early androgens on gender

role expression. Additionally, the effects of having a chronic medical condition and its

management on physical appearance and body image could also be involved, and the

possibility that the correlation results from a combination of these and possibly other factors

cannot be ruled out.152

Finally, animal experiments have demonstrated that early exposure to sex hormones during

steroid-sensitive periods of brain development has long-lasting effects on neurocognitive

function and sex-dimorphic behaviours.153 However, few studies have addressed the

possibility of sex-hormone-independent effects on brain structure and function.154,155

Genetically modified mice have been used to separate gonadal phenotypes from

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chromosome complement. This separation is accomplished by moving Sry, which

determines testes development, from the Y chromosome to an autosome. This paradigm has

enabled investigators to consider whether observed sex differences in brain and behaviour

are consequent to hormone exposure, genetic factors or both.154,155 Little evidence exists to

support a role for genetic factors acting independently of sex hormones in shaping aspects of

human psychosexual differentiation, such as gender identity, gender role or sexual

orientation. Nonetheless, studies in mice have demonstrated sex differences in physiology

and behaviour with similarities in humans that are unaccounted for by differential hormone

exposure.155 Examples include autoimmune diseases such as multiple sclerosis, which are

more common in female individuals,156 and more rapid escalation of substance abuse to the

point of addiction in women than in men.157

Conclusions

The classification of patients with DSDs has been revolutionized by use of multidisciplinary

approaches and large research networks that seek to better understand the pathophysiology

of DSDs. By providing a consistent framework for diagnosis, clinicians can begin to identify

patients with similar phenotypes and better understand the medical, social, and

psychological factors that contribute to the overall well-being of patients with DSDs. Given

the rarity of individuals with DSDs, multicenter studies are essential to identifying the

methods that will lead to consistent diagnosis and optimal medical care for these people.

Despite the focus on the identification of genetic mutations that result in disruptions to the

processes of sex determination and sex differentiation, genetics alone cannot completely

explain the full range of health or psychological issues that might be experienced by an

individual with a DSD. However, mutations identified in critical developmental genes have

provided much needed insight into pathophysiological mechanisms of DSDs as well as a

means of classification, which can be used to follow the long-term effects of genetic

mutations and treatment outcomes in these individuals.

Emerging technological advances have transformed the ability to identify mutations and

CNVs in individuals with DSDs; however, the interpretation and validation of identified

mutations that are the direct cause of the observed phenotypes remains challenging. As

sequencing technologies advance and our appreciation of the degree of variation within the

human genome continues to expand, the distinction between causative mutations versus

normal genetic variation will increase. The complexity of the human genome is evident in

multiple layers of regulation, including at the level of DNA, messenger and long non-coding

RNA transcripts, post- transcriptional and post-translational regulation, as well as within

non-protein-coding genomic regions and the epigenome. Thus, while a large proportion of

patients with DSDs have been diagnosed with a genetic lesion within known genes, much of

the remaining variation that contributes DSDs have yet to be fully explored. Studying

embryonic gonads in humans is ethically and technically very difficult, as these

investigations require using tissues appropriate to the stage in development being examined.

For example, fetal gonads of around 7 weeks of gestation would be needed to assess the

epigenetic status, levels of various RNA species and mechanisms of post-translational

regulation if sex determination were to be investigated.

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Our understanding of the mechanisms within sex determination processes is heavily

dependent on knowledge of the human patients with DSDs and studies in mouse models,

which can be manipulated to determine the effects of individual genetic mutations.

Understanding the multiple layers of genetic regulation that govern gonadal development

and how these layers interact using next-generation sequencing tools coupled with careful

studies in both animal models and in affected individuals might unlock the key to fully

appreciating and treating the health needs of people with DSDs.

Acknowledgments

Funding for this project was from the Doris Duke Foundation and the National Institute of Child Health and Human Development RO1HD06138 DSD-TRN (Platform for Basic and Translational Research) grant to E.V. and D.E.S, University of California Los Angeles institutional funds to V.A.A. and Patient-Centered Outcomes Research Institute contract funds to D.E.S.

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Key points

• Disorders of sex development (DSDs) are defined as congenital conditions in which development of chromosomal, gonadal, or anatomic sex is atypical

• Mutations in genes that encode transcription factors, signalling components and epigenetic modifiers that are involved in sex determination can result in 46,XX

and 46,XY DSDs

• At 6–8 weeks post-conception in human fetal development, upregulated expression of SRY in the bipotential gonad promotes testis determination,

whereas activation of WNT4 and RSPO1 signalling promotes ovary

determination

• Gonadal phenotypes in patients with DSDs range from gonadal dysgenesis (in which the gonads are fibrous streak gonads) to varying degrees of ovotesis (in

which both ovary and testicular tissue are present)

• The complexity and interrelatedness of factors that contribute to the aetiology and the medical and psychological outcomes of DSDs demand a

multidisciplinary team approach to health care

• In contrast to gender differences in activities and interests, associations between prenatal exposure to androgens and development of gender identity or sexual

orientation are unclear

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Review criteria

A search for articles published between 1991 and 2014 and focusing on disorders of sex

development was performed in PubMed and MEDLINE. The search terms used were

“sex determination”, “disorders of sex development”, “intersex”, “quality of life”,

“gender”, “gender identity”, “gender role”, “sexual differentiation” and “psychosexual

differentiation”. Specific search terms for genes were also used (“SRY”, “SOX9”,

“WT1”, “NR5A1”, “NR0B1”, “WNT4”, “RSPO1”, “FOXL2”, “DMRT1”, “MAP3K1”,

and “CBX2”). All articles identified were English-language full-text papers and abstracts.

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Figure 1. Genetic pathophysiology of human sex determination. Within the developing gonad,

regulation of gene transcription occurs through cellular signalling pathways (WNT4–RSPO1

in ovary determination, Map-kinase in testis determination) that activate genes through

alteration of chromatin structures and modulation of epigenetic factors or by direct

activation of transcriptional networks. In 46,XX individuals, WNT4 and RSPO1 act through

Frizzled or LRP5–LRP6 receptors to activate β-catenin (CTNNB1) transcription. β-catenin

and FOXL2 promote expression of ovary-specific genes while inhibiting the expression of

testis factors such as SOX9. In 46,XY individuals, Map-kinase signalling through MAP3K1

may alter chromatin conformation indirectly through histone modifications (dotted arrow).

Map-kinase signalling also increases phosphorylation of transcription factors such as

GATA4, which is thought to alter chromatin (dotted arrow) upstream of SRY, and was

shown to directly bind to SRY promoter (solid arrow) to activate transcription. Within the

nucleus, transcription factors GATA4 and ZFPM2 bind and transactivate SRY and SOX9.

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Other important factors are CBX2 that has been shown to directly bind the SRY promoter

and that, in conjunction with the NR5A1 protein, binds to the SOX9 promoter. The SRY

protein can then turn on downstream genes such as SOX9, which initiates the testis gene

expression network and represses ovarian-specific genes such as RSPO1 and β-catenin.

Ovary-promoting transcription factors are noted in orange and testis-promoting factors are

noted in green. Abbreviations: ORF, open reading frame; P, phosphate.

Arboleda et al. Page 27

Nat Rev Endocrinol. Author manuscript; available in PMC 2015 October 01.

A u th

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Arboleda et al. Page 28

T a b

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ow th

r et

ar da

ti on

.

Nat Rev Endocrinol. Author manuscript; available in PMC 2015 October 01.

A u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

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u th

o r M

a n u scrip

t A

u th

o r M

a n u scrip

t

Arboleda et al. Page 29

Table 2

Genetic mutations associated with disorders of sex development

Gene involved in sex determination Genomic region of mutation Inheritance

Noncoding ORF

MAP3K1 No Yes Autosomal dominant, gain-of-function

WNT4 No Yes Autosomal recessive

RSPO1 No Yes Homozygous recessive

CBX2 No Yes Autosomal recessive

SRY Yes Yes Sex-linked recessive

SOX9 Yes Yes Autosomal dominant

NR5A1 (SF-1) Yes Yes Autosomal dominant

GATA4 Yes Yes Autosomal dominant

ZFPM2 (FOG2) No Yes Autosomal dominant

NR0B1 Yes Yes Sex-linked recessive

FOXL2 Yes Yes Autosomal dominant

DMRT1 Yes Yes Autosomal dominant

Abbreviation: ORF, open reading frame.

Nat Rev Endocrinol. Author manuscript; available in PMC 2015 October 01.