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Research Unit in Developmental Biology (P.C., D.S., J.P.M.), Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México, Distrito Federal 06725, México; and Department of Pathology (E.R.), Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán", México, Distrito Federal 14000, México
Address all correspondence and requests for reprints to: Juan Pablo Méndez, M.D., Unidad de Investigación Médica en Biología del Desarrollo, Coordinación de Investigación en Salud, Coahuila 5, Apartado Postal A-047, Colonia Roma, C.P. 06703, México, D.F., México. E-mail: jpmb{at}servidor.unam.mx.
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Abstract |
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DHH was sequenced using genomic DNA from paraffinembedded gonadal tissue from six patients with complete 46,XY PGD. Mutations were found in three patients: a homozygous mutation in exon 2, responsible for a L162P, and a homozygous 1086delG in exon 3.
Mutated individuals displayed 46,XY complete PGD, differentiating from the only previously described patient with a homozygous DHH mutation, who exhibited a partial form of PGD with polyneuropathy, suggesting that localization of mutations influence phenotypic expression.
This constitutes the first report where mutations of DHH are associated with the presence of 46,XY complete PGD, demonstrating that the genetic origin of this entity is heterogeneous and that disorders in other genes, different from SRY, involved in the testis-determining pathway are implicated in abnormal testicular differentiation in humans. These data extend previous reports demonstrating DHH is a key gene in gonadal differentiation.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Gonadal dysgenesis encompasses a heterogeneous group of different chromosomal, gonadal and phenotypic abnormalities (2, 3). Sex reversal in XY females is a consequence of failure of testis determination or differentiation. Individuals with the complete form of 46,XY pure gonadal dysgenesis (PGD) present a 46,XY karyotype, bilateral streak gonads, normally developed Müllerian ducts, and female external genitalia (2). It has been estimated that the presence of the Y chromosome in 46,XY PGD patients increases (10–30%) the risk of developing gonadal tumors, i.e. gonadoblastoma or dysgerminoma (4, 5).
Mutations of the SRY gene are the cause of 46,XY sex reversal in approximately 10–15% of patients with PGD. In the remaining individuals, a precise cause has not been determined, and it has been suggested that they may bear mutations in the SRY regulatory elements or in other genes involved in the testis-determining pathway (6, 7). One of these genes is Desert hedgehog (DHH), a member of the hedgehog family of signaling proteins, which also includes Sonic hedgehog and Indian hedgehog (8). In humans, DHH is located in 12q12
q13.1, is composed of three exons, and encodes a protein of 396 amino acids (9).
In mice, Dhh has a sexually dimorphic expression. Analysis of gene transcripts demonstrated that expression of Dhh is observed in fetal testes at 11.5 d postcoitum, whereas no transcripts are detected in fetal ovaries (10). The product of the Dhh gene is specifically expressed in Sertoli cells and Schwann cells along peripheral nerves (11, 12). The importance Dhh has in testicular morphology was originally described by Clark et al. (13) in a study in which the majority of Dhh null male mice developed into pseudohermaphrodites. Likewise, it was demonstrated that the differentiation of peritubular myoid cells and the consequent formation of testis cords is regulated by Dhh (13, 14). Furthermore, Hung-Chang et al. (1) suggested that Dhh/Ptch1 signaling is a positive regulator of the differentiation of steroid-producing Leydig cells in the fetal testis. Dhh is expressed in Sertoli cells, being the only mammalian hedgehog protein expressed in the gonad between 11.5 and 13.5 d postcoitum. Ptch1, one of the hedgehog receptors, is expressed in interstitial cells. In conclusion, Dhh/Ptch1 signaling triggers Leydig cell differentiation by up-regulating Steroidogenic Factor 1 and P450 Side Chain Cleavage enzyme expression in Ptch1-expressing precursor cells, which are located outside the testis cords.
In 2000, Umehara et al. (15) reported a homozygous missense mutation of the DHH gene in one patient with 46,XY partial gonadal dysgenesis associated with minifascicular neuropathy. The authors suggested that DHH is a key molecule that intervenes in both male gonadal differentiation and perineural formation in peripheral nerves.
Here we describe the first report in which in three cases of 46,XY complete PGD, homozygous mutations of the DHH gene are associated with the disorder.
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Patients and Methods |
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The study was approved by the Institute’s Human Research Committee. Gonadal tissue obtained from gonadectomies between 1983 and 1998, from six unrelated phenotypic females with 46,XY complete PGD, was molecularly studied. All patients had a Mexican-mestizo ethnic origin, and in all cases family history was negative for consanguinity. All individuals had a nonmosaic 46,XY karyotype in at least 50 cells (peripheral blood leukocytes). Clinical and histopathological findings in these patients are shown in Table 1
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Genomic DNA was isolated from peripheral blood leukocytes in control subjects by standard techniques (16) and from paraffin-embedded gonadal tissue from the patients and three controls using the MagneSil genomic fixed tissue system (Promega Corp., Madison, WI), following the conditions recommended by the manufacturer. DNA was amplified by PCR in 50 µl of reaction mixture containing 0.3 µg genomic DNA, 0.1 mM dNTPs, 2.0 U of thermostable DNA polymerase (AmpliTaq, Applied Biosystems, Foster City, CA), and 250 nM of each specific set of DHH primers. The sequences of the DHH primers for all three exons and the PCR conditions were described by Umehara et al. (15) (GenBank accession number AB010581 for exon 1, AB010993 for exon 2, and AB010994 for exon 3).
After amplification, PCR products were electrophoresed in a 1.2% agarose gel and afterward purified using the QIAEX II gel extraction kit (QIAGEN GmbH, Hilden, Germany). These products were then sequenced (0.1 µg DNA template reaction) on an ABI 377 automated DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA) using the BigDye terminator cycle sequencing ready reaction kit (Perkin-Elmer). For all exons, both strands were sequenced and compared. Each mutation was confirmed in three independent PCR amplifications and sequencings.
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Results |
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Genetic defects in the DHH gene were found in three of six patients. In patient 4, a homozygous mutation in exon 2 consisting of a T-to-C substitution at position 485 was observed. This mutation was responsible for a leucine (CTG) into proline (CCG) substitution at codon 162 (Fig. 1). Exons 1 and 3 showed no sequence variations. In patients 2 and 6, a nucleotide deletion was found in exon 3, at position 1086, which comprises the third nucleotide (guanine) of codon 362. This deletion caused a stop codon (TAG), four codons after the deletion was located (Fig. 2). No sequence variations were observed in the first two exons of the gene.
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One hundred normal male individuals (200 alleles) did not harbor any of the mutations, being homozygous wild type.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In mammals, testis development is initiated in response to the expression of Sry in Sertoli cell precursors. In Sertoli cells, the activation of Dhh transcription occurs immediately after the initiation of Sry expression (11). Bitgood et al. (10) generated knockout mice genetically null for the Dhh gene, observing male sterility. Likewise, Clark et al. (13) studied the testes from adult and prepubertal mice lacking Dhh, demonstrating peritubular cell defects that may be indicative of the role these cells have in the development of tubular morphology, Leydig cell differentiation, and spermatogenesis. Umehara et al. (15) analyzed the DHH gene in peripheral blood from one subject with 46,XY partial PGD and polyneuropathy who presented premature female genitalia, a blinded vagina and immature uterus, as well as a testis in one side and a streak gonad on the other. These authors found a missense mutation at the initiation codon of exon 1, which abolished initiation of translation at the normal start site, suggesting that failure of translation of the DHH gene may disturb the differentiation of male gonads and may result in 46,XY partial PGD.
In the present study, we analyzed all three exons of the DHH gene in six patients with 46,XY complete PGD, finding homozygous mutations in three of them. Mutated individuals displayed a 46,XY complete PGD (female external genitalia, bilateral fallopian tubes with infantile uterus, and streak gonads). To date, the only DHH mutation described in 46,XY PGD is the one by Umehara et al. (15); comparing this mutation with our cases, we can affirm that the phenotypic spectrum of 46,XY PGD patients with mutations in the DHH gene is variable, ranging from a partial form of PGD with polyneuropathy (15) to complete PGD without polyneuropathy. All of our patients had normal motor functions of both extremities and superficial and deep sensations; reflexes were also normal, there was no presence of symptoms or signs that suggested polyneuropathy, and mental function was not impaired. The phenotypic differences observed between our patients and the one reported by Umehara et al. (15) suggest that the localization of the mutations as well as a variety of other factors influence the expression of the phenotype. The presence of DHH mutations in patients with 46,XY partial PGD, as well as in 46,XY complete PGD, is similar to what has been observed regarding the SRY gene, where mutations have been shown to induce 46,XY complete PGD or partial PGD (18).
The L162P mutation exhibited by patient 4 is located in the mature amino-terminal domain of the DHH protein, constituting the first spontaneous mutation described in this domain. This mutation led to a nonconservative amino acid substitution, changing a highly conserved residue (Table 2A). We assume that the L162P mutation affected DHH function considering that the mature amino-terminal domain of Hh proteins has been shown to be essential for all the known long- and short-range activities of this protein (19, 20), and perhaps this mutation could alter the ability to bind with the transmembrane protein Ptc, which is required for cellular responsiveness to DHH (21).
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). Taking into consideration that two of six patients exhibited this mutation and although there is no known relationship between them, the mutation could derive from a common ancestor by a founder gene effect. An alternative explanation is that the identical deletion observed might indicate the presence of a hot spot in this region of the DHH gene. This deletion is located in the carboxyl-terminal domain of the DHH protein. Lee et al. (19) examined the effects of several distinct types of mutations in the carboxyl-terminal domain of Drosophila melanogaster, demonstrating that deletion or alteration of residues within this domain is associated with absence or reduced efficiency of autoproteolysis of the hedgehog. Furthermore, Porter et al. (22) observed that the autoprocessing activity of the carboxy-terminal domain influences the cellular localization of the amino-terminal domain. As it has been proposed for the SRY gene (18), we assume that a given mutation in DHH may produce sufficient DHH activity to reach the threshold required for testis formation. However, the same mutation on a different genetic background may reduce DHH activity, preventing testis development. Bitgood et al. (10) observed that the severity of the phenotype presented in their colony of Dhh-null mice varied depending upon the genetic background. Those on an inbred background of the 129/Sv strain developed germ cells up to primary spermatocytes, whereas germ cells in some of the 129/Sv-C57BL/6JF1 hybrids developed through meiosis to become step-15 spermatids. Furthermore, Clark et al. (13) reported that in their colony of Dhh-null mice bred on a mixed genetic background, the phenotypic outcome of the Dhh-null condition was more severe than the ones previously described.
We identified mutations of the DHH gene in only three of the six patients studied. The true prevalence of such mutations is difficult to assess because of the rarity of this entity. Larger samples of patients will need to be studied to determine the true prevalence of DHH mutations in humans. The absence of mutations in the other patients studied indicates that molecular defects in such patients could be present in the untranslated regulatory regions of the DHH gene or within introns; besides, defects in other gene(s) could explain the disorder.
In conclusion, to our knowledge, this constitutes the first report where mutations of the DHH gene are associated with the presence of 46,XY complete PGD. These data demonstrate that the genetic origin of 46,XY complete PGD is heterogeneous and that disorders in other genes, different from SRY, that are involved in the testis-determining pathway are directly implicated in abnormal testicular differentiation in humans. Likewise, these data extend previous reports in humans and other species demonstrating that DHH constitutes a key gene in gonadal differentiation.
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Acknowledgments |
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Footnotes |
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Abbreviation: PGD, Pure gonadal dysgenesis.
Received May 11, 2004.
Accepted June 16, 2004.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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q13.1 by in situ hybridization. Cytogenet Cell Genet 88:93–94[CrossRef][Medline]
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