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Haplotype-defined linkage region for gPRA in Schapendoes dogs.

Lippmann T, Jonkisz A, Dobosz T, Petrasch-Parwez E, Epplen JT, Dekomien G - Mol. Vis. (2007)

Bottom Line: Detailed pedigree and ophthalmological data were assembled in selected Schapendoes pedigrees.Genotyping data of the microsatellite genome scan evidenced a peak two-point lod score of 4.78 for marker REN93E07 on CFA20.Although the mutation causing gPRA in Schapendoes dogs has not yet been identified, we established indirect DNA testing for gPRA in this breed based on linkage analysis data.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Ruhr-University, Bochum, Germany. tanja.lippmann@t-online.de <tanja.lippmann@t-online.de>

ABSTRACT

Purpose: In order to determine the molecular basis of canine generalized progressive retinal atrophy (gPRA), we initiated whole-genome scanning for linkage in gPRA-informative pedigrees of the Schapendoes breed.

Methods: Detailed pedigree and ophthalmological data were assembled in selected Schapendoes pedigrees. A whole-genome scan was initiated by two-point linkage analysis using microsatellite markers in combination with haplotype analyses. Mutation screening was carried out in respective candidate genes by DNA sequencing of amplified products and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).

Results: Genotyping data of the microsatellite genome scan evidenced a peak two-point lod score of 4.78 for marker REN93E07 on CFA20. Haplotype analyses inferred the gPRA locus in a 5.6 megabase (Mb) region between markers FH3358 and TL336MS. Mutation screening in the genes CACNA2D3, HT017, and WNT5A revealed no causative sequence deviations. In addition, CACNA2D3 mRNA levels were equivalent in retinas of affected and healthy dogs.

Conclusions: By genome-wide linkage analysis a region for gPRA was identified and fine-localized in Schapendoes dogs. Although the mutation causing gPRA in Schapendoes dogs has not yet been identified, we established indirect DNA testing for gPRA in this breed based on linkage analysis data.

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Related in: MedlinePlus

Schematic overview of the critical region on chromosome 20 in which the gPRA locus maps in Schapedoes dogs. On the left hand side of the chromosome the analyzed genes and their genomic location are shown, on the right hand side genotyped microsatellite markers are depicted. Location ascertained from UCSC Genome Browser (assembly: dog May 2005).
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f3: Schematic overview of the critical region on chromosome 20 in which the gPRA locus maps in Schapedoes dogs. On the left hand side of the chromosome the analyzed genes and their genomic location are shown, on the right hand side genotyped microsatellite markers are depicted. Location ascertained from UCSC Genome Browser (assembly: dog May 2005).

Mentions: Genotyping of ten microsatellite markers for all 57 dogs of the five pedigrees revealed that the "2-2-3-2-2-2-2" haplotype (marker REN149D23 to TL327MS) segregates with the gPRA trait and has a frequency of 50% in the analyzed pedigrees (Figure 1). Analysis of this haplotype placed the gPRA locus in a region between marker FH3358 and TL195IIMS. A potential double recombination event in the dog SD90 of family SD5 may be interpreted to confine the size of the critical haplotype to 5.6 Mb flanked by markers FH3358 and TL336MS. For the "recombined region" of dog SD90 no canine gene has been published in the different gene banks so far. Comparison of the canine DNA sequence of the critical region with the human genome in UCSC Genome Browser (assembly: human May 2004) shows homology with chromosome 3p. In man this region comprises candidate genes for retinitis pigmentosa (RP)-and thus also for gPRA in Schapendoes. The marker with the peak lod score REN93E07 is located in intron 7 of candidate gene CACNA2D3 (Figure 3) which spans a genomic region of about 830 kb (kb) and consists of 37 exons. The mRNA of this gene encodes the calcium channel α2δ3 subunit, which is mainly expressed in brain [19] and also in the eye UniGene. In intron 26 of the human CACNA2D3 gene the HT017/LRTM1 gene is located (Figure 3). This gene encodes the leucine-rich repeat and transmembrane domain 1. Upstream of CACNA2D3 the WNT5A gene is situated (Figure 3) encoding member 5A of the wingless-type MMTV integration site family. Sequencing of the candidate genes HT017 and WNT5A did not reveal any pathogenic mutations, neither in the coding sequences nor in intron/exon junctions of affected individuals. Five single nucleotide polymorphisms (SNPs) were detected in the CACNA2D3 gene: IVS16-7T>C, IVS23-51A>T, IVS29+18A>G, IVS29+57C>T, IVS30-57T>C. Yet, these SNPs occur in homozygous state not only in gPRA-affected dogs, but also in healthy Schapendoes (data not shown). Thus these SNPs do not cause gPRA in Schapendoes. Additionally, a thymin-insertion in intron 6 (IVS6-38_-34insT) was identified in comparison to the UCSC dog genome sequence (assembly: dog May 2005). Further investigations revealed that this insertion was present in homozygous state in healthy dogs of other breeds, implying a non-pathogenic polymorphism. Furthermore, sequencing of the CACNA2D3 cDNA from a diseased Schapendoes eye revealed no sequence deviations, thus excluding "hidden" mutations affecting the splicing process. In addition, altogether 25 kb in introns 5, 7 and 8 of the CACNA2D3 gene comprising evolutionarily conserved sequences were analyzed without any hint on the gPRA mutation in question (data not shown).


Haplotype-defined linkage region for gPRA in Schapendoes dogs.

Lippmann T, Jonkisz A, Dobosz T, Petrasch-Parwez E, Epplen JT, Dekomien G - Mol. Vis. (2007)

Schematic overview of the critical region on chromosome 20 in which the gPRA locus maps in Schapedoes dogs. On the left hand side of the chromosome the analyzed genes and their genomic location are shown, on the right hand side genotyped microsatellite markers are depicted. Location ascertained from UCSC Genome Browser (assembly: dog May 2005).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533032&req=5

f3: Schematic overview of the critical region on chromosome 20 in which the gPRA locus maps in Schapedoes dogs. On the left hand side of the chromosome the analyzed genes and their genomic location are shown, on the right hand side genotyped microsatellite markers are depicted. Location ascertained from UCSC Genome Browser (assembly: dog May 2005).
Mentions: Genotyping of ten microsatellite markers for all 57 dogs of the five pedigrees revealed that the "2-2-3-2-2-2-2" haplotype (marker REN149D23 to TL327MS) segregates with the gPRA trait and has a frequency of 50% in the analyzed pedigrees (Figure 1). Analysis of this haplotype placed the gPRA locus in a region between marker FH3358 and TL195IIMS. A potential double recombination event in the dog SD90 of family SD5 may be interpreted to confine the size of the critical haplotype to 5.6 Mb flanked by markers FH3358 and TL336MS. For the "recombined region" of dog SD90 no canine gene has been published in the different gene banks so far. Comparison of the canine DNA sequence of the critical region with the human genome in UCSC Genome Browser (assembly: human May 2004) shows homology with chromosome 3p. In man this region comprises candidate genes for retinitis pigmentosa (RP)-and thus also for gPRA in Schapendoes. The marker with the peak lod score REN93E07 is located in intron 7 of candidate gene CACNA2D3 (Figure 3) which spans a genomic region of about 830 kb (kb) and consists of 37 exons. The mRNA of this gene encodes the calcium channel α2δ3 subunit, which is mainly expressed in brain [19] and also in the eye UniGene. In intron 26 of the human CACNA2D3 gene the HT017/LRTM1 gene is located (Figure 3). This gene encodes the leucine-rich repeat and transmembrane domain 1. Upstream of CACNA2D3 the WNT5A gene is situated (Figure 3) encoding member 5A of the wingless-type MMTV integration site family. Sequencing of the candidate genes HT017 and WNT5A did not reveal any pathogenic mutations, neither in the coding sequences nor in intron/exon junctions of affected individuals. Five single nucleotide polymorphisms (SNPs) were detected in the CACNA2D3 gene: IVS16-7T>C, IVS23-51A>T, IVS29+18A>G, IVS29+57C>T, IVS30-57T>C. Yet, these SNPs occur in homozygous state not only in gPRA-affected dogs, but also in healthy Schapendoes (data not shown). Thus these SNPs do not cause gPRA in Schapendoes. Additionally, a thymin-insertion in intron 6 (IVS6-38_-34insT) was identified in comparison to the UCSC dog genome sequence (assembly: dog May 2005). Further investigations revealed that this insertion was present in homozygous state in healthy dogs of other breeds, implying a non-pathogenic polymorphism. Furthermore, sequencing of the CACNA2D3 cDNA from a diseased Schapendoes eye revealed no sequence deviations, thus excluding "hidden" mutations affecting the splicing process. In addition, altogether 25 kb in introns 5, 7 and 8 of the CACNA2D3 gene comprising evolutionarily conserved sequences were analyzed without any hint on the gPRA mutation in question (data not shown).

Bottom Line: Detailed pedigree and ophthalmological data were assembled in selected Schapendoes pedigrees.Genotyping data of the microsatellite genome scan evidenced a peak two-point lod score of 4.78 for marker REN93E07 on CFA20.Although the mutation causing gPRA in Schapendoes dogs has not yet been identified, we established indirect DNA testing for gPRA in this breed based on linkage analysis data.

View Article: PubMed Central - PubMed

Affiliation: Human Genetics, Ruhr-University, Bochum, Germany. tanja.lippmann@t-online.de <tanja.lippmann@t-online.de>

ABSTRACT

Purpose: In order to determine the molecular basis of canine generalized progressive retinal atrophy (gPRA), we initiated whole-genome scanning for linkage in gPRA-informative pedigrees of the Schapendoes breed.

Methods: Detailed pedigree and ophthalmological data were assembled in selected Schapendoes pedigrees. A whole-genome scan was initiated by two-point linkage analysis using microsatellite markers in combination with haplotype analyses. Mutation screening was carried out in respective candidate genes by DNA sequencing of amplified products and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).

Results: Genotyping data of the microsatellite genome scan evidenced a peak two-point lod score of 4.78 for marker REN93E07 on CFA20. Haplotype analyses inferred the gPRA locus in a 5.6 megabase (Mb) region between markers FH3358 and TL336MS. Mutation screening in the genes CACNA2D3, HT017, and WNT5A revealed no causative sequence deviations. In addition, CACNA2D3 mRNA levels were equivalent in retinas of affected and healthy dogs.

Conclusions: By genome-wide linkage analysis a region for gPRA was identified and fine-localized in Schapendoes dogs. Although the mutation causing gPRA in Schapendoes dogs has not yet been identified, we established indirect DNA testing for gPRA in this breed based on linkage analysis data.

Show MeSH
Related in: MedlinePlus