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.

1. Mansour, S., Hall, C. M., Pembrey, M. E. and Young, I. D. (1995) A clinicaland genetic study of campomelic dysplasia. J. Med. Genet., 32, 415­420.
2. Tommerup, N., Schempp, E., Meinecke, P., Pedersen, S., Bolune, L., Brandt,C., Goodpasture, C., Guldberg, P., Held, K. R., Reinwein, H., Saugstad, O. D.,Scherer, G., Skljeldsl, O., Toder, R., Westvik, J., van der Hagen, C. B. andWolf, U. (1993) Assignment of an autosomal sex reversal locus (SRA1) and campomelic dysplasia (CMPD1) to 17q24.3­q25.1. Nature Genet., 4,170­174.
3. Foster, J. W., Dominguez-Steglich, M. A., Guioli, S., Kwok, C., Weller, P. A.,Stevanovic, M., Weissenbach, J., Mansour, S., Young, I. D., Goodfellow, P.N., Brook, J. D. and Schafer, A. J. (1994) Campomelic dysplasia and autosomal sex-reversal caused by mutations in an SRY-related gene. Nature,372, 525­530.
4. Wagner, T., Wirth, J., Meyer, J., Zabel, B., Held, M., Zimmer, J. J. P.,Bricarelli, F. D., Keutel, J., Hustert, E., Wolf, U., Tommerup, N., Schempp, W.and Scherer, G. (1994) Autosomal sex reversal and campomelic dysplasia arecaused by mutatuions in and around the SRY-related gene SOX-9. Cell, 79,1111­1120.
5. Kwok, C., Weller, P. A., Guioli, S., Foster, J. W., Mansour, S., Zuffardi, O.,Punnett, H. H., Dominguez-Steglich, M. A., Brook, J. D., Young, I. D.,Goodfellow, P. N. and Schafer, A. J. (1995) Mutations in SOX-9, the generesponsible for campomelic dysplasia and sex reversal. Am. J. Hum. Genet.,57, 1028­1036.
6. Maraia, R., Saal, H. M. and Wangsa, D. (1991) A chromosome 17q de novo paracentric inversion in a patient with campomelic dysplasia; case report andetiologic hypothesis. Clin. Genet., 39, 401­408.
7. Young, I. D., Zuccollo, J. M., Maltby, E. L. and Broderick, N. J. (1992)Campomelic dysplasia associated with a de novo 2q­17q reciprocal translocation. J. Med. Genet., 29, 251­252.
8. Chatters, S. and Whitecross, N. (1994) Campomelic dysplasia with sex-reversal associated with an apparently balanced paracentric inversion within the long arm of chromosome 17. Clin. Cytogenet. Bull., 2, 12­13.
9. Wirth, J., Wagner, T., Meyer, J., Pfeiffer, R. A., Tietze, H. U., Schempp, W.and Scherer, G. (1996) Translocation breakpoints in three patients with campomelic dysplasia and autosomal sex reversal map more than 130 kb from SOX9. Hum. Genet., 97, 186­193.
10. Ninomiya, S., Isomura, M., Narahara, K., Seino, Y. and Nakamura, Y. (1996)Isolation of a testis-specific cDNA on chromosome 17q from a region adjacent to the breakpoint of t(12;17) observed in a patient with a campomelic dysplasia and sex reversal. Hum. Mol. Genet., 5, 69­72.
11. Shafai, T. and Schwartz, L. (1976) Campomelic syndrome in siblings. J.Pediatr., 89, 512­513.12. Mellows, H. J., Pryse-Davies, J., Bennet, M. J. et al. (1980) The camptomelic syndrome in two female siblings. Clin. Genet., 18, 137­141.
13. Winter, R., Rosenkranz, W., Hoffmann, H. et al. (1985) Prenatal diagnosis of campomelic dysplasia by ultrasonography. Prenat. Diagn., 5, 1­8.
14. Prader Von, A. (1954) Der genitalbefund beim Pseudohermaproditismus femininus des kongenitalen adrenogenitalen Syndroms. Morphologie, Hausfigkeit, Entwicklung und Vererbung der verschiedenen Genitalformen.Helv. Pediatr. Acta., 9, 231­248.
15. Braun, A., Kammerer, S., Cleve, H., Lohrs, U., Schwarz, H.-P. and Kuhnle, U.(1993) True hermaphroditism in a 46,XY individual, caused by a post zygoticsomatic point mutation in the male gonadal sex-determining locus (SRY):Molecular genetics and histological findings in a sporadic case. Am. J. Hum.Genet., 52, 578­585.
16. Neely, E. K. and Rosenfeld, R. G. (1996) Turner syndrome. Pediatr.Endocrinol., 267­280.
17. McKusick, V.A. (1992) Camptomelic dwarfism, MIM no. 211970. InMendelian Inheritance in Man. Johns Hopkins University Press, Baltimore.
18. Hall, J. G. (1988) Somatic mosaicism: observations related to clinical genetics. Am. J. Hum. Genet., 43, 355­363.
19. Murray, E. W., Giles, A. R. and Lillicrap, D. (1992) Germ-line mosaicism for a valine-to-methionine substitution at residue 553 in the glycoprotein Ib-binding domain of von Willebrand factor, causing type IIb von Willebrand disease. Am. J. Hum. Genet., 50, 199­207.
20. Lázaro, C., Ravella, A., Gaona, A., Volpini, V. and Estivill, X. (1994)Neurofibromatosis type 1 due to germ-line mosaicism in a clinically normal father. N. Engl. J. Med., 331, 1403­1407.
21. MacDonald, M. E., Barnes, G., Srinidhi, J., Duyao, M. P., Ambrose, C. M.,Myers, R. H., Gray, J., Conneally, P. M., Young, A., Penney, J., Shoulson, I.,Hollingsworth, Z., Koroshetz, W., Bird, E., Vonsattel, J. P., Bonilla, E.,Moscowitz, C., Penchaszadeh, G., Brzustowicz, J., Alvir, J., Bickham Conde,J., Cha, J.-H., Dure, L., Gomez, F., Ramos-Arroyo, M., Sanchez-Ramos, J.,Snodgrass, S. R., de Young, M., Wexler, N. S., MacFarlane, H., Anderson, M.A., Jenkins, B. and Gusella, J. F. (1993) Gametic but not somatic instability of CAG repeat length in Huntington's disease. J. Med. Genet., 30, 982­986.
22. Eicher, E. M., Washburn, L. L., Whitney, J. B. and Morrow, K. E. (1982) Mus Poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. Science, 217, 535­537.
23. Washburn, L L., Lee, B. K. and Eicher, E. M. (1990) Inheritance ofT-associated sex reversal in mice. Genet. Res., 56, 185­191.
24. Sinclair, A. H. (1995) New genes for boys. Am. J. Hum. Genet., 57,998­1001.25. Hawkins, J. R. (1994) Sex determination. [Review] Hum. Mol. Genet., 3,1463­1467.
26. Morrison, K. E., Daniels, R. J., Suthers, G. K., Flynn, G. A., Francis, M. J.,Buckle, V. J. and Davies, K. E. (1992) High-resolution genetic map around the spinal muscular atrophy (SMA) locus on chromosome 5. Am. J. Hum.Genet., 50, 520­527.
27. Wright, D. K. and Manos, M. M. (1990) Sample preparation from paraffin-embedded tissues. In Innis, M. A., Gelfand, D. H., Sninsky, J. J. andWhite, T J. (eds). PCR Protocols. A guide to methods and applications.Academic Press, San Diego, pp.153­158.
28. Miller, S. A., Dykes, D. D. and Polesky, H. F. (1988) A simple salting outp rocedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215.