Sintesi di: A novel germ line mutation in SOX9 causes familial
campomelic dysplasia and sex reversal
Fergus J. Cameron, Robyn M. Hageman, Claire Cooke-Yarborough ,
Cheni Kwok ,Linda L. Goodwin , David O. Sillence and Andrew H.
Sinclair Human Molecular Genetics, 1996, Vol. 5, No. 10
Campomelic dysplasia (CD) is one of several syndromes that
can result in XY gonadal dysgenesis. The syndrome is characterisedby
the radiological features of bowed femora and tibiae,hypoplastic
scapulae and pelvic bones and non-mineralised thoracic pedicles.
Clinically, an affected infant exhibits bowed lower limbs with
pretibial skin dimpling, dolichocephaly,micrognathia, cleft palate,
a flat nasal bridge, low set ears, talipes equinovarus and congenital
dislocation of the hips (1). Patients usually succumb in the neonatal
period due to respiratory insufficiency. Necropsy findings may
also demonstrate cardiac,renal and CNS anomalies including absence
of the olfactory bulbs. Three-quarters of karyotypic males have
external genitalia which lie on a spectrum between those of an
unambiguous female to that of hypospadias with a bifid scrotum
(1). Various combinations of internal Mullerian and Wolffian duct
structures have been reported. Gonadal morphology in these patients
is similar to that seen in XY gonadal dysgenesis (ranging from
dysplastic testicular tissue to poorly differentiated ovarian
tissue with a few primordial follicles).
Analysis of patients with CD and de novo chromosome 17 translocations
mapped the locus to 17q24.3q25.1 (2). Two groups independently
identified the SOX9 gene as responsible for CD (3,4). The distribution
and variety of mutations reported in SOX9 suggest that CD is caused
by haploinsufficiency of functional SOX9. Mutations in one allele
of SOX9 have been caused by splice acceptor and donor changes;
missense and frame-shift mutations and deletions. There has been
one reported case of compound heterozygosity, with two separate
mutations in the one patient (4), and one reported case of an
unaffected parent sharing the same mutation as an affected infant
(4). To date 13 mutations (35) and 10 translocations (2,510)
have been described in 24 patients with CD. There have been no
reported cases of patients having both a balanced 17q translocation
and a mutation (5). Nine of these mutations were sex-reversing
in46,XY affected patients.
One mutation was seen in two unrelated46,XY patients with CD,
it being sex-reversing in one and not in the other (5). Families
with two normal parents and more than one CD affected sibling
have been described (1,1113) and consequently the condition
was previously thought to be transmitted in an autosomal recessive
fashion. However, this assumption was never tested as none of
the families were described genotypically.The Authors describe
the genotype, gonadal phenotype and mode ofinheritance of three
affected sibs with CD.
The family described in this paper is informative for several
reasons.
1-they possess a novel mutation in SOX9(insertion C at position
1096 in exon 3) which causes CD.
Figure1. Patient 1. (a) Genitalia, sagittal schematic view.
Phallus 1.5 cm in length. (b) Genitalia, coronal schematic view.
Blind ending uterus and vagina with unilateral fallopian tube.
Right gonad at the internal inguinal ring, left gonad in normal
relationship to
the ipsilateral fallopian tube . Patient 2. (c), (d) Genitalia
showing sagittal and
coronal schematic views. Normal female internal and external genitalia.(Nota
bene i pazienti erano fratelli e percio' avevano la stessa mutazione)
This brings the total number of different mutations causing CD to 14 (10 of these resulting in 46,XY sex-reversal fig2).Gonad-specific mutations in SRY have been reported in other forms of gonadal dysgenesis (15). To eliminate the possibility of an organ specific mutation they analysed SOX9 sequences derived from both a phenotypically affected tissue (gonad) and a tissue phenotypically unaffected by the campomelic syndrome (liver). The SOX9 mutation was present in both tissues.
2-, this study is the first report of true hermaphroditism
associated with 46,XY CD. A confounding issue in classifying
gonadal dysgenesis histologically is that morphology may be constantly
changing. Consequently, the timing of patient
examination will determine the gonadal phenotype described. For
example, Turners syndrome patients have morphologically
normal ovarian stroma and germ cells at 12 weeks gestation but
have streak gonads by the time of puberty (16).
Figure 2. Mutations in SOX9. Sex reversing mutations are indicated below the diagram while non-sex reversing mutations are shown above. References for the mutations are cited.
Similarly, the authors observed an XY individual with partially
virilised external genitalia and ovaries at birth. In this instance
it suggests there may have been testicular material present at
an earlier stage and implies that the 'ovaries' may originally
have been ovotestes. If this patient had been examinedat 812
weeks gestation he may have been classified as a true hermaphrodite.
Thus the real incidence of true hermaphroditismin this form of
46,XY gonadal dysgenesis may have been previously unrecognised.
This underlies the more general concept that in 46,XY gonadal
dysgenesis there is a spectrum from dysplastic testes to ovotestis
and dysplastic ovaries.
Prior to the isolation and mutation analysis of SOX9, the mode
of inheritance for CD was not clear. The observation of affected
offspring with normal parents implied that CD may be inherited
in an autosomal recessive manner (17). With the discovery that
CD patients were heterozygous for mutations in SOX9 it was postulated
that transmission was autosomal dominant (3,4).Previous studies
have analysed parental genomic DNA obtained from lymphocytes;
however, none has examined parental germ cells. While it was impossible
to obtain maternal oocytes,examination of paternal germ cells
revealed mosaicism for the familial SOX9 mutation. This mutation
was not seen in paternal lymphocyte DNA. This study suggests that
a mutation in SOX9 arising in a mosaic germ cell line can be transmitted
in a dominant fashion and result in familial CD. This finding
explains the earlier observations of unaffected parents with CD
affected offspring.While germ cell specific mutations have been
implicated in a number of other genetic disorders (18) they have
only been unequivocally demonstrated in familial von Willebrand
disease(19), neurofibromatosis type 1 (20) and triplet repeat
expansion disorders such as Huntington's disease (21). The data
reported in this paper are the first to confirm that CD can be
an autosomal dominantly inherited syndrome which may be caused
by a mosaic, de novo germ cell mutation. However, the Authors
only examined the father's lymphocyte derived DNA: the possibility
exists that other somatic tissues may carry the SOX9 mutation.
3-, this study has shown that the same mutation in SOX9,in two
individuals, can cause varying degrees of 46,XY sex-reversal.
This finding is consistent with previous studies (5).In that studies
the authors found two unrelated, 46,XY CD patients with identical
mutations in SOX9, characterised by insertion of an adenine residue
following nucleotide position 1462. One patient had external male
genitalia with hypospadias and the other normal female external
genitalia. In neither patient was gonadal morphology or internal
genital duct structure reported. In this study, the two 46,XY
CD siblings had different gonadal morphologies with consequently
varying genital phenotypes. The range of gonadal morphologies
observed may be explained by several possible mechanisms such
as variable penetrance of the SOX9 mutation, increased activity
of the non-mutant SOX9 allele or stochastic environmental factors.
Studies in mice have also indicated that genetically identical
individuals can have varying gonadal phenotypes (22,23).
It is apparent that both intact SRY and SOX9 are necessary for
embryonal testis determination (24). Mutations in either of these
genes can disrupt testis development and cause a sex-reversed
phenotype. While mutations in SRY almost always cause failure
of normal testicular differentiation, this is not so with SOX9.
6Twenty-five per cent of 46,XY patients with CD are not sex-reversed.
There are now two examples of the same SOX9 mutations causing
variable sexual phenotypes. All but one of the mutations in SRY
have been found to lie within the HMG-boxdomain (25), while those
of SOX9 span virtually the entire open reading frame. Phenotypic
and mutational analyses indicate that there is no portion of SOX9
that is specifically associated with testis or skeletal development.
The ability of the SRY protein to bind and bend the DNA helix
appears to be critical to its function. SOX9 has the appearance
of a classical transcription factor with the HMG box DNA binding
domain and a proline rich region which could act as an activation
domain. However, mutation studies indicate that a CD phenotype
can still occur even when SOX9 has both these apparently critical
regions intact. SRY is thought to be expressed in pre-Sertoli
cells and may be responsible for recruiting other cell types necessary
for testicular determination. SOX9 is expressed in mesenchymal
cells that are the precursors for a number of developing tissues
including gonad and bone. In the testis these cells are responsible
for testis cord formation. Haploinsufficiency of SOX9 may either
prevent migration of these cells from the mesonephros into the
developing testis or may cause these cells to be dysfunctional
after they arrive.Genotypic and phenotypic analysis of this family
has allowed the mode of inheritence of CD to be established and
provided new insights into the differing roles of SOX9 and SRY
in mammalian testis determination.
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