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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.In addition, CACNA2D3 mRNA levels were equivalent in retinas of affected and healthy dogs.

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

Haplotypes of the gPRA Schapendoes families SD1-5 as established by microsatellite markers for chromosome 20. Affected dogs are represented by black, unaffected by white and those with known carrier status are represented by half-filled symbols. Circles represent females and squares represent males. Genotype that could not be ascertained are scored as "0". Black bars indicate the affected haplotype. In the box, genotyped markers and the disease haplotype are indicated. The observed recombination events evidence the disease causing locus in the region between markers FH3358 and TL195IIMS.
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f1: Haplotypes of the gPRA Schapendoes families SD1-5 as established by microsatellite markers for chromosome 20. Affected dogs are represented by black, unaffected by white and those with known carrier status are represented by half-filled symbols. Circles represent females and squares represent males. Genotype that could not be ascertained are scored as "0". Black bars indicate the affected haplotype. In the box, genotyped markers and the disease haplotype are indicated. The observed recombination events evidence the disease causing locus in the region between markers FH3358 and TL195IIMS.

Mentions: For the WGS highly informative autosomal microsatellite markers were analyzed from the minimal screening set 2 (MSS-2) [12]. Microsatellites for fine mapping (Table 1, Figure 1) were identified using published dog markers [15], the dog genome sequence (May 2005) and the Tandem Repeats Finder included in the UCSC Genome Browser. Only microsatellites with a repeat length exceeding 15 units were selected. PCR primers were designed using Primer Express software (PE Biosystems). For PCR we used the "tailed primer PCR" as described before [16]. This method requires three oligonucleotides for amplification: 1. tailed forward primer (tailed F), 2. reverse primer and 3. labeled primer (labeled F) corresponding to the 5'-tail sequence of tailed F. PCR conditions were as follows: 1-PCR buffer (Genecraft, Lüdinghausen, Germany), 0.2 mM each dNTP, 1.5 to 3 mM MgCl2, 0.2 pmol tailed F, 2.5 pmol labeled F, 2.5 pmol reverse primer, 0.5 U BioTherm DNA Polymerase (Genecraft) and 50 ng DNA. PCRs were performed in 96-well microtiter plates (Thermowell Costar Corning, NY). Each well contained a reaction volume of 10 μl. A "touchdown" PCR procedure was applied in a thermocycler (Biometra, Göttingen, Germany): initial denaturation (5 min at 95 °C), two initial cycles 6 °C and 3 °C above the annealing temperature, 40 cycles of 95 °C (30 s), annealing temperature of 53 °C (30 s), elongation at 72 °C (30 s) and a final elongation step at 72 °C (3 min).


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)

Haplotypes of the gPRA Schapendoes families SD1-5 as established by microsatellite markers for chromosome 20. Affected dogs are represented by black, unaffected by white and those with known carrier status are represented by half-filled symbols. Circles represent females and squares represent males. Genotype that could not be ascertained are scored as "0". Black bars indicate the affected haplotype. In the box, genotyped markers and the disease haplotype are indicated. The observed recombination events evidence the disease causing locus in the region between markers FH3358 and TL195IIMS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Haplotypes of the gPRA Schapendoes families SD1-5 as established by microsatellite markers for chromosome 20. Affected dogs are represented by black, unaffected by white and those with known carrier status are represented by half-filled symbols. Circles represent females and squares represent males. Genotype that could not be ascertained are scored as "0". Black bars indicate the affected haplotype. In the box, genotyped markers and the disease haplotype are indicated. The observed recombination events evidence the disease causing locus in the region between markers FH3358 and TL195IIMS.
Mentions: For the WGS highly informative autosomal microsatellite markers were analyzed from the minimal screening set 2 (MSS-2) [12]. Microsatellites for fine mapping (Table 1, Figure 1) were identified using published dog markers [15], the dog genome sequence (May 2005) and the Tandem Repeats Finder included in the UCSC Genome Browser. Only microsatellites with a repeat length exceeding 15 units were selected. PCR primers were designed using Primer Express software (PE Biosystems). For PCR we used the "tailed primer PCR" as described before [16]. This method requires three oligonucleotides for amplification: 1. tailed forward primer (tailed F), 2. reverse primer and 3. labeled primer (labeled F) corresponding to the 5'-tail sequence of tailed F. PCR conditions were as follows: 1-PCR buffer (Genecraft, Lüdinghausen, Germany), 0.2 mM each dNTP, 1.5 to 3 mM MgCl2, 0.2 pmol tailed F, 2.5 pmol labeled F, 2.5 pmol reverse primer, 0.5 U BioTherm DNA Polymerase (Genecraft) and 50 ng DNA. PCRs were performed in 96-well microtiter plates (Thermowell Costar Corning, NY). Each well contained a reaction volume of 10 μl. A "touchdown" PCR procedure was applied in a thermocycler (Biometra, Göttingen, Germany): initial denaturation (5 min at 95 °C), two initial cycles 6 °C and 3 °C above the annealing temperature, 40 cycles of 95 °C (30 s), annealing temperature of 53 °C (30 s), elongation at 72 °C (30 s) and a final elongation step at 72 °C (3 min).

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.In addition, CACNA2D3 mRNA levels were equivalent in retinas of affected and healthy dogs.

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