Genetics of Ambiguous Genitalia in Newborns

Objectives
Genetics of Ambiguous
Genitalia in Newborns
1. X and Y ch
ch.;
.; how sexual differences are encoded?
‫ﮔﺮوه ژﻧﺘﯿﮏ ﭘﺰﺷﮑﯽ‬
‫داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﮑﯽ ﺗﻬﺮان‬
2. common sex ch
ch.. disorders
3. Understand what sex
sex--reversal is and how it is caused.
Hermaphroditism,, Pseudohermaphroditism
Hermaphroditism
Seyed Mohammad Akrami [MD, PhD]
[email protected]
Dey 1389
for reproduction biology PhD students
http://www.microscopix.co.uk/chromosomes/16306.gif
Disorders of gonadal development; a wide clinical, cytogenetic and histopathological spectrum
Neonatal Emergency
Genetic Determinants of Ambiguous Genitalia
psychosocial problem, …
Chromosomal abnormalities
 47XXY or 45X
Single gene disorders
 Smith-Lemli-Optiz syn.;
Danger;
1. CAH; salt loosing
MR, abnormality of face & skeletal,
cryptorchidism, Hypospadias in males
2. 11
11β
β - 17
17α
α; hypertension
Multifactorial disorders
Iatrogenic, …
3. XY pure gonadal dysgenesis
dysgenesis;; germinoma
germinoma,, Wilm’s
tumour
Causes of abnormalities of sexual
differentiation
Klinefelter syndrome
Virilization of a genetic female with ovaries



Fetal androgens - e.g. CAH, adrenal adenoma or hyperplasia
Maternal androgens - e.g. ovarian or adrenal tumors
Iatrogenic - exogenous androgens or progestagens with androgenic activity
Turner syndrome
Incomplete virilization of a genetic male with testes





Defect of testis development (termed gonadal dysgenesis leading to impaired AMH and testosterone
production) - e.g. loss of SRY (Y-linked XY dysgenesis), additional DSS (X-linked dysgenesis), loss of SOX9
(autosomal-linked dysgenesis). Dysgenetic testes are at increased risk of malignant transformation and,
therefore, removal is recommended
Leydig cell hypoplasia - e.g. loss of function mutation of the LH receptor. AMH is still produced so the
phenotype is female with no Müllerian duct system
Impaired testosterone production - e.g. defects in synthetic enzymes including 3β-hydroxysteroid
dehydrogenase or 17α-hydroxylase
Androgen insensitiv ity syndrome - may be variable severity
5α-reductase deficiency.
True hermaphroditism
- both external and internal structures show gradations between normal male and female. The
initial presentation is with genital ambiguity but more rarely isolated cliteromegaly or penile
hypospadias. Virtually all have a urogenital sinus and uterus. Most are raised as males
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Major numerical disorders of sex chromosomes;
•
Klinefelter syndrome (47, XXY)
•
47XYY and 47 XXX
•
Turner syndrome (45, X)
Most of the autosomal abnormalities can be diagnosed at
birth but most sex chromosome abnormalities (with the
exception of Turner syndrome) are not recognized until
puberty.
1
Sex Chromosomes
The Y chromosome
50 yrs ago……
The finding that 45X individuals were
female and that XXY individuals were
male established the chromosomal basis
of gender—i.e., the Y is crucial for
male development.
X and Y are structurally
distinct
thought to have common
progenitor

Y chromosome lost many of the
genes (remains ~50 genes)
Some genes retain copies on
both X and Y

Found at the ends of each arm
– pseudoautosomal regions

Pairing and meiotic
recombination occurs in these
location
A normal baby boy, >2.5 cm penis
6th week of development
Fallopian tubes,
Uterus,
Upper vagina
Male genital ducts,
Seminiferous tubules
The mesonephric ducts regress
The paramesonephric ducts
develop into the female duct
system. In the absence of testis,
female external
genitalia develop
regardless of whether
an ovary is present
Seminiferous tubules
Leydig cells
HCG from placenta
induces Leydig cells
to make
Sertoli cells
Paramesonephric ducts
Diagram of the development of the external genitalia
Factors necessary for normal sexual development
Testosterone,
dihydroxytestosterone
Fertilization of an egg by a sperm
containing the first chromosome results
in an XX male;
fertilization of an egg by
a sperm containing the second
chromosome is an XY female because
there is no SRY, however, the ovaries
will regress because 2 doses of X-linked
genes are necessary for ovarian function
and oogenesis
centromeres
Bill Daniel’s Medical Genetics Study Guide
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Sex reversal
2
Turner syndrome
1/4000 of female births
• Survival to term is rare – 99
99%
% of
fetuses spontaneously abort
• 50
50%
% are 45
45,X
,X
• 25
25%
% mosaics
AZF-azoospermia factor
AZFDAZ--deleted in azoospermia
DAZ
Azoospermia--no sperm
Azoospermia
• Usually caused by paternal
error in Meiosis
Clinical Features
webbed neck and lymphedema of
the hands and feet
Turner Syndrome
Clinical Features
Turner syn.
• short stature,
• gonadal dysgenesis (streak gonads), infertile
webbed neck, broad chest,
• elevated frequency of renal and
cardiovascular anomalies
•Intelligence – avg. or above
•Deficiency in spatial perception,
fine motor execution
Emery and Rimoin’s
Principles and practice of Medical Genetics
Emery and Rimoin’s Principles and practice of Medical Genetics
47,XXY
47
,XXY - Klinefelter syndrome
1/1000 male births
Clinical Features
tall, thin, long legs, hypogonadism,
underdeveloped secondary sex
characteristics, gynecomastiagynecomastiaexcessive development of the male
mammary glands
.
usually infertile
Disorders of Gonadal and Sexual Development
role of various X-linked and autosomal genes in
ovarian and testicular development
Ambiguous Genitalia in Newborns
Mild hypospadias in malesmales-the urethra opens on the
underside of the penis or on the perineum
Hermaphroditism-- both ovarian and testicular tissue is present.
Hermaphroditism
Verbal comprehension and ability
slightly lower than average
Increased risk of learning difficulties,
esp. in reading
Karyotype!
Several variants – 48
48,XXYY;
,XXYY;
48,XXXY;
48
,XXXY; 49
49,XXXXY
,XXXXY – additional Xs cause more abnormal phenotype
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Camptomelic Dysplasia
Role of autosomal and X linked genes in conversion of the
• Autosomal dominant disorder with
biopotential gonad to either a testis or ovary.
usually lethal bone and cartilage
• Deletion of SRY by recombination leads to XY females.
malformations
• due to mutations in the SOX9 gene on 17q
• However, there are XY females in whom the
SRY gene was not deleted.
• These females have a duplication of a portion
of the X chromosome that contains the DAX 1 genes
• 2/3 of 46 XY patients with this disorder
are phenotypic females because SOX9
2X
• DAX1 plays a dosage-sensitive role in determination
is required for normal testis formation.
of gonadal sex.
• In the absence of Sox 9--the default ovarian
• An excess of DAX1 resulting from gene duplication
pathway is followed and these individuals are sex-reversed
can suppress the normal male-determining function of SRY and
ovarian development ensues.
These females are referred to as sex reversed,
XY female
3 ways to be XY female:
• Delete SRY
• 2X DAX1
DAX1
• 1x SOX9
SOX9
2 ways to be XY female:
Delete SRY
2X DAX1
DAX1
Sox9
Sox
9 duplication
=XX male
Other autosomal loci implicated in
gonadal development
1. WT1
WT1 gene in 11
11p
p13 encodes a transcription factor
involved in interactions between Sertoli and Leydig cells
dominant WT1
WT1 mutations disrupt normal testicular devel.
result: XY males have female or ambiguous genitalia
Denys--Drash syndrome
Denys
2. 9p deletion syndrome
9p24 encodes a transcription factor DMRT1
DMRT1 expressed
in the developing gonad
result: 46 XY females
• Duplication of Sox9 can lead to XX sex
reversal, suggesting that overproduction of
Sox9 in the absence of SRY can lead to testis
formation.
Sex reversed, 46
46,, XY female:
•
•
•
•
•
Delete SRY
2X DAX1
DAX1
1x SOX9
SOX9
dominant WT1
WT1 mutation
9p24 deletion
Pseudohermaphroditism
Pseudohermaphrodites have gonadal tissue of only one sex.
Female pseudohermaphrodites, 46 XX, normal ovarian tissue
but ambiguous or male genitalia-genitalia--usually
usually due to
Congenital Adrenal Hyperplasia (CAH)
autosomal recessive (AR) disorder
Sex reversed, 46
46,, XX male:
• presence of SRY on X due to recombination
• arises from specific defects in enzymes of the adrenal
cortex required for cortisol biosynthesis
Results in virilization of female infants (Masculinisation;
• 3X SOX9
SOX9
increased body or facial hair, change in voice, clitoral
enlargement and malemale-type baldness)
Most common defect: deficiency of 21
21--hydroxylase.
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Congenital Adrenal
Hyperplasia (CAH)
Deficiency of 21-hydroxylase
90-95% of cases
Deficiency of 11?-hydroxylase
5 - 8% of cases
Other defects constitute < 1% of all reported cases
A group of AR disorders
resulting from the deficiency of
one of the five enzymes
required for the synthesis of
cortisol and aldosterone ( salt retaining hormone )
3? - Hydroxysteroid-dehydrogenase
18 -Hydroxylase
17α - Hydroxylase
Deficiency of steroidogenic acute regulatory
protein (STAR) → Lipoid hyperplasia
Pathways of Steroid Biosynthesis in the Adrenal Cortex
Importance:
The most common cause of ambiguous genitalia in the newborn
female is a deficiency of the 21
21--hydroxylase -cortisol biosynthesis
• masculinized genitalia: clitoral enlargement
labial fusion to form a scrotum-like structure
• Normal ovarian development
• Because affected females have the capacity for an
entirely normal female sex role, including child
bearing, it is very important to recognize this
disorder in newborns.
• Also important to recognize because 75% have a
salt-losing type that may lead to neonatal death.
Treated with hormone replacement.
Classification
Classic 21-hydroxylase deficiency
Simple virilizing type
Salt – wasting type
severe form
1 in 16000 birth
Non-classic form
most common type ~0.2 of the general white population
but is more frequent (1-2%) in certain population, such
as Jews of Eastern European origin.
Genetic
AR
CYP21 ( CYP21A2 ) gene, 6p21.3; located in the highly
polymorphic HLA, major histocompatibility complex
(MHC)
Along with a pseudogene, CYP21P
(CYP21A1P)
Clinical Manifestations
Salt wasting
Ambiguous genitalia
Postnatal virilization
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CYP21
6p21.3
CYP11B1 8q24.3
21-Hydroxylase
1/10-18000
11β-Hydroxylase
1/100000
CYP11B2 8q24.3 18-Hydroxylase
(Aldosterone synthase)
Rare
CYP17
10q24.3 17α-Hydroxylase
Rare
HSD3B2 1p13.1 3β-Hydroxysteroid
dehydrogenase
Rare
STAR
Rare
8p11.2
Lipoid hyperplasia
CYP21 gene
Common point mutations in the CYP21 gene
8bp deletion (∆707) in exon 3
Nucleotide
Position
Position in
Gene
Sequence
Change
Protein
Change
Clinical
Phenotype
A frame shift in exon 7
89
exon 1
CCG  CTG
Pro31  Leu
655
intron 2
A or C  G
Splice mutation
Salt wasting
999
exon 4
ATC  AAC
Ile 173  Asn
Simple virilising
1683
exon 7
GTG  TTG
Val 282  Leu
Non-classical
1994
exon 8
CAG  TAG
Gln 319  stop
2108
exon 8
CGG  TGG
Arg 357  Trp
2578
exon 10
CCC  TCC
Pro 454  Ser
Nonsense mutation in exon 8
3bp change in exon 6
Salt wasting
~75% represent deleterious mutations found in
the pseudogene that are transferred to CYP21
during mitosis by “gene conversion.”
principles of management & treatment
Avoid the use of gender-specific pronouns or the term intersex (that promulgates the idea
of the neonate being of neither sex) and simply make reference to ‘your baby‘.
Avoid an early provisional opinion prior to a complete assessment.
The sex of rearing is independent of the genetic sex (or karyotype).
Decision on sex of rearing should be taken jointly by a multidisciplinary team including
endocrinologist, gynecologist or urologist and the parents. Anatomy and biochemical test
results must be considered.
Virilized females with CAH should in general be raised as females. Long-term outcomes
for these patients (in terms of a full female role) may be less than ideal.
Genetic males with severe genital abnormalities and testosterone insensitivity should also
be raised as females. Low radio-labeled androgen binding or poor clinical response to
administered testosterone (e.g. 50 mg monthly for 3 months) would support this
decision.
Cultural, ethical and social issues or the special case of puberty in patients with 5αreductase deficiency may affect the final decision.
Recombinations between CYP21 & CYP21P
(gene conversions or deletions)
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Medical approach
Examination; palpable gonad, skin
pigmentation
Systematic illness; BP
Clinical investigation of ambiguous genitalia
1.
2.
3.
4.
Elicit family history and any consanguinity
Examine for dysmorphic features
Take blood for karyotype (chromosome analysis)
Palpate gonads:


Paraclinic approach
Electrolytes
Hormones
Sonography
Karyotype, molecular study
Diagnosis
Classic 21 - hydroxylase deficiency is
characterized by markedly elevated serum
levels of 17-hydroxyprogesterone .
10,000 ng/dl (300 nmol/L) in affected infants .
levels in normal newborns are below 100 ng/dl
(3 nmol/L)
This difference makes it possible to screen
newborns .
Treatment

If none palpable, patient is likely to be female with virilization, most likely
due to CAH. Measure blood 17α-hydroxyprogesterone and 11-deoxycortisol
concentrations
If two palpable, patient is likely to be male with incomplete virilization,
most likely due to impaired testosterone biosynthesis, androgen
insensitivity or 5α-reductase deficiency. Perform a 3 day hCG test (1000 U
daily for 3 consecutive days) with blood taken on days 0 and 4 for
testosterone, dihydrotestosterone, dehydroepiandrosterone and
androstendione analysis. Consider androgen binding studies, molecular
studies and pelvic ultrasound
If one palpable, patient is likely to have a form of gonadal dysgenesis or
true hermaphroditism. Perform an hCG test and gonadal biopsy. Consider
pelvic ultrasound and laparoscopy to define anatomy
Molecular Diagnosis
ARMS –PCR
Followed by Nested PCR
CYP21 and CYP21P with high homology
Male pseudohermaphroditism
46,, XY Testes but female in appearance
46
Glucocorticoids
Mineralocorticoids
Management of ambiguous genitalia
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1. Deficiency of 5 -reductase
2. Androgen Insensitivity Syndrome (AIS);
testicular feminization
7
Male pseudohermaphroditism
Deficiency of 5 -reductase
Deficiency of 5 -reductase
Autosomal recessive (AR)
5  -reductase
testosterone
Dihydroxytestosterone
(active form)
• Cannot form DHT in adequate amounts
• encoding gene; type 1 (SRD
(SRD5
5A1): 5p15
type 2 (SRD
(SRD5
5A2): 2p23 (responsible for this disease)
• May be born with ambiguous genitalia normal testes, small
penis, blind vaginal pouch-pouch --initially
initially reared as females
• Pubertal testosterone surge can lead to sex change
in male
• They lack a prostate, thus, are sterile
Different mutations are more common in different ethnic
groups
The ethnic predilection of recurring mutations are shown on the right
Emery and Rimoin’s Principles and practice of Medical Genetics
Male pseudohermaphroditism, 46 XY
Androgen insensitivity syndrome= testicular feminization
Androgen Insensitivity Syndrome (AIS)
46, XY
• X-linked rec.
1/20
20,,000
• A female phenotype despite the presence of bilateral testes,
absence of prostate & axillary hair, sparse pubic hair and
breast development (gynecomastia).
Testes are present and secreting androgen but there is no
androgen receptor, thus XY appears female but is sterile.
•a short, blind ending vagina and no uterus or uterine tubes.
Androgen binds its receptor, enters nucleus, binds DNA, activates transcription
AR gene; Xq11.2-12
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Summary
1. Neonatal emergency
Urol Clin North Am. 2010 May;
May;37
37((2):
):195
195--205
205..
A practical approach to ambiguous genitalia in the newborn period.
Lambert SM,
SM, Vilain EJ
EJ,, Kolon TF
TF..
The evaluation and management of neonates with ambiguous genitalia requires sensitivity, efficiency, and
accuracy. The approach to these neonates is facilitated by a multidisciplinary team including urology,
2. Importance of rule out any chromosomal abnormality
endocrinology, genetics, and psychiatry or psychology. Disorders of sex development (DSD) encompass
chromosomal DSD, 46,XX DSD, and 46,XY DSD. The 46,XX DSD is the most common DSD and in the
3. Female pseudohermaphrodites (46
(46,, XX) are caused by
majority of these children congenital adrenal hyperplasia is the underlying etiology. The 46,XY DSD is a
heterogeneous disorder that often results from a disruption in the production or response to testosterone,
congenital adrenal hyperplasia (CAH)
dihydrotestosterone, or Mullerian inhibitory substance. Chromosomal DSD includes conditions resulting from
abnormal meiosis, including Klinefelter syndrome (47, XXY) and Turner syndrome. The evaluation of children
3. Male pseudohermaphrodites (46
(46,, XY) are caused by
deficiencies in 5 -reductase and androgen insensitivity
with DSD demands a thorough physical examination, medical history, karyotype, metabolic panel, 17-OH
progesterone, testosterone, luteinizing hormone, follicle stimulation hormone, and urinalysis. A radiographic
evaluation should begin with an abdominal and pelvic ultrasound but may include magnetic resonance
imaging, endoscopy, or laparoscopy.
syndrome (AIS).
4. Team work; pediatrics, endocrinologist, laboratory, surgeon,
[email protected]
psychologist, …
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