AMD-associated variants at the chromosome 10q26 locus and the stability of ARMS2 transcripts

Emily A. Minor, Brenda L. Court, Sander Dubovy, Gaofeng Wang

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Purpose. To analyze the effect of variants including age-related macular degeneration (AMD)-associated combinative insertion/deletion polymorphism (indel) at 3′UTR of ARMS2 and possibly associated R38X on the stability of ARMS2 transcripts. Methods. ARMS2 transcription from minigene vectors carrying different alleles at variants R38X and the indel were assessed in mouse embryonic fibroblasts (MEFs). Dual luciferase assays were applied to evaluate the effect of the indel on gene expression. RT-PCR and quantitative RT-PCR (qRT-PCR) were used to measure the two ARMS2 transcripts (isoform A and isoform B) in MEFs and human retina-RPE-choroid samples (n = 83). Results. Allele X at variant R38X decreased exogenous ARMS2 transcripts in MEFs compared to allele R. In contrast, the indel did not change the level of exogenous ARMS2 transcripts. After blocking transcription by actinomycin D, R38X appeared to accelerate the degradation of ARMS2 transcripts, while the indel did not obviously affect the stability of ARMS2 transcripts compared to the wild-type (WT) allele. Dual luciferase assays further indicated that the indel did not influence gene expression. Quantitative RT-PCR results showed that there was no significant difference in two ARMS2 transcript splice isoforms among retina-RPE-choroid samples carrying different genotypes at variants R38X and the indel. Conclusions. Variant R38X, not the indel, decreases the stability of ARMS2 transcripts in vitro. However, genotypes at R38X and the indel do not obviously affect the level of ARMS2 transcripts in retina-RPE-choroid samples. These results suggest that variants R38X and the indel are less likely to play a pathogenic role in AMD by changing the level of ARMS2 transcripts.

Original languageEnglish
Pages (from-to)5913-5919
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume54
Issue number8
DOIs
StatePublished - Sep 4 2013

Fingerprint

Macular Degeneration
Choroid
Chromosomes
Alleles
Retina
Protein Isoforms
Fibroblasts
Luciferases
Polymerase Chain Reaction
Genotype
Gene Expression
Dactinomycin

Keywords

  • Age-related macular degeneration
  • ARMS2
  • Gene expression
  • Transcript stability
  • Variants

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

AMD-associated variants at the chromosome 10q26 locus and the stability of ARMS2 transcripts. / Minor, Emily A.; Court, Brenda L.; Dubovy, Sander; Wang, Gaofeng.

In: Investigative Ophthalmology and Visual Science, Vol. 54, No. 8, 04.09.2013, p. 5913-5919.

Research output: Contribution to journalArticle

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abstract = "Purpose. To analyze the effect of variants including age-related macular degeneration (AMD)-associated combinative insertion/deletion polymorphism (indel) at 3′UTR of ARMS2 and possibly associated R38X on the stability of ARMS2 transcripts. Methods. ARMS2 transcription from minigene vectors carrying different alleles at variants R38X and the indel were assessed in mouse embryonic fibroblasts (MEFs). Dual luciferase assays were applied to evaluate the effect of the indel on gene expression. RT-PCR and quantitative RT-PCR (qRT-PCR) were used to measure the two ARMS2 transcripts (isoform A and isoform B) in MEFs and human retina-RPE-choroid samples (n = 83). Results. Allele X at variant R38X decreased exogenous ARMS2 transcripts in MEFs compared to allele R. In contrast, the indel did not change the level of exogenous ARMS2 transcripts. After blocking transcription by actinomycin D, R38X appeared to accelerate the degradation of ARMS2 transcripts, while the indel did not obviously affect the stability of ARMS2 transcripts compared to the wild-type (WT) allele. Dual luciferase assays further indicated that the indel did not influence gene expression. Quantitative RT-PCR results showed that there was no significant difference in two ARMS2 transcript splice isoforms among retina-RPE-choroid samples carrying different genotypes at variants R38X and the indel. Conclusions. Variant R38X, not the indel, decreases the stability of ARMS2 transcripts in vitro. However, genotypes at R38X and the indel do not obviously affect the level of ARMS2 transcripts in retina-RPE-choroid samples. These results suggest that variants R38X and the indel are less likely to play a pathogenic role in AMD by changing the level of ARMS2 transcripts.",
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N2 - Purpose. To analyze the effect of variants including age-related macular degeneration (AMD)-associated combinative insertion/deletion polymorphism (indel) at 3′UTR of ARMS2 and possibly associated R38X on the stability of ARMS2 transcripts. Methods. ARMS2 transcription from minigene vectors carrying different alleles at variants R38X and the indel were assessed in mouse embryonic fibroblasts (MEFs). Dual luciferase assays were applied to evaluate the effect of the indel on gene expression. RT-PCR and quantitative RT-PCR (qRT-PCR) were used to measure the two ARMS2 transcripts (isoform A and isoform B) in MEFs and human retina-RPE-choroid samples (n = 83). Results. Allele X at variant R38X decreased exogenous ARMS2 transcripts in MEFs compared to allele R. In contrast, the indel did not change the level of exogenous ARMS2 transcripts. After blocking transcription by actinomycin D, R38X appeared to accelerate the degradation of ARMS2 transcripts, while the indel did not obviously affect the stability of ARMS2 transcripts compared to the wild-type (WT) allele. Dual luciferase assays further indicated that the indel did not influence gene expression. Quantitative RT-PCR results showed that there was no significant difference in two ARMS2 transcript splice isoforms among retina-RPE-choroid samples carrying different genotypes at variants R38X and the indel. Conclusions. Variant R38X, not the indel, decreases the stability of ARMS2 transcripts in vitro. However, genotypes at R38X and the indel do not obviously affect the level of ARMS2 transcripts in retina-RPE-choroid samples. These results suggest that variants R38X and the indel are less likely to play a pathogenic role in AMD by changing the level of ARMS2 transcripts.

AB - Purpose. To analyze the effect of variants including age-related macular degeneration (AMD)-associated combinative insertion/deletion polymorphism (indel) at 3′UTR of ARMS2 and possibly associated R38X on the stability of ARMS2 transcripts. Methods. ARMS2 transcription from minigene vectors carrying different alleles at variants R38X and the indel were assessed in mouse embryonic fibroblasts (MEFs). Dual luciferase assays were applied to evaluate the effect of the indel on gene expression. RT-PCR and quantitative RT-PCR (qRT-PCR) were used to measure the two ARMS2 transcripts (isoform A and isoform B) in MEFs and human retina-RPE-choroid samples (n = 83). Results. Allele X at variant R38X decreased exogenous ARMS2 transcripts in MEFs compared to allele R. In contrast, the indel did not change the level of exogenous ARMS2 transcripts. After blocking transcription by actinomycin D, R38X appeared to accelerate the degradation of ARMS2 transcripts, while the indel did not obviously affect the stability of ARMS2 transcripts compared to the wild-type (WT) allele. Dual luciferase assays further indicated that the indel did not influence gene expression. Quantitative RT-PCR results showed that there was no significant difference in two ARMS2 transcript splice isoforms among retina-RPE-choroid samples carrying different genotypes at variants R38X and the indel. Conclusions. Variant R38X, not the indel, decreases the stability of ARMS2 transcripts in vitro. However, genotypes at R38X and the indel do not obviously affect the level of ARMS2 transcripts in retina-RPE-choroid samples. These results suggest that variants R38X and the indel are less likely to play a pathogenic role in AMD by changing the level of ARMS2 transcripts.

KW - Age-related macular degeneration

KW - ARMS2

KW - Gene expression

KW - Transcript stability

KW - Variants

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