Journal of Heredity Advance Access originally published online on November 2, 2005
Journal of Heredity 2005 96(7):759-763; doi:10.1093/jhered/esi129
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Exclusion of Candidate Genes for Canine SRY-Negative XX Sex Reversal
From the J. A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Kothapalli, Pujar, Van Wormer, Meyers-Wallen); and the Institute for Genomic Research, Rockville, MD 20850 (Kirkness)
Address correspondence to V. N. Meyers-Wallen at the address above, or e-mail: vnml{at}cornell.edu.
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Abstract |
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 Top Abstract FOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.
In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of SRY. Specifically, testes or ovotestes develop in XX human patients and domestic animals with SRY-negative XX sex reversal. Autosomal genes causing this disorder in the majority of human patients as well as dogs, pigs, and horses are presently unknown. Transgenic models of SRY-negative XX sex reversal have been produced by introducing mutations in known genes, but there is no naturally occurring mouse model.
In the canine model, SRY-negative XX sex reversal segregates as an autosomal recessive trait with expression limited to 78,XX individuals that are homozygous for the sex reversal trait. Affected dogs have ovotestes (XX true hermaphrodites) or testes (XX males), a complete bicornuate uterus, and clitoral enlargement with bone formation characteristic of canine penile differentiation (Meyers-Wallen and Patterson 1988). With allowance for species differences in secondary sex characteristics, these phenotypes are strikingly similar to those observed in human patients. Furthermore, both XX true hermaphrodites and XX males occur within the same canine family, as has been reported in some human families (Sarafoglou and Ostrer 2000). Thus, the canine model of SRY-negative XX sex reversal could provide a novel gene in the mammalian sex determination pathway and increase our understanding of the mechanism of testis induction in the absence of SRY. Candidate genes for this disorder include autosomal genes known to have a role in the emergence of the bipotential gonad or those involved in testis or ovarian differentiation in humans, rodents, or other animal models (Table 1).
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FOXL2 and PISRT1 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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In the polled intersex (PIS) goat model, SRY-negative XX sex reversal and absence of horns are caused by a large deletion that affects transcription of FOXL2 and PISRT1, which are located nearby. Mutations in FOXL2 have been found in patients with the blepharophimosis/ptosis/epicanthus inversus syndrome and/or premature ovarian failure but not in association with sex reversal. The region homologous to the PIS goat mutation has been excluded by marker analysis, indicating that mutations involving FOXL2 and PISRT1 are not responsible for the canine disorder (Kothapalli et al. 2003).
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WT1 and DMRT1 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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Mutations in the Wilms' tumor gene cause renal and XY gonadal dysgenesis in humans (WAGR, Denys-Drash, and Frasier syndromes). WT1 encodes a zinc finger protein, and experimental evidence suggests that it upregulates SRY transcription by directly binding to DNA (Hossain and Saunders 2001). DMRT1, the double sex- and mab3-related transcription factor 1, encodes a protein with a zinc fingerlike DNA-binding motif (Pask et al. 2003; Raymond et al. 1999). DMRT1 is conserved among vertebrates, being involved in testis differentiation in mammals, birds, reptiles, amphibians, and fish (Aoyama et al. 2003) and is associated with Sertoli cell maturation (Raymond et al. 2000). Deletions involving DMRT1 result in XY sex reversal in some animals. In humans, the minimal deletion in 9p24.3 that results in XY sex reversal removes both DMRT1 and DMRT2, but no significant mutations in either were found in a survey of sex-reversed patients (Raymond et al. 1999). Regions homologous to WT1 and DMRT1 have been excluded by marker analysis as candidates in the canine model (Kothapalli et al. 2004, in press).
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GATA4 and FOG2 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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These genes have been attributed to the testis determination pathway in humans and mice. GATA4 encodes a transcription factor containing a zinc finger, and FOG2 is its cofactor. Both promote SRY expression in the XY gonad and are required for gonadal differentiation in mice (Tevosian et al. 2002). GATA4 is expressed in somatic cells of both XX and XY genital ridges and regulates testis DMRT1 expression (Lei and Heckert 2004).
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LHX1 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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Lim homeobox 1, also known as LIM1, encodes a transcription factor with a DNA-binding domain. LHX1 is expressed at various times in several tissues involved in sexual development, including the intermediate mesoderm, mesonephros, fetal gonads, and the Mullerian and Wolffian ducts. Knock-in and knock-out experiments confirmed that LHX1 is essential to development of Mullerian ducts in females (Kobayashi et al. 2003). Although the majority of LHX1 null mice died, the few that survived to a stage sufficient for ascertainment developed normal testes or ovaries, but Mullerian ducts were absent in XX mice.
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SF1 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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Steroidogenic factor 1 (SF1) has multiple roles in sex determination, including development of the pituitary, hypothalamus, and bipotential gonad and in transcriptional regulation of Mullerian-inhibiting substance and enzymes controlling steroid hormone synthesis. SF1 is also capable of regulating SRY transcription by transactivation of the promoter (Pilon et al. 2003). Both XX and XY mice with SF1 null mutations fail to develop gonads (Luo et al. 1994), as do human patients with similar mutations (Achermann et al. 1999).
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SOX9 |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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SRY-box containing gene 9 is an autosomal gene, which, like SRY, is a member of the high-mobility group, nonhistone proteins that associate with DNA. Transcriptional activation of SOX9 is critical to testis induction in all vertebrates, whether activated genetically (as in mammals) or environmentally (as in reptiles). In mammals it is likely that the function of SRY is to directly or indirectly upregulate SOX9. Initial SOX9 expression occurs in bipotential XX and XY gonads. After SRY is expressed, SOX9 is expressed only in XY gonads, becoming limited to pre-Sertoli cells (Moreno-Mendoza et al. 2003). Sertoli cell differentiation is critical to further testis organization and differentiation. SOX9 has the ability to induce the bipotential gonad to follow the testis determination pathway in the absence of SRY, as demonstrated in transgenic mice and human patients in which SOX9 overexpression results in testis differentiation in XX individuals (Bishop et al. 2000; Huang et al. 1999).
The objective of this study was to determine whether microsatellite marker alleles within GATA4, FOG2, LHX1, SF1, and SOX9 are associated with the affected phenotype in a subset of the canine SRY-negative XX sex reversal pedigree.
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Materials and Methods |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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Pedigree
The multigenerational canine pedigree of SRY-negative XX sex reversal was founded upon one American cocker spaniel sire that was outcrossed to beagles to produce F1, F2, and F1 backcross offspring (Meyers-Wallen and Patterson 1988). Affected animals were identified by two criteria: the presence of testicular tissue in serial histologic sections of the gonads and the absence of SRY in genomic DNA (Meyers-Wallen et al. 1995). A subset of informative animals (n = 20) from the multigenerational pedigree was genotyped. These individuals included affected dogs (n = 10) and their direct ancestors back to the founding sire of the pedigree.
Genes
Canine genes were identified previously or by query with human homologues to the 1.5x canine genome database (Kirkness et al. 2003) or the 7.6x canine genome sequence (http://www.ncbi.nlm.nih.gov/genome/guide/dog). To generate microsatellite markers, nucleotide repeats were identified within each canine gene. Flanking polymerase chain reaction (PCR) primers (Table 1) were designed with Primer Select software (DNASTAR, Madison, WI). The forward primer of each pair was 5'-end labeled with a 6-FAM fluorescent tag (IDT, Coralville, IA).
PCR Conditions
Amplification was performed from genomic DNA templates (50 ng) in a reaction volume of 30 µl containing 1µM of each primer, 0.25 mM dNTPs, 3 µl of 10x PCR buffer (Perkin-Elmer Life Sciences, Foster City, CA), 1.5–3 mM MgCl2, and 1.5 U Taq Polymerase (Ampli Taq II, Perkin-Elmer Life Sciences).
For SOX9 reactions, initial denaturation was 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 70°C for 1 min, 72°C for 1 min, and final extension at 72°C for 2 min. For FOG2, LHX1, and SF1 reactions, the annealing step was 60°C, 64 °C, and 54 °C, respectively, for 1 min in 35 cycles. Touch down PCR was used for GATA4 reactions, wherein annealing was performed between 57°C and 60°C for 1 min in 35 cycles.
A 1.5 µl portion of each amplified product was pooled with 0.5 µl of TAMRA standard (PRISM Genescan-500, Applied Biosystems, Warrington, UK) and 13 µl deionized formamide. Samples were denatured at 95°C for 5 min and analyzed on the ABI310 Genetic Analyzer with filter set C (Applied Biosystems, Foster City, CA). The size of reaction products relative to the internal TAMRA standard was determined by software (GENESCAN 2.1, Perkin-Elmer Life Sciences). All reaction products for each gene were run in the same assay and read relative to the same standard.
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Results |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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All comparisons were made to human or canine sequences deposited at www.ensembl.org, www.genome.ucsc.edu, or www.ncbi.nlm.nih.gov.
GATA4
A pentanucleotide (caaaa) repeat in canine GATA4 was identified in the genomic sequence. This region maps to CFA25 (Table 1). Two alleles ranging from 314 to 319 bp and 3 different genotypes were found in the pedigree subset (Figure 1A). Genotypes in affected dogs were 1/1, 1/2, and 2/2. Seven affected dogs had genotypes that were different from those of the founding sire, and in four, neither allele was inherited from the founding sire.
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FOG2
A tetranucleotide (cttt) repeat in FOG2 was identified in the canine genome. This region maps to CFA13 (Table 1). Five alleles ranging from 315 to 363 bp and eight different genotypes were found in the pedigree subset (Figure 1B). Genotypes in affected dogs were 1/1, 1/3, 1/4, 1/5, and 2/5. Six affected dogs had genotypes at this locus that were different from those of the founding sire, and in two, neither allele was inherited from the founding sire. In those two, allele 5 can be traced to dog C3. Allele 5 (363 bp) in C3 is likely to be derived from the paternal allele because it differs by one repeat (4 bp) of the tetranucleotide (cttt) from allele 1 (359 bp). The simplest explanation for the occurrence of allele 5 in C3 is that a duplication of one repeat in the microsatellite region occurred in allele 1, which was transmitted by the sire. Allele 3 (335 bp) in C3 is likely to be inherited from the dam (B2) given that it is also present in other beagles, such as B3.
LHX1
A pentanucleotide (gtttt) repeat was identified in the canine genomic sequence of LHX1. This region maps to CFA9 (Table 1). Two alleles ranging from 289 to 294 bp and a total of two different genotypes at the LHX1 marker locus were identified in the pedigree (Figure 1C). Genotypes in affected dogs were 1/1 (n = 4) and 1/2 (n = 6). Six affected dogs had genotypes at this locus that were different from those of the founding sire. Due to the paucity of alleles at this locus, phase could not be determined with certainty for all affected dogs; however, four affected dogs (C9, C10, C18, C19) had at least one allele that was not inherited from the founding sire.
SF1
A trinucleotide (agt) repeat was identified in the genomic sequence of canine SF1. This region maps to CFA9 (Table 1). Three alleles ranging from 466 to 475 bp and a total of five different genotypes at the SF1 marker locus were identified in the pedigree (Figure 1C). Genotypes in affected dogs were 1/1 (n = 1), 1/2 (n = 4), 2/3 (n = 3), and 3/3 (n = 2). Six affected dogs had genotypes at this locus that were different from those of the founding sire, and in two, neither allele was inherited from the founding sire.
SOX9
A trinucleotide (cca) repeat was identified in the genomic sequence of canine SOX9. This region maps to CFA18 (Table 1). Two alleles ranging from 340 to 343 bp and a total of two different genotypes at the SOX9 marker locus were identified in the pedigree (Figure 1D). Genotypes in affected dogs were 1/1 (n = 3) and 1/2 (n = 7). Three affected dogs had genotypes at this locus that were different from those of the founding sire. Due to the paucity of alleles at this locus, phase could not be determined with certainty for all affected dogs; however, two affected dogs (C18, C19) had at least one allele that was not inherited from the founding sire.
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Discussion |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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Previous breeding studies have indicated that SRY-negative XX sex reversal in this pedigree segregates as an autosomal recessive trait with expression limited to 78,XX individuals that are homozygous for the sex reversal trait (Meyers-Wallen and Patterson 1988). Given that all affected dogs inherited this trait from C1, the founding sire of the pedigree (Figure 1), the disease-causing mutation should be identical by descent in all affected dogs. For each of the candidate gene markers noted here, the genotypes varied between affected dogs. Furthermore, assuming autosomal recessive inheritance of the sex reversal trait, one would expect that both alleles in affected dogs would be inherited from the founding sire of the pedigree. For each aforementioned candidate gene, a minimum of two affected dogs had at least one marker allele that was not inherited from the founding sire of the pedigree. These results indicate that it is unlikely that autosomal recessive mutations in or near GATA4, FOG2, LHX1, SF1, and SOX9 contain the causative mutation for SRY-negative XX sex reversal in this model.
The goat model is the only model in which a causative mutation for naturally occurring SRY-negative XX sex reversal has been identified (Pailhoux et al. 2001). However, the region homologous to goat PIS has been excluded as a candidate for the canine disorder (Kothapalli et al. 2003). No human patients with SRY-negative XX sex reversal have been identified with mutations in the regions homologous to the PIS-deleted region or in WT1, DMRT1, GATA4, FOG2, LHX1, or SF1. Mutations causing overexpression of SOX9 in XX individuals have resulted in SRY-negative XX sex reversal in humans and mice; however, few human patients with such mutations have been reported. Taken together with results of the present study, this suggests that mutations responsible for this type of sex reversal in humans and other animals remain to be identified. Thus, the canine model could lead to the discovery of novel genes in the mammalian sex determination pathway. In addition to examining other candidate genes, we are analyzing results from a genome-wide screen of the multigenerational canine pedigree in order to identify the causative gene.
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Acknowledgments |
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These studies were supported by the National Institutes of Health (R01 HD40351). This article was presented at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: William Murphy
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References |
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TopAbstractFOXL2 and PISRT1WT1 and DMRT1GATA4 and FOG2LHX1SF1SOX9Materials and MethodsResultsDiscussionReferences |
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