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Journal of Heredity Advance Access originally published online on April 6, 2005
Journal of Heredity 2005 96(4):452-454; doi:10.1093/jhered/esi058
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© The American Genetic Association. 2005. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org.

Brief Communication

Exclusion of Lhx9 as a Candidate Gene for Sry-Negative XX Sex Reversal in the American Cocker Spaniel Model

S. Pujar, K. S. Kothapalli, E. Kirkness, R. H. Van Wormer, and V. N. Meyers-Wallen

From the J. A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Pujar, Kothapalli, Van Wormer Meyers-Wallen); Institute for Genomic Research (TIGR), Rockville, MD 20850 (Kirkness)

Address correspondence to V. N. Meyers-Wallen at the address above, or e-mail: vnm1{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
XX sex reversal is known in 17 breeds of dogs. In the American cocker spaniel, it segregates as an autosomal recessive trait, and the affected animals lack the testis determining Sry gene. In the search for an autosomal gene that causes this trait, we considered the possibility of Lhx9, a gene encoding LIM homeobox containing transcription factor 9, as a candidate gene. An American cocker spaniel pedigree showing Sry-negative XX sex reversal phenotype was genotyped with an intronic Lhx9 microsatellite marker. Segregation of the Lhx9 marker in the pedigree indicated that a mutation in canine Lhx9 is not likely to be the cause of Sry-negative XX sex reversal. In addition, using the recently available 7.6X canine genomic sequence, we report the location and genomic organization of canine Lhx9.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sry-negative XX sex reversal is known in at least 17 breeds of dogs (Kuiper et al. 2004; Melniczek et al. 1999; Meyers-Wallen 1999). Though Sry-negative XX sex reversal has been shown to be inherited as an autosomal recessive trait in the American cocker spaniel, with affected animals invariably lacking the Sry gene (Meyers-Wallen 1999), the causal gene remains elusive. An obvious approach is to determine whether there is linkage between the XX sex reversal phenotype and genes known to play a role in sex determination.

Lhx9, which belongs to the LIM homeobox domain family of transcription factors, has been described as essential to gonad formation in mice (Birk et al. 2000). In rats, Lhx9 expression is limited to the somatic cell lineage, which gives rise to the Sertoli and granulosa cells. Its expression diminishes in both sexes when these cells begin to differentiate. In the chick, Lhx9 is expressed in the ovarian cortex and, following hatching, in follicular cells of the earliest follicles (Oreal et al. 2002).

The objective of this study was to determine whether an Lhx9 marker allele segregates with the affected phenotype within this pedigree, as a first indication of whether a mutation in Lhx9 is causative of Sry-negative XX sex reversal in the American cocker spaniel model. This note describes the genotyping of a subset of the canine pedigree of Sry-negative XX sex reversal with a polymorphic marker designed from a canine sequence that showed homology to human LHX9. In addition, we report the location and presumptive sequence of canine Lhx9.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pedigree
A brief history of the pedigree used in this study is given in Kothapalli et al. (2003). The founder sire in the pedigree is C1, an American cocker spaniel. Because Sry-negative XX sex reversal is inherited as an autosomal recessive trait and the causal mutation originates solely from this founder sire, alleles for a linked marker should be identical by descent in all affected animals.

Primers
Canine Lhx9 sequence was initially obtained from GenBank (AACN010306525) and its identity verified by sequence similarity to the human gene (GenBank AY273889). Primers flanking a microsatellite (caaaa)5 in the canine Lhx9 genomic sequence were designed with PrimerSelect software (DNASTAR): forward 5'-TGG TGA GCA GGA CAA AGA A-3' and reverse 5'-CAG CCG TAA ATA ATA ATC ATA A-3'. The forward primer was 5'-end labeled with a 6-FAM fluorescent tag (IDT).

Subsequently, the online 7.6X canine genome sequence became available, and the presumptive canine Lhx9 exons were identified (Figure 1) based on homology to those of human LHX9 (Ensembl transcript #ENST00000271609). The primers shown amplify the intronic canine Lhx9 marker located between nucleotide positions 7543727 and 7544011 on CFA7.



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  		Figure 1.. (A) Presumptive genomic organization of canine Lhx9, spanning nucleotide positions 169025 to 180619 of the canine contig #13294 on CFA7 (GenBank accession number AAEX01013295). E1 to E6 are exons 1 to 6. The hatched box (M) indicates the microsatellite marker location in intron 3. (B) Genomic organization of human LHX9 (Ensembl transcript ID #ENST00000271609). E1 to E6 are exons 1 to 6 (located between positions 195169921 and 195186648 on HSA 1).

 
PCR
A 30-µl reaction contained 50 ng DNA template, 1 µM of each primer, 3 µl of 10x polymerase chain reaction (PCR) buffer (Perkin-Elmer Life Sciences), 3.0 mM MgCl2, 0.1 mM each of dNTP and 1.5 U of Taq DNA polymerase (Ampli Taq II, Perkin-Elmer Life Sciences). The PCR was carried out in a thermal cycler (PCR Express, Hybaid) with an initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 51°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 2 min.

Genotyping
A 3-µl aliquot of the PCR reaction was denatured along with 1 µl of TAMRA size standard (PRISM Genescan-500, Applied Biosystems) and 11 µl deionized formamide. Following capillary electrophoresis of denatured samples (ABI-310 genetic analyzer, Applied Biosystems), PCR product sizes relative to the internal TAMRA standard were determined using software (Genescan version 2.1, Perkin-Elmer Life Sciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Location and Composition of Canine Lhx9
Based on homology to human LHX9, the canine Lhx9 was found to be located between nucleotide positions 169025 and 180619 in canine contig #13294 on CFA7 (GenBank accession number AAEX01013295). Based on sequence similarity to the human gene, the location of canine Lhx9 is between nucleotide positions 7535698 and 7552383 on CFA7 in the canine genome database (www.ensembl.org/canis_familiaris). The canine Lhx9 microsatellite marker was identified in intron 3 of the presumptive canine Lhx9 between nucleotide positions 172306 and 172589 (Figure 1) of the above-mentioned contig. In silico PCR (http://genome.ucsc.edu/cgi-bin/hggateway) placed the marker between nucleotide positions 7543727 and 7544011 on CFA7.

When individual exons of human LHX9 were compared to the canine Lhx9 sequence, the latter was found to be comprised of six exons (as in human LHX9) with five of the exons (E2 through E6) showing 95%, 97%, 94%, 94%, and 97% sequence similarity, respectively, to those of human LHX9. 5' and 3' untranslated regions and upstream regulatory regions were not included in the sequence comparison. Canine and human LHX9 introns showed homology to a lesser extent, but only in some regions (data not shown).

LHX9 maps to human chromosome 1q31.3, which is syntenic to the region between markers REN97M11 and FH2266 of canine autosome 7 (CFA7; Guyon et al. 2003).

Polymorphism
Two Lhx9 marker alleles (269 bp and 279 bp) were found in the pedigree (Figure 2). The smaller allele was present in homozygosity in C1, the founder sire. Of the 10 affected dogs screened in this pedigree subset, only 3 (C8, C18, and C19) had the same genotype as the founder sire at this locus. Three other affected dogs (C9, C14, and C16) were homozygous for the larger allele, and the remaining four affected dogs were heterozygous at this locus.



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  		Figure 2.. Pedigree showing the segregation of canine Lhx9 marker alleles. Genotypes at the marker locus are given below the symbol of each animal as 1 (for the 269-bp allele) or 2 (for the 279-bp allele). The symbol ? indicates an unavailable sample. C1 is American cocker spaniel founder sire and the source of sex reversal trait in the pedigree. B1–B4 are beagles.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have deduced the position and exon composition of canine Lhx9 based on sequence similarity of a canine contig with human LHX9. The exons of the human and canine genes show a high degree of sequence similarity. A microsatellite marker present in intron 3 of canine Lhx9 was used to genotype a canine pedigree segregating for the Sry-negative XX sex reversal phenotype. The sex reversal trait in this pedigree is transmitted solely from the founder sire, and thus the causative mutation should be identical by descent in affected dogs. Seven of 10 affected dogs screened had a genotype at the marker locus that was different from that of the founder sire. In addition, affected animals had all possible combinations of alleles (1, 1; 1, 2; 2, 2) at this locus. Therefore, we conclude that an Lhx9 mutation is unlikely to be causative of Sry-negative XX sex reversal in this model.

While more candidate genes are under consideration, it is possible that a hitherto unknown gene could be involved. As a first step to search for such a gene throughout the canine genome, we have carried out an initial genome-wide scan using microsatellite markers and are presently analyzing these data.


   Acknowledgments
 
We thank Anita Hesser for manuscript preparation. These studies were supported by a grant from the National Institutes of Health (R01 HD40351).


   Footnotes
 
Corresponding Editor: Elaine Ostrander

Received October 20, 2004
Accepted January 3, 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

    Birk OS, et al., 2000. Nature 403(6772):909–913.[CrossRef][Medline]

    Guyon, et al., 2003. Proc Natl Acad Sci USA 100(9):5296–5301.[Abstract/Free Full Text]

    Kuiper, et al., 2004. Vet J (in press).

    Mazaud S, et al., 2002. Gene Express Pat 2:373–377.

    Melniczek JR, et al., 1999. J Vet Intern Med 13:564–569.[CrossRef][Web of Science][Medline]

    Meyers-Wallen VN, 1999. Mol Reprod Dev 53:266–273.[CrossRef][Web of Science][Medline]

    Oreal E, et al., 2002. Dev Dynam 225:221–232.[CrossRef][Web of Science][Medline]


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