Forgetting RSPO1

9 04 2010

To the Editor:

We have read with much interest the article by Kim et al. (1) dealing with the clinical, cytogenetic and molecular analysis of a SRY-negative 46,XX sex reversal male.

Actually this clinical condition, which is extremely rare, is very interesting for researchers involved in studies regarding sex determination process since the molecular analysis of such patients could lead to the discovery of genes involved in testis differentiation in absence of the master gene SRY. Nevertheless the article includes a number of inaccuracies and omissions regarding the state of the art on clinical features and genetic studies of this condition in human patients and animal models. Consequently some aspects of it ought to be revised or clarified as follows:

1. To explain one of the possible causes of induction of testicular tissue in SRY- negative patients, the authors reported the “Z” gene model, first suggested by McElreavey and colleagues in 1993 (2), but they completely misrepresented it. Indeed they wrote “a regulatory autosomal recessive gene, termed ‘‘Z’’ gene, whose product inhibits SRY that normally inhibits the male pathway.”

This statement seems to indicate that 1st – “Z” gene inhibits SRY and 2nd- SRY inhibits the male pathway, whilst the original hypothesis proposes that “the wild-type Z product is a negative regulator of male determination and is functional in wild-type females. In males, SRY product represses or negatively regulates Z and thereby allows male sex determination.” The “Z” gene model is very interesting, as it fits the hypothesis that different kind of mutations, i.e. loss or gain of function mutations, in the same gene can respectively lead to XX or XY sex reversal.

2. The authors in their discussion reported that increased expression of SOX9 and DAX1 genes could lead to 46, XX female-to-male sex reversal. It is worth reminding that, while it has been proposed that Dax1 is required for testis determination in mice, nevertheless in humans DAX1 duplications lead to XY-SRY+ sex reversal phenotype (not XX maleness) (3).

Regarding the hypothetical role of SOX9 over expression in XX maleness the authors omitted to report a genomic duplication containing SOX9 (4). Moreover the authors suggested the requirement of transgenic experiments to confirm the involvement of these genes in SRY negative XX sex maleness. XX SOX9 transgenic mice already exist (resulting in XX SR) (5) whilst in mice overexpression of DAX1 leads to XY SR (6).

3. Regarding the literature review about 46,XX sex reversal in the absence of SRY, the role of RSPO1 gene in this condition and in sex determination process has also been omitted. Mutations in RSPO1 have been found to be responsible for 46,XX female-to-male sex reversal in 4 brothers from a consanguineous family (7). These patients had been already described in 2005 and reported to show Leydig cell hyperplasia, (8). RSPO1 is the first gene, and the unique to date, in which null mutations lead to complete 46,XX maleness in the absence of the SRY gene and its fundamental role in sex determination process has been confirmed by animal models (9) and broadly discussed in a number of reviews. Moreover RSPO1 homozygous mutations are responsible for the phenotype of the patient described in Vernole et al., 2000 (cited in Kim et al. article) and for a case of true hermaphroditism (10).

Dr. Pietro Parma,
Dept. Of Animal Science
University of Milan
Milan, Italy

Dr. Orietta Radi,
Dept. of Human and Hereditary Pathology
University of Pavia
Pavia, Italy

1. Kim et al. 2010. Y-negative 46,XX infertile male with Leydig cell hyperplasia: clinical, cytogenetic, and molecular analysis and review of the literature • CASE REPORT , Available online 12 March 2010

2. McElreavey et al., 1993. A regulatory cascade hypothesis for mammalian sex determination: SRY represses a negative regulator of male development. Proc Natl Acad Sci U S A. 90:3368-72.

3. Bardoni et al. 1994. A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal. Nature Genet. 7: 497-501.

4. Huang et al. 1999. Autosomal XX sex reversal caused by duplication of SOX9. J. Am J Med Genet. 87(4):349-53.

5. Vidal et al., 2001. Sox9 induces testis development in XX transgenic mice. Nature Genet. 28: 216-7.

6. Swain et al., 1998. Dax1 antagonizes Sry action in mammalian sex determination. Nature 391: 781-6.

7. Parma et al., 2006. R-spondin 1 is essential in sex determination, skin differentiation and malignancy. Nature Genet. 38: 1304-9.

8. Radi et al. 2005. XX sex reversal, palmoplantar keratoderma, and predisposition to squamous cell carcinoma: genetic analysis in one family. Am J Med Genet A.138A(3):241-6.

9. Chassot et al., 2008. Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum Mol Genet. 17(9):1264-77.

10. Tomaselli et al., 2008. Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation. Hum Mutat. 29(2):220-6.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.04.021

The Authors Respond:

We are sincerely grateful of the interests and comments made by Dr. Parma and Dr. Radi, whose previous articles provided exceptionally valuable information such as R-Spondin1 (RSPO1).

As you understand, SRY-negative 46,XX males were extremely rare clinical condition. Up to now, several genes including SRY, SOX9, DAX-1, WT1, RSPO1, etc, have been known to be associated with mammalian sex-determination. However, the molecular mechanism of sex reversal, especially, complete sex reversal condition like the case presented by us, is not fully understood.

RSPO1 mutations were identified in two independent families and one sporadic individual with 46,XX sex reversal (1). In addition, Rspo1-/- mice also showed hermaphroditism not complete sex reversal (2). However, the patient in our study is completely different from the one who had RSPO1 mutations. First of all, our patient has not had any dermatologic problems such as keratosis or other symptoms in Huriez syndrome. Secondly, he was not a true hermaphrodite since abdominal and pelvic CT as well as ultrasound evaluation did not detect any abnormalities. In detail, the andrologist requested a thorough examination of his abdomen and pelvis in the search of any rudimentary female organs that were reviewed by a group of expert radiologists and our patient with no ovarian tissues revealed by histopathologic examination.

For the above-mentioned reasons, we did not pay attentions to the RSPO1 gene and missed some articles when searching for the references. On the other hand, we briefly mentioned SOX9 and DAX-1 gene in our report. It has been reported that SOX9 and DAX-1 gene expression were altered in 46,XX males, the increased SOX9 expression and the decreased DAX-1 levels (3). So, we have thought these two genes should be checked to elucidate the molecular mechanism of the SRY negative XX male sex reversal.

Lastly, in this case report, we did not study SOX 9, DAX-1 or any other related genes such as RSPO1 but SRY, and are willing to look into the genes. Again, we would like to reemphasize that, phenotypically, he was a male without ambiguous genitalia, other physical abnormalities except the ones described in the article or family history, unlike the case reported by Dr. Parma and Dr. Radi, and, hence, our case is still unique. The fact that it has not been clearly understood the underlying mechanisms of sexual differentiation despite of endeavor by researchers and renders all of us further study.

Ji Won Kim M.D.a,b
Chong Won Bak M.D.a,c
Sung Han Shim Ph.D.a,d
aFertility Center of CHA Gangnam Medical Center
bDepartment of Obstetrics and Gynecology,
cDepartment of Urology
dGenetic Laboratory
CHA University
Seoul, South Korea

1. Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nature Genetics 2006;38:1304-9.

2. Chassot AA, Gregoire EP, Magliano M, Lavery R, Chaboissier MC. Genetics of ovarian differentiation: Rspo1, a major player. Sexual development 2008;2:219-27.

3. Kojima Y, Hayashi Y, Mizuno K, Sasaki S, Fukui Y, Koopman P et al. Up-regulation of SOX9 in human sex-determining region on the Y chromosome (SRY)-negative XX males. Clinical endocrinology 2008;68:791-9.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.04.022




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