Sintesi di :Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations.

Jobst Meyer, Peter Südbeck, Marika Held, Thomas Wagner, M. Lienhard Schmitz, Franca Dagna Bricarelli, Ephrem Eggermont, Ursula Friedrich, Oskar A. Haas, Albrecht Kobelt, Jules G. Leroy, Lionel Van Maldergem, Erik Michel, Beate Mitulla, Rudolf A. Pfeiffer, Albert Schinzel, Heinrich Schmidt and Gerd Scherer.
Human Molecular Genetics, 1997, Vol. 6, No. 1

Campomelic dysplasia (CD) is a rare, often lethal, dominantly inherited, congenital osteochondrodysplasia, associated with male-to-female autosomal sex reversal in two-thirds of the affected karyotypic males. Prominent features of CD are bowing of femora and tibiae, hypoplastic scapulae, 11 instead of 12 pairs of ribs, Robin sequence [per sequenza si intende una serie di malformazioni che si ritiengono legate da una comune pathway nello sviluppo, in questo caso sono delle malfolmazioni del viso], pelvic malformations and bilateral clubfeet. The majority of CD patients die neonatally due to respiratory distress (1,2). By positional cloning in combination with positional candidate information, the SOX9 gene on chromosome 17q was isolated from the vicinity of breakpoints in CD patients with reciprocal de novo translocations (3,4). Proof of SOX9 being responsible for both CD and XY sex reversal came from the demonstration of de novo heterozygous loss-of-function mutations within the SOX9 coding region in non-translocationCD patients (3­5). Unexpected and still unexplained remains the observation that the breakpoints in all six translocation patients studied do not interrupt the SOX9 gene but map 50 kb or more 5' to the gene (3,4,6,7). In line with the generalized defect in skeletal development seen in CD patients, the mouse Sox9 gene has been shown to be expressed in mesenchymal condensations before and during embryonic cartilage deposition, consistent with a primary role for SOX9/Sox9 in skeletal formation (8).
Like the Y-located testis-determining gene SRY, SOX9 is a member of the SOX gene family of transcription factors. SOX proteins share an amino acid sequence identity of 60% or more in their high-mobility group (HMG) domain with the HMG domain present in SRY (9). The HMG domain is an 80 amino acid DNA-binding and bending motif that characterizes, besides the SOX proteins, a whole class of transcription factors (10). A partfrom the HMG domain, the 509 residue SOX9 protein contains two additional protein motifs: first, a stretch of 41 residues(residues 339­379) composed solely of proline, glutamine an dalanine (PQA motif), the function of which is unknown; secondly, a C-terminal transcription activation domain rich in serine,proline and glutamine, ranging from residues 402­509 (11).Up to now, a total of 13 SOX9 mutations have been published(3­5). As these mutations are all heterozygous and appear to cause loss of function of SOX9, CD can be regarded as a haploinsufficiency syndrome. To gain more insight into the mutational spectrum in CD and to see whether or not some genotype/pheno-typecorrelations emerge regarding XY sex reversal, survival or severity of disease, the authors have extended their previous study to 12 new CD cases with and without XY sex reversal. The authors have thus identified 10 novel SOX9 mutations and one recurrent mutation,and have analysed some of these mutations in functional assays. In addition, they have screened DNA samples from patients with XYgonadal dysgenesis (Swyer syndrome) known to have an intact SRY gene for mutations in SOX9.
The authors present 10 novel mutations and one recurrent mutation in SOX9, the gene responsible for both CD and autosomal XY sex reversal. Including 13 SOX9 mutations previously reported (3­5),altogether 24 SOX9 mutations are now known (see Fig. 3). Nine of them are located within the HMG box, which encodes the DNAbinding domain of the protein. These nine mutations include all six amino acid substitutions described so far in CD. As the aminoacid sequences of SOX9 proteins from human (3,4), mouse (8) and chicken (GenBank accession no. U12533) are highly conserved over the entire N-terminal half of the protein, includingthe HMG domain, and within the C-terminal TA domain (11), it appears that the amino acid sequence of the HMG domain isparticularly critical for correct function.
All four patients carrying the missense mutations P108L,W143R, R152P and P170R died within 6 months after birth. The authors have analysed these mutations with respect to their effects onDNA binding of the resulting mutant HMG domains. The P108Land W143R mutations cause complete loss of binding, while the HMG domains with the R152P and P170R mutations show reduced DNA binding (Fig. 2). The observation that the latter two mutations result in protein/DNA complexes moving slower or faster than the wild-type complexes may indicate that these aminoacid complexes also result in an altered DNA bending angle, as has been demonstrated for one amino acid substitution in the HMG domain of SRY found in an XY female with gonadal dysgenesis (19). However, SOX9 has not yet been shown to be a DNA binding protein.
The W143R mutation affects the most conserved amino acid residue among the HMG domain proteins. This residue is present at a corresponding position in 39 of 44 members of this protein family and is present in all SOX proteins (20). It is not surprising,therefore, that the W143R mutation shows complete loss of DNAbinding. Interestingly, replacement of the corresponding tryptophan residue by arginine in the HMG domain of the HMG1 protein results in an altered protein structure and affects many, but not all,of its DNA binding properties (21,22).
Of all SOX9 mutations that are not missense mutations, only there current nonsense mutation Y440X found in patients S.P. (3) andN.Z. and the frameshift mutation at codon 507 in patientGM04329 (5) leave part or almost all of the TA domain intact(Fig. 3). The authors have demonstrated in functional transfection assays that the truncated SOX9 protein resulting from the Y440X mutation retains some transactivation function, while the mutant SOX9 protein resulting from the codon 507 frameshift shows no transactivation, probably due to instability of the protein, or itsmRNA. These findings correlate with the clinical course of the patients involved, as S.P. and N.Z. survived the neonatal period, whereas patient GM04329 died shortly after birth. 95

Figure . Summary of presently known SOX9 mutations in campomelic dysplasia and autosomal XY sex reversal. Exons 1­3 of SOX9 are drawn to scale, with exon 3 being truncated. Coding sequences are indicated by green, red (HMG domain), blue (PQA motif) or black boxes (transactivation domain), untranslated regions by white boxes. Numbers indicate codons or amino acid residues, given as single letter code. The nature of the mutations is indicated at left. The number of bases inserted or deleted in frameshift mutations is given in brackets. Mutations found in XY and XX females are marked by filled and open circles, respectively, while mutations present in XY males are indicated by open squares. Data are from this report ; ref. 3 (E148X, Y440X, 329(+1), splice donor GT"AT); ref. 4 (Q195X,261(+1), 286(+1); and ref. 5 (F112L, A119V, 368(+1), 368(+1), 507(+4), splice acceptor AG"CG).

The other nonsense and frameshift mutations described so far, and the two splice mutations, result in SOX9 proteins that entirely lack the TA domain; additionally, some mutant SOX9 proteins lack part or all of the HMG domain (Fig). As might be expected,most of the patients having such severely impaired SOX9 proteins died in the neonatal period. This is the case for most of the patients previously described (3­5), and for five of the six such patients with nonsense mutations W86X, Q375X andE400X, and those with frameshift mutations at codons 277 and357. However, patient J.N.carrying the nonsense mutation Q117X at the N-terminus of theHMG domain is now doing well at the age of 12 years. Four SOX9 mutations in phenotypic male CD patients have now been reported. Two are amino acid substitutions in the HMG domain, F112L (5) and P170R (see Fig). Whereas the F112L mutation has not been tested functionally, the P170R mutation in patient T.L. has been shown to retain some DNAbinding ability (Fig. 2). While it is tempting to correlate this with the lack of sex reversal seen in this patient, authors are reluctant to do so in view of the fact that residual DNA binding has also been documented for some amino acid substitutions in the HMG domain of SRY found in XY females with gonadal dysgenesis(18,19,23,24). The E400X mutation in the male patient P.G.described here results in a truncated protein completely lacking the transactivating domain at its C-terminus (11). As nonsense mutations flanking codon 400 on either side are found in sex-reversed XY females (Q375X and Y440X), no correlation between the position of a stop codon in SOX9 and the sexual phenotype is apparent.
Finally, a SOX9 mutation described in a male CD patient is a single A insertion in codon 368 (5), but an identical mutation was also found in a sex reversed XY female(5) (Fig), providing a particularly clear example that XY sex reversal in SOX9 is subject to variable penetrance. None of the four phenotypic and karyotypic male patients with known SOX9 mutations survived beyond the neonatal period. However, a caseof a 17 year old male long-term-survivor is documented (1,25).Although cases of CD without male to female sex reversal are common, no case of sex reversal without skeletal malformations caused by a SOX9 mutation has been reported to date. As both SRY and SOX9 act within the sex determination/differentation pathway, one could assume that SOX9 mutations may also cause gonadal dysgenesis and XY sex reversal (Swyer syndrome), as domutations in SRY. However, the authors detected no SOX9 mutations in 18patients with Swyer syndrome carrying an intact SRY gene. As only 91% of the gene has been analysed by SSCP in these 18 cases, they cannot completely rule out that SOX9 mutations may occasionally cause Swyer syndrome in other patients.
In patient R.R., as in three patients previously studied(3,5), no SOX9 mutations and no chromosomal rearrangements could be detected. In all CD cases with chromosomal aberrations analysed so far, the translocation breakpoints are located at remarkable distances of 50 kb to more than 130 kb 5' from SOX9,providing evidence for an extended control region of the SOX9 gene (3,4,6,7). In the four CD cases with no detectable SOX9 structural gene mutation, the mutations may be located within the putative far upstream regulatory element(s) affected by the translocations. Alternatively, the mutations may reside within intronic sequences of the SOX9 gene, or may affect the putative expressed sequence 5' to SOX9 described by Ninomiya et al. ( 26). As discussed above, a major conclusion from this and from previous reports is the lack of correlation between the type and position of mutation within the gene with the resulting phenotype.It appears that both XY sex reversal and disease severity are a matter of penetrance of a mutation rather than the result of a specific type of mutation: a patient may have a nonsense mutation removing 80% of the protein and survive for several years, as inpatient J.N. with the Q117X mutation, while nonsense or frameshift mutations leaving a much larger segment of SOX9 intact are found in patients who died in the neonatal period. The only tentative genotype/phenotype correlation the authors can formulate concerns the Y440X mutation, where the retention of some transactivation potential may be causally related with the fact that the two unrelated patients carrying this mutation survived for several years. Future mutational studies of SOX9 in CD patients may show whether this correlation linking residual trans-activating activity of a mutant SOX9 protein to an increased survival rate will hold.

abbreviations
CAT, chloramphenicol acetyltransferase; HMG, high-mobility group (box, domain);PQA, proline-gluta-mine-alanine (motif); SSCP, single strand conformation poly-morphism;TA, transcription activation (domain); TAT, tyrosine aminotransferase.

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