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Assessment of canine BEST1 variations identifies new mutations and establishes an independent bestrophinopathy model (cmr3).

Zangerl B, Wickström K, Slavik J, Lindauer SJ, Ahonen S, Schelling C, Lohi H, Guziewicz KE, Aguirre GD - Mol. Vis. (2010)

Bottom Line: Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function.In contrast, cmr1 is found in multiple related breeds.These results indicate that cmr has a larger impact on the general dog population than was initially suspected.

View Article: PubMed Central - PubMed

Affiliation: Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. bzangerl@vet.upenn.edu

ABSTRACT

Purpose: Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype-phenotype associations.

Methods: Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype.

Results: Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso.

Conclusions: A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the novel cmr3 mutation is predicted to be based on a distinctly different molecular mechanism. So far cmr2 and cmr3 are exclusive to a single dog breed each. In contrast, cmr1 is found in multiple related breeds. Additional sequence alterations identified in exon 10 of cBEST1 in other breeds exhibit potential disease-causing features. The inherent genetic and phenotypic variation observed with retinal disorders in canines is complicated further by cmr3 being one of four distinct genetic retinal traits found to segregate in LH. Thus, a combination of phenotypic, molecular, and population analysis is required to establish a strong phenotype-genotype association. These results indicate that cmr has a larger impact on the general dog population than was initially suspected. The complexity of these models further confirms the similarity to human bestrophinopathies. Moreover, analyses of multiple canine models will provide additional insight into the molecular basis underlying diseases caused by mutations in BEST1.

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Mutations identified in LH affected with canine multifocal retinopathy (cmr3). A C1388del/Pro463fs mutation (A, boxed) and linked G1466T/Gly489Val nucleotide substitution (B, boxed) were identified in canine bestrophin 1 (cBEST1) exon 10 of cmr3-affected Lapponian herder (LH). Wild-type sequence and resulting amino acids are identified on top (codons are separated by dotted lines), while the mutations and resulting amino acid changes are noted on the bottom. Note that the Gly489Val substitution leads to a stop codon within the Pro463fs altered reading frame. The carrier sequence in the middle is heterozygous for both changes. WT=wild type, C=carrier, A=cmr3 affected. C: Conservation of the nucleotide sequence between species. D: Comparison of partial bestrophin exon 10, amino acid 436 to 507 between different species demonstrates the conservation of identified variants. Positions 438, 440, 463, 473, 489, and 505 are highlighted in red. Dog=Canis familiaris, Cow=Bos taurus, Pig=Sus scrofa, Human=Homo sapiens, Chimp=Pan troglodytes, Macaca=Macaca fascicularis. “.”=position identical to Dog.
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f3: Mutations identified in LH affected with canine multifocal retinopathy (cmr3). A C1388del/Pro463fs mutation (A, boxed) and linked G1466T/Gly489Val nucleotide substitution (B, boxed) were identified in canine bestrophin 1 (cBEST1) exon 10 of cmr3-affected Lapponian herder (LH). Wild-type sequence and resulting amino acids are identified on top (codons are separated by dotted lines), while the mutations and resulting amino acid changes are noted on the bottom. Note that the Gly489Val substitution leads to a stop codon within the Pro463fs altered reading frame. The carrier sequence in the middle is heterozygous for both changes. WT=wild type, C=carrier, A=cmr3 affected. C: Conservation of the nucleotide sequence between species. D: Comparison of partial bestrophin exon 10, amino acid 436 to 507 between different species demonstrates the conservation of identified variants. Positions 438, 440, 463, 473, 489, and 505 are highlighted in red. Dog=Canis familiaris, Cow=Bos taurus, Pig=Sus scrofa, Human=Homo sapiens, Chimp=Pan troglodytes, Macaca=Macaca fascicularis. “.”=position identical to Dog.

Mentions: Initially, affected animals from selected breeds with typical cmr changes and three LH that were obligate cmr carriers (Table 1) had the complete cBEST1 coding region and corresponding exon–intron boundaries sequenced. Several known and novel (NCBI ss250608388-ss250608389, ss250608394-ss250608401) polymorphisms were identified in these samples (Table 2). Of these, two coding changes that differ from the WT cBEST1 sequence were found homozygous in affected LH; a deletion at nucleotide position 1,388 of the open reading frame (Figure 3A; NCBI ss250608397), and a substitution at nucleotide position 1,466 (Figure 3B; NCBI ss250608399). The C1388del results in a frame shift (Pro463fs) introducing a new stop codon at amino acid 490. The G1466T substitution by itself leads to a conservative change in the amino acid sequence (Gly489Val), which is predicted to change the protein function with only marginal significance (SIFT p=0.05; Table 3). In combination with the C1388del, however, the G1466T substitutions results in an additional stop codon at amino acid position 489 within the shifted reading frame (Gly489X). Since the mutations have only been found in complete linkage disequilibrium, we conclude that the combination of changes results in the disease we now refer to as cmr3. Notably, both positions appear highly conserved in the BEST1 gene of different species at the nucleotide (Figure 3C) and amino acid level (Figure 3D).


Assessment of canine BEST1 variations identifies new mutations and establishes an independent bestrophinopathy model (cmr3).

Zangerl B, Wickström K, Slavik J, Lindauer SJ, Ahonen S, Schelling C, Lohi H, Guziewicz KE, Aguirre GD - Mol. Vis. (2010)

Mutations identified in LH affected with canine multifocal retinopathy (cmr3). A C1388del/Pro463fs mutation (A, boxed) and linked G1466T/Gly489Val nucleotide substitution (B, boxed) were identified in canine bestrophin 1 (cBEST1) exon 10 of cmr3-affected Lapponian herder (LH). Wild-type sequence and resulting amino acids are identified on top (codons are separated by dotted lines), while the mutations and resulting amino acid changes are noted on the bottom. Note that the Gly489Val substitution leads to a stop codon within the Pro463fs altered reading frame. The carrier sequence in the middle is heterozygous for both changes. WT=wild type, C=carrier, A=cmr3 affected. C: Conservation of the nucleotide sequence between species. D: Comparison of partial bestrophin exon 10, amino acid 436 to 507 between different species demonstrates the conservation of identified variants. Positions 438, 440, 463, 473, 489, and 505 are highlighted in red. Dog=Canis familiaris, Cow=Bos taurus, Pig=Sus scrofa, Human=Homo sapiens, Chimp=Pan troglodytes, Macaca=Macaca fascicularis. “.”=position identical to Dog.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008713&req=5

f3: Mutations identified in LH affected with canine multifocal retinopathy (cmr3). A C1388del/Pro463fs mutation (A, boxed) and linked G1466T/Gly489Val nucleotide substitution (B, boxed) were identified in canine bestrophin 1 (cBEST1) exon 10 of cmr3-affected Lapponian herder (LH). Wild-type sequence and resulting amino acids are identified on top (codons are separated by dotted lines), while the mutations and resulting amino acid changes are noted on the bottom. Note that the Gly489Val substitution leads to a stop codon within the Pro463fs altered reading frame. The carrier sequence in the middle is heterozygous for both changes. WT=wild type, C=carrier, A=cmr3 affected. C: Conservation of the nucleotide sequence between species. D: Comparison of partial bestrophin exon 10, amino acid 436 to 507 between different species demonstrates the conservation of identified variants. Positions 438, 440, 463, 473, 489, and 505 are highlighted in red. Dog=Canis familiaris, Cow=Bos taurus, Pig=Sus scrofa, Human=Homo sapiens, Chimp=Pan troglodytes, Macaca=Macaca fascicularis. “.”=position identical to Dog.
Mentions: Initially, affected animals from selected breeds with typical cmr changes and three LH that were obligate cmr carriers (Table 1) had the complete cBEST1 coding region and corresponding exon–intron boundaries sequenced. Several known and novel (NCBI ss250608388-ss250608389, ss250608394-ss250608401) polymorphisms were identified in these samples (Table 2). Of these, two coding changes that differ from the WT cBEST1 sequence were found homozygous in affected LH; a deletion at nucleotide position 1,388 of the open reading frame (Figure 3A; NCBI ss250608397), and a substitution at nucleotide position 1,466 (Figure 3B; NCBI ss250608399). The C1388del results in a frame shift (Pro463fs) introducing a new stop codon at amino acid 490. The G1466T substitution by itself leads to a conservative change in the amino acid sequence (Gly489Val), which is predicted to change the protein function with only marginal significance (SIFT p=0.05; Table 3). In combination with the C1388del, however, the G1466T substitutions results in an additional stop codon at amino acid position 489 within the shifted reading frame (Gly489X). Since the mutations have only been found in complete linkage disequilibrium, we conclude that the combination of changes results in the disease we now refer to as cmr3. Notably, both positions appear highly conserved in the BEST1 gene of different species at the nucleotide (Figure 3C) and amino acid level (Figure 3D).

Bottom Line: Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function.In contrast, cmr1 is found in multiple related breeds.These results indicate that cmr has a larger impact on the general dog population than was initially suspected.

View Article: PubMed Central - PubMed

Affiliation: Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. bzangerl@vet.upenn.edu

ABSTRACT

Purpose: Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype-phenotype associations.

Methods: Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype.

Results: Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso.

Conclusions: A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the novel cmr3 mutation is predicted to be based on a distinctly different molecular mechanism. So far cmr2 and cmr3 are exclusive to a single dog breed each. In contrast, cmr1 is found in multiple related breeds. Additional sequence alterations identified in exon 10 of cBEST1 in other breeds exhibit potential disease-causing features. The inherent genetic and phenotypic variation observed with retinal disorders in canines is complicated further by cmr3 being one of four distinct genetic retinal traits found to segregate in LH. Thus, a combination of phenotypic, molecular, and population analysis is required to establish a strong phenotype-genotype association. These results indicate that cmr has a larger impact on the general dog population than was initially suspected. The complexity of these models further confirms the similarity to human bestrophinopathies. Moreover, analyses of multiple canine models will provide additional insight into the molecular basis underlying diseases caused by mutations in BEST1.

Show MeSH
Related in: MedlinePlus