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Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice.

ABSTRACT: In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes.DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT-PCR. Tumor necrosis factor, alpha- induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3(+/-) mice were tested for ageRD.Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT-PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3(+/-) and Tnfaip3(+/+) mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age.The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD.

SUBMITTER: Ogando DG

PROVIDER: S-EPMC2890552 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice.

Ogando Diego G DG Dahlquist Kam D KD Alizadeh Mitra M Kunchithapautham Kannan K Li Jun J Yu Nicole N LaVail Matthew M MM Rohrer Bärbel B Vollrath Douglas D Danciger Michael M

Molecular vision 20100605

<h4>Purpose</h4>In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes.<h4>Me ...[more]

PMID: 20577653

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Project description:The first goal of the study was to find the genes that explain the difference in progression of age-related retinal degeneration (ageRD) between the inbred strains C57BL/6J-c2J (B6a) and BALB/cByJ (C). The specific goal was to find the candidate genes that explain the chromosome 6 and chromosome 10 quantitative trait loci (QTL) found in a previous study. Genes differentially expressed in retina between both strains and that map to the QTL are candidate genes. By integrating different approaches: microarray study, knowledge-based candidate gene search and screening of genetic variants, we found 35 candidate genes for chromosome 6 QTL and 14 candidate genes for chromosome 10 QTL. Five genes for each QTL were selected for sequencing and qPCR analysis. One gene, Tnfaip3 was selected for phenotypic testing and was excluded as the quantitative trait gene for chr10 QTL. The second goal of the study was to find biological processes and pathways associated with ageRD by Gene Ontology analysis of the microarray data. We concentrated on GO terms overrepresented in either strain at 8 months; the age when there is a difference in the phenotype (outer nuclear layer thickness). The GO analysis revealed that transcripts associated with inflammation, mitochondrial envelope, DNA repairing and oxidative stress were increased in the ageRD sensitive C strain with age (8 months). In the ageRD resistant B6a strain transcripts associated with antioxidant response, regulation of blood vessel size and growth factor activity were increased with age. Transcripts associated with cell remodeling and lipid metabolism were increased in either strain with age. This study aims to identify gene expression differences in posterior eyecups between the strains C and B6a at 4 months and at 8 months. Twelve animals were used per strain and time point for a total of 48 mice. Total RNA for 3 animals (6 eyes) were pooled giving a total of 4 biological replicates per strain and time point. The 4-month C samples labeled with Cy5 were hybridized with the 4-month B6a samples labeled with Cy3 (4 biological replicates); the 8-month C samples labeled with Cy5 were hybridized with the 8-month B6a samples labeled with Cy3, for 8 arrays (4 biological replicates). A second set of eight arrays were used to analyzed the same RNA samples but with the dye orientation reversed, yielding a total of 16 arrays in the experiment. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.

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Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice.

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Candidate genes for chromosomes 6 and 10 quantitative trait loci for age-related retinal degeneration in mice.

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