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Original Studies |
Department of Pediatrics, Keio University School of Medicine (K.M., T.Ok., T.Og.), Tokyo 160-8582; Department of Genetics, National Children’s Medical Research Center (T.Ok.), Tokyo 154-8509; Divisions of Neonatology (K.G.) and Clinical Pathology (Y.O.), Nagano Children’s Hospital, Toyoshina 399-8288; Department of Pediatrics, Kurashiki Central Hospital (S.F., J.K.), Kurashiki 710-8602; Division of Endocrinology, Chiba Children’s Hospital (K.S.), Chiba 266-0007; Department of Pediatrics, Toyohashi Municipal Hospital (Y.S.), Toyohashi 441-8570; Mitsubishi Kagaku Bioclinical Laboratories, Inc. (H.T.), Tokyo 174-8555; and Departments of Orthopedics (H.G.) and Hygiene and Medical Genetics (K.W., Y.F.), Shinshu University School of Medicine, Matsumoto 390-8621, Japan
Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: t-ogata{at}po.iijnet.or.jp
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Abstract |
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The results provide further support for the presence of a sex-determining gene(s) on distal 9p and favor the possibility of DMRT1 and/or DMRT2 being the sex-determining gene(s). Furthermore, as hemizygosity of the 9p sex-determining region was associated with a wide spectrum of gonadogenesis from agonadism to testis formation in karyotypic males and with primary hypogonadism regardless of karyotypic sex, it is inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The sex-determining gene(s) has been located to a roughly 250-kb region distal to D9S1779 at 9p24.3, on the basis of molecular studies in XY sex-reversed patients with partial 9p monosomy (8, 10, 13, 14, 15, 16, 17). Recently, Raymond et al. identified evolutionary conserved, sex-determining genes with a novel DNA-binding domain (DM domain) (18), and mapped their human homologous gene DMRT1 (formerly called DMT1) to 9p24.3 distal to D9S1779 (17). DMRT1/Dmrt1 is conserved across the phyla, including Caenorhabditis elegans, Drosophila melanogaster, chicken, alligator, mouse, and human (18, 19), and shows a male-dominant, sexually dimorphic expression pattern in at least the alligator, chicken, and mouse (19, 20). In mammals, mouse Dmrt1 is expressed exclusively in the genital ridge before gonadal sex determination and shows a testis-dominant expression with gonadal sex development (19, 20), and human DMRT1 shows a testis-specific expression in adults (17, 18). Furthermore, Raymond et al. identified a second DM domain gene, DMRT2, expressed in adult testes and mapped it to 9p24.3 distal to D9S1779 (17). These findings argue that DMRT1 and DMRT2 represent excellent candidates for the sex-determining gene(s) on 9p.
However, several matters remain uncertain for the 9p sex-determining gene(s). First, there has been no formal evidence equating DMRT1 and/or DMRT2 with the sex-determining gene(s) (17). Second, it remains to be clarified whether sex reversal in 9p monosomy is caused by haploinsufficiency of a single gene, hemizygosity of plural genes, or unmasking a recessive mutation(s) on the normal chromosome 9 (5, 7, 8, 17). Third, it is unknown whether male sex development in 9p monosomy has occurred under terminal 9p deletions missing the sex-determining region or interstitial 9p deletions preserving that region (17). Lastly, the role of the 9p sex-determining gene(s) in the fetal gonadogenesis remains to be determined.
In this paper we report on clinical and molecular findings in six patients with partial 9p monosomy and discuss the unresolved issues for the 9p sex-determining gene(s).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This study consisted of six Japanese patients with distal 9p
monosomy. Age, karyotype, and nongenital features of each patient are
shown in Table 1
. Cases 1–5 were
karyotypic males, and case 6 was a karyotypic female. Case 4 has been
reported previously (8). All cases shared monosomy of 9p24 in common.
The parents of cases 1 and 4 had normal karyotypes, the sister of case
3 had a 46,XX, der(4)t(4;9)(p15;p23) karyotype, and the father of case
6 had a 46,XY, t(9;16)(p24;q22) karyotype. Physical growth was variable
among patients, ranging from low normal growth (case 3) to profound
growth failure (case 2). Developmental retardation was clinically
obvious in all cases. Multiple congenital minor anomalies were detected
in all cases together with major anomalies in cases 2–4 and 6. Renal
and urinary tract anomalies were absent in all cases. The general
condition was severely impaired in case 2 and relatively well preserved
in the remaining cases.
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. Cases 1 and 2 had female external
genitalia, and magnetic resonance imaging delineated uterus and
undetected Wolffian and gonadal structures; however, a direct gonadal
examination was not performed, so it was uncertain whether cases 1 and
2 had dysgenetic gonads or agonadism. Case 3 manifested female external
genitalia with hypoplastic labia majora, and macroscopic exploration at
the time of gonadectomy at 15 months of age disclosed left streak gonad
with Wolffian structures and right agonadism with Müllerian
structures. Histological examination showed left dysgenetic gonad with
gonadoblastomas and confirmed right agonadism (Fig. 1). Case 4 exhibited ambiguous genitalia,
and macroscopic and microscopic examinations at the time of gonadectomy
at 12 months of age revealed bilateral hypoplastic testes with Wolffian
structures. Case 5 had male external genitalia with left cryptorchidism
and right intrascrotal testis of approximately 15 mL; however, it was
not determined whether the left gonad was testis-like, dysgenetic, or
absent, because of parental refusal for further studies. Case 6
exhibited female external genitalia; however, gonadal structures was
not examined, because of parental refusal for further
investigations.
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. The results indicated that cases 1, 3,
and 6 had primary hypogonadism. Case 2 was unremarkable for the
baseline values. Case 4 showed fairly good testicular function. Case 5
was not available for endocrine studies.
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Lymphocyte metaphase spreads of cases 1–6 and the father of
case 6 were hybridized with a bacterial artificial chromosome (BAC)
probe containing DMRT1, but lacking DMRT2; a yeast artificial
chromosome (YAC) probe containing D9S1858, DMRT1, and DMRT2 (765H2); a
YAC probe containing D9S1779, DMRT1, and DMRT2 (757A1); and a P1 phage
artificial chromosome (PAC) probe containing D9S1136 (34H2) (25). The
BAC probe was obtained by screening of a BAC library with primers for
DMRT1 (forward, 5'-AGGCATTCAGCAAGCC CTCTA-3'; reverse,
5'-AGACGCTTTGCCAAAGC-3') and was shown to lack DMRT2 by PCR with the
JK7/JK8 primers for DMRT2 (17). The 765H2 YAC containing D9S1858 (13)
and the 757A1 YAC containing D9S1779 (13) were confirmed to harbor both
DMRT1 and DMRT2 by PCR amplification with the above primers for DMRT1
and DMRT2. For an internal signal control, metaphase spreads were
concomitantly hybridized with a satellite probe for D9Z1 (Oncor, Inc.,
Gaithersburg, MD) or a BAC probe for APBA1. The chromosomal location of
the examined loci is shown in Table 4. In
addition, whole chromosome 9 painting (Oncor, Inc.) was performed in
case 1, 4p telomere FISH was carried out with a B31 cosmid probe (25)
in cases 3 and 5, and 16q telomere FISH was performed with a D3b1
cosmid probe (25) in case 6 and her father. The DMRT1 BAC probe, 765H2,
757A1, 34H2, and 9p painting probe were labeled with digoxigenin and
were detected by rhodamine antidigoxigenin. The remaining probes were
labeled with biotin and were detected by avidin conjugated to
fluorescein isothiocyanate.
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Genomic DNA was extracted from peripheral leukocytes of cases
1–4 and 6, the parents of cases 1 and 4, and the father of case 6 and
was amplified by PCR with primers defining 18 loci on chromosome 9
(Table 4
). Amplification was performed in a reaction volume of 20 µL
containing 0.1 µg genomic DNA, 8 pmol fluorescently labeled forward
primer, 8 pmol unlabeled reverse primer, 0.25 mmol/L deoxy-NTPs, and 1
U Taq polymerase. The primer sequences and the PCR
conditions are described in the Genome database. The PCR products were
determined for fragment size on an autosequencer (ABI PRISM 310,
PE Applied Biosystems, Perkin-Elmer Corp.,
Foster City, CA) using GeneScan software 2.1. In case 1, PCR
products of different cycles were examined for area under curve to
allow for semiquantitative analysis.
Sequence analysis
Leukocyte genomic DNA of cases 1–4 and 6 was examined for the DMRT1 and DMRT2 sequences, and that of cases 1–4 was also analyzed for the SRY sequence. In brief, each exon of DMRT1, the DM domain of DMRT2, and the coding region of SRY were amplified by PCR, and the PCR products were subjected to direct sequencing from both directions on the autosequencer (it was impossible to examine each exon of DMRT2, because exon-intron boundaries have not been determined for DMRT2). The primer sequences and the PCR conditions were described previously (17, 28).
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Results |
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Representative results are shown in Fig. 2, and the data are summarized in Table 4. In all cases, the DMRT1 BAC probe, the 765H2 probe, and the 757A1
probe detected positive signals on the normal chromosome 9 alone,
demonstrating that the 9p sex-determining region distal to D9S1779,
including both DMRT1 and DMRT2, was deleted from the abnormal
chromosome 9. The 34H2 probe detected positive signals on both the
normal and the abnormal chromosome 9 in cases 1 and 5 and on the normal
chromosome 9 alone in the remaining cases. In the father of case 6, the
four probes delineated positive signals on the der(16) chromosome and
on the normal chromosome 9. D9Z1 and/or APBA1, used as internal
controls, were present in two copies, one on the normal chromosome 9
and the other on the abnormal chromosome 9, in all cases and in the
father of case 6. The chromosome 9 painting probe detected positive
signals on the entire add(9)(p24) chromosome in case 1, indicating that
the extra chromosomal material added to 9p24 was derived from
chromosome 9. The B31 probe detected positive signals on the der(9)
chromosome and the normal chromosome 4 in cases 3 and 5, confirming the
4p;9p translocations. The D3b1 probe identified positive signals on the
der(9) chromosome and the normal chromosome 16 in case 6 and her father
together with week cross-hybridization signals on a few chromosomes,
demonstrating 9p;16q translocations.
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Representative results are shown in Fig. 3, and the data are summarized in Table 4. In cases 1, 4, and 6, genotyping analysis confirmed distal 9p
monosomy, with the breakpoints between D9S1871 and D9S143 in case 1,
between D9S168 and D9S288 in case 4, and between IFNA and D9S288 in
case 6. In cases 2 and 3, single peaks only were detected for all of
the 9p loci distal to IFNA and D9S169, respectively, providing further
support for distal 9p monosomy in both cases. In addition, comparison
of the area under the curve between paternally and maternally derived
peaks in case 1 indicated duplication of five paternally derived
alleles on 9p12–9p24 (for details, see Fig. 3), suggesting that the
add(9) chromosome was generated by abnormal sister chromatid exchange
during paternal meiosis and was associated with deletion of the tip of
9p and inverted duplication of most part of 9p.
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The sequences of each exon of DMRT1 were normal in cases 1–4 and
6, with a previously reported polymorphism being identified in cases 1
and 3 (A
T variation at a nucleotide position 133 from the start
codon at the exon 1) (17). The DM domain sequence of DMRT2 was also
normal in cases 1–4 and 6, as was the SRY sequence in cases 1–4.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cases 1–4 had variable degrees of sex reversal and distal 9p monosomy. Morphological and/or endocrine studies indicated defective testis formation in cases 1, 3, and 4, and molecular studies showed hemizygosity of the 9p sex-determining region distal to D9S1779 and loss of both DMRT1 and DMRT2 from the abnormal chromosome 9 in cases 1–4. Although endocrine studies failed to show hypergonadotropic hypogonadism in case 2, this could primarily be due to severe malnutrition. Indeed, patients with anorexia nervosa are known to have hypogonadotropic hypogonadism with malnutrition (31). Thus, the results provide further support for the presence of the sex-determining gene(s) distal to D9S1779 and favor the possibility of DMRT1 and/or DMRT2 being the sex-determining gene(s) (17, 18, 19, 20), although it remains to be determined whether DMRT1 and/or DMRT2 is truly the sex-determining gene(s).
Sex reversal in cases 1–4 occurred with normal sequences of each exon of DMRT1 and the DM domain of DMRT2 together with the intact SRY sequence. This implies that the examined sequences of DMRT1 and DMRT2 were intact on the normal chromosome 9, although it might be possible that a mutation existed in the promoter region of DMRT1 or in the promoter or coding region other than the DM domain of DMRT2. Thus, if DMRT1 and/or DMRT2 is the sex-determining gene(s), it is likely that sex reversal is caused by haploinsufficiency of a single gene or both genes, rather than by unmasking a recessive mutation(s) on the normal chromosome 9.
Case 5 showed normal male external genitalia, although he had unilateral cryptorchidism. The development of male external genitalia has also been described in karyotypic males with distal 9p monosomy (1, 32). Here, his 46,XY, der(9)t(4;9)(p13;p24) karyotype indicates a terminal, rather than an interstitial, 9p deletion, and the FISH results are consistent with loss of the 9p sex-determining region distal to D9S1779 (13). In addition, it is unlikely that a mutation(s) existed on the normal chromosome 9, because of male external genitalia in case 5. Thus, it is suggested that male external genitalia can occur in patients with 9p deletions, under hemizygosity of the 9p sex-determining region.
Case 3 had unilateral agonadism with Müllerian derivatives, and case 6 showed primary hypogonadism. These findings are informative to deduce the role of the 9p sex-determining gene(s) in the fetal gonadogenesis. Fetal gonadogenesis is divided into three steps: 1) formation of indifferent gonad from zygote, 2) development of fetal testis from indifferent gonad, and 3) development of fetal ovary from indifferent gonad (33). The first step is common to both sexes and forms a bipotential gonad, the second step is specific to males and is characterized by the development of Sertoli cells for seminiferous tube formation and Leydig cells for testosterone production, and the third step is specific to females and is characterized by the development of oocytes with meiosis. In this regard, right agonadism with Müllerian derivatives in case 3 would suggest failure in the formation of indifferent gonad. The defect after the formation of indifferent gonad should cause dysgenetic testis rather than agonadism, and the possibility of testicular regression is unlikely because of ipsilateral Müllerian development and Wolffian regression. Furthermore, primary hypogonadism in case 6 would also argue for defective formation of indifferent gonad common to both sexes, although it is unknown whether the patient had dysgenetic ovaries, streak gonad, or agonadism. Thus, it is inferred that the 9p sex-determining gene(s) is operating in the formation of indifferent gonad, and that haploinsufficiency of the 9p sex-determining gene(s) hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females.
As a whole, the present study implies that karyotypic males with haploinsufficiency of the 9p sex-determining gene(s) have a wide range of sex development, with variable gonadal phenotype between patients and between gonads in a patient. By analogy, it is conceivable that similarly affected karyotypic females would also have variable sex development, although there has been no previous report describing molecular or endocrine data in karyotypic females with distal 9p monosomy. Such phenotypic variability is not specific to haploinsufficiency of the 9p sex-determining gene(s). It is known that haploinsufficiency of genes involved in human development frequently shows a wide range of penetrance and expressivity (34). For example, haploinsufficiency of SOX9 results in a wide spectrum of sex development in karyotypic males, ranging from nearly normal male phenotype to nearly complete female phenotype (35). Similarly, gonadal phenotype in familial sex reversal and true hermaphroditism can vary between patients and between gonads in a patient (33, 36). Probably, the clinical effect of haploinsufficiency would be subject to the modification of multiple genetic and/or environmental factors.
Murine Dmrt1 is predominantly expressed in the developing genital ridge of both sexes and in the Sertoli cells and germ cells of developing testis (20). Provided that Dmrt1 is relevant to sex determination, this expression pattern would suggest two possibilities. First, Dmrt1 may be involved in both the formation of indifferent gonad and the development of the fetal testis. In this case, sex reversal in karyotypic males with 9p monosomy could be due not only to defective formation of indifferent gonad but also to impaired development of the fetal testis. Second, Dmrt1 may have distinct biological functions at different developmental stages. In this case, Dmrt1 may be required for the formation of indifferent gonad and then for some unknown testicular function, such as interaction between Sertoli cells and germ cells (37). In support of the latter possibility, Sf-1 is known to play an essential role in the formation of indifferent gonad and in the production of testicular hormones (38). Further studies, including knockout mouse experiments, will serve to clarify the role of Dmrt1 in the gonadal development and function.
In summary, although the 9p sex-determining gene(s) is still elusive, the results suggest that haploinsufficiency of the 9p sex-determining gene(s) primarily hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females. Accumulation of clinical and molecular data in similarly affected patients will permit better elucidation of the nature of the 9p sex-determining gene(s).
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Acknowledgments |
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Footnotes |
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Received January 10, 2000.
Revised April 21, 2000.
Accepted May 24, 2000.
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References |
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