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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Exp Eye Res. 2011 Nov 22;94(1):187–191. doi: 10.1016/j.exer.2011.11.005

A novel ARMS2 Splice Variant is identified in Human Retina

Gaofeng Wang 1,*, William K Scott 1, Patrice Whitehead 1, Brenda L Court 1, Jaclyn L Kovach 2, Stephen G Schwartz 2, Anita Agarwal 3, Sander Dubovy 2, Jonathan L Haines 4, Margaret A Pericak-Vance 1
PMCID: PMC3259277  NIHMSID: NIHMS340894  PMID: 22138417

Age-related macular degeneration (AMD, MIM 603075) is the major cause of irreversible visual impairment in developed countries. AMD damages photoreceptors located in the central region of the retina (the macula), resulting in an impairment of central visual acuity (Jager et al., 2008). A locus at chromosome 10q26 has been consistently linked to the disease and represents one of the two strongest genetic effects discovered (Weeks et al., 2001; Majewski et al., 2003; Weeks et al., 2004; Kenealy et al., 2004; Fisher et al., 2005). However, it has been difficult to identify with certainty the genetic variant at this locus biologically responsible for modulating risk of AMD. Studies have shown that the AMD-associated haplotype includes thesingle nucleotide polymorphisms (SNPs) rs10490924 in gene ARMS2 (Age-related maculopathy susceptibility 2, previously known as hypothetical LOC387715) and rs11200638 in the promoter region of HTRA1 (High-temperature requirement protein A1) (Jakobsdottir et al., 2005; Rivera et al., 2005; Schmidt et al., 2006; Yang et al., 2006; Dewan et al., 2006; Kanda et al., 2007; Fritsche et al., 2008). Unfortunately, the variations in genes ARMS2 and HTRA1 are in such strong linkage disequilibrium (LD) that their effects are indistinguishable using statistical analysis. While studies have suggested a functional effect for each SNP, evidence that the HTRA1 polymorphism is functional and influences gene expression is inconsistent (Yang et al., 2006; Chan et al., 2007; Kanda et al., 2007; Chowers et al., 2008; Tuo et al., 2008; Kanda et al., 2010; Wang et al., 2010; Yang et al., 2010; Friedrich et al., 2011). This inconsistency, combined with the observation that the ARMS2 rs10490924 variant changes the corresponding amino acid sequence of the protein, suggests that ARMS2 is the more likely AMD gene in this region, although this continues to be debated in the literature.

The basic function of ARMS2 in human retina remains largely unknown. One major reason is the lack of a homologous gene in commonly used model animals such as mouse or Drosophila (Ding et al., 2009). Currently ARMS2 is only annotated in the genomes of humans and higher primates (Francis et al., 2008). For an accurate gene annotation, expressed sequence tags (EST) are required in addition to sequence similarity comparisons and computational predictions (Mount 2000; Ashurst et al., 2003). However, in genome databases, all ARMS2 ESTs are from the placenta and HT1080 cell (a fibrosarcoma cell line) and no EST from the human retina is available, suggesting that the annotation of the ARMS2 gene in human retina needs experimental verification. Part of ARMS2 transcripts has been observed using RT-PCR by previous studies (Rivera et al., 2005; Kanda et al., 2007; Fritsche et al., 2008; Wang et al., 2010; Yang et al., 2010). We therefore examined the full-length ARMS2 transcript in human retinas.

The 5′ and 3′ ends of gene transcripts provide important information about the transcription start site and stop site respectively. To verify the sequence of the ARMS2 transcript, and to search for alternative ARMS2 transcripts, we performed 5′ RACE (rapid amplification of complementary DNA ends) and 3′ RACE assays. The premade adaptor-ligated Marathon-Ready human retina cDNA (Clontech) was used as a template to perform both 5′ RACE and 3′ RACE. The cDNA library is a pool of 99 Caucasian samples. In combination with AP1 and AP2 primers (provided with the cDNA by Clontech), the two reverse primers that were used in the nested PCR cycles are 5′-GGATGATAGACAGTGTCAGGTGGT-3′ and 5′-TCAGTTACCATCGGTCCTGGGTATAGGCGCAGCAT-3′. The 5′ RACE PCR product showed one band at ~400bp and the sequence of the band was perfectly aligned to the ARMS2 region (Figure 1). PCR bands were cloned into pCR4-topo vectors. Eight independent colonies from the band and eight independent colonies from the smears were sequenced. Analysis of the sequences of the smear below the ~400bp band showed that they are non-specific PCR products. The annotated transcription start site (TSS) for ARMS2 in the database (UCSC Genome Browser, NCBI GRCh37) is at Chr10:124204169, which is largely based on the sequence of ESTs from placenta. The 5′ RACE data showed that the aligned sequence starts at Chr10:124203949. This result indicates that the TSS for ARMS2 in the retina is 220bp upstream from the TSS annotated in the genome databases.

Figure 1.

Figure 1

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The initial transcription site of ARMS2 gene in human retina is 220bp upstream than annotated in UCSC genome browser. A, gel image of 5′ RACE B, sequence of the 5′ RACE band (arrow); the Marathon cDNA adaptor sequence is in italic and primer sequences for nested PCR are underlined; C, Sequence alignment of 5′ RACE results and ARMS2 gene region.

We further used the Marathon-Ready human retina cDNA (Clontech) for 3′ RACE. In combination with AP1 and AP2 primers, the two forward primers that were used in the first and the second PCR cycles are 5′-CCACATTATGTCCCTGTACCCTACAT-3′ and 5′-CACTGTCTATCATCCACACTGCAGCA-3′. The 3′ RACE PCR produced one band at ~500bp and the sequence of the band was perfectly aligned to the ARMS2 region (Figure 2). The 3′ RACE confirmed that the polyadenylation site of ARMS2 transcript is indeed at Chr10:124206858 as annotated in the database (UCSC Genome Browser, NCBI GRCh37).

Figure 2.

Figure 2

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Validation of polyadenylation site of ARMS2 transcript in human retina. A, gel image of 3′ RACE; B, sequence of the 3′ RACE band; the Marathon cDNA adaptor sequence is in italic and primer sequences for nested PCR are underlined; C, Sequence alignment of 3′ RACE results and ARMS2 gene region.

To examine the full-length ARMS2 transcripts, we designed “ARMS2-full” primers (Forward: 5′-TTTTTCAAATCCCTGGGTCTCT -3′; Reverse: 5′-AGAGAAAGGAGGGCAAGAAAAC-3′) near the 5′ and 3′ ends of the transcripts based on the RACE data. Using human retinal cDNA, the RT-PCR showed a specific band at ~800 bp which is smaller than the expected size of 966 bp. We then sequenced the ~800 bp band. Aligned to the UCSC genome database by BLAT (BLAST-like alignment tool), the sequence of the band indicated: (1) it matched the extended 5′ end of the ARMS2 transcript obtained from RACE assays, suggesting the ARMS2 transcript in retina is indeed 220bp longer at the 5′ end compared to the RNA sequence annotated in the database; (2) the first 187 nucleotides of exon 2 were spliced out. This result was replicated by RT-PCR using the “ARMS2-full” primers and by sequencing 4 additional human retinal cDNA samples. No deletion in the corresponding region of exon 2 was found by sequencing genomic DNA of the retinas (data not shown). This result suggests a novel splice variant of ARMS2 transcripts.

To further characterize ARMS2 transcription, we designed “ARMS2-part” primers (Forward: 5′-CCCTACATGCTGCGCCTATACCCA-3′; Reverse: 5′-TGCACAGAGCAGAAGATGCATCCAATTG-3′) to capture both splice variants of ARMS2. RT-PCR of human retinal cDNA produced two bands at ~400 bp and ~600 bp. The sequence of the bigger band completely matched the annotated cDNA sequence (designated as isoform-A), while the sequence of the smaller band lacked the same 187 nucleotides of the 5′ end of exon 2 compared to the annotated ARMS2 transcript. This result strongly supports a novel splice variant (designated as isoform-B) of ARMS2. By sequence alignment using BLAT, we found that both isoforms use the splice donor site GT at genomic DNA position chr10:124,214,541–124,214,542 (NCBI GRCh37). Isoform-A uses the conventional splice acceptor site AG at the genomic DNA position chr10:124,216,421–124,216,422. Isoform-B uses the alternate splice acceptor site TG at the genomic DNA position chr10:124,216,608–124,216,609 (Figure 3). TG dinucleotides have been reported as alternative 3' splice sites (Szafranski et al., 2007). The sequence of one ARMS2 EST (BG194076) also suggests the same splice site (Harrington et al., 2001). These results indicate that ARMS2 expression is more complicated than previously thought, with multiple transcripts.

Figure 3.

Figure 3

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The RT-PCR gel image and sequence trace showing the novel splicing site for the ARMS2 transcripts which result in isoform-A and isoform-B mRNAs.

The presence of the annotated ARMS2 transcript in retinas has been evaluated by quantitative RT-PCR or other methods (Rivera et al., 2005; 2006; Kanda et al., 2007; Fritsche et al., 2008, Wang et al., 2010; Yang et al., 2010; Friedrich et al., 2011). However, the expression of the novel splice variant of ARMS2 and more importantly, the ratio of the two ARMS2 isoforms has not been reported previously. We used quantitative RT-PCR to quantify the ARMS2 transcripts in human retina. The quantitative RT-PCR assays were custom designed to selectively amplify cDNA fragments of both exons. We first measured ARMS2 transcript isoforms in punched eye tissues containing choroid/retinal pigment epithelium (RPE)/retina from 5 donor eyes without any clinical history of eye disease by quantitative RT-PCR. The results show that isoform-B is expressed at much higher levels than the isoform-A (Figure 4A, showing the amplification curve of ARMS2 isoforms). The Δcycle is about 3-4. Hence, the concentration of isoform A is about1/8 (2-3) to 1/16 (2-4) of the concentration of isoform B (Kinoshita et al., 1992). This result suggests that the isoform-B is the major transcript form of ARMS2 in human retinas.

Figure 4.

Figure 4

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Preferential expression of ARMS2 in choroid/RPE. A, Amplification curves of quantitative RT-PCR show that ARMS2 isoform-B is expressed at a much higher level than the isoform-A in 5 donor eyes without any known eye disease history. B, ARMS2 (total and isoform-B) is preferentially expressed in choroid/RPE than retina (* P<0.05). The data was presented as mean ± SD (standard deviation).

The expression pattern of ARMS2 transcripts in retinal layers has not has not yet been presented in the peer-reviewed literature. Previous findings on the distribution of ARMS2 expression in human retina were performed at the protein level using custom-made antibodies and the results are inconsistent and inconclusive (Fritsche et al., 2008; Kortvely et al., 2010). To examine ARMS2 expression in retina, we carefully separated retina from choroid/RPE in 5 donor eyes without any clinical history of eye disease. Quantitative RT-PCR (Figure 4B) showed that ARMS2 is preferentially expressed in choroid/RPE compared to retina (28.3-fold difference, p<0.05). This difference is due to significant differences in isoform-B expression (30.3-fold difference, p<0.05 by Student t test), but not isoform-A (4.7-fold difference, not significant). This result suggests that ARMS2 (particularly isoform-B) is expressed in a higher level in choroid/RPE than in retina.

Accurate gene annotation is critical to functional analysis of associated genetic variants in AMD. Little is known about the basic function of ARMS2 due to lack of complete knowledge of ARMS2 transcription. In this study, we found that the ARMS2 gene transcription starts 220bp upstream from the annotated start site. Importantly, we identified a novel ARMS2 splice variant which uses TG at the genomic DNA position chr10:124,216,608–124,216,609 as an alternate splice acceptor site. The novel ARMS2 splice variant is the major transcript isoform in retina, which is expressed in a higher level in choroid/RPE than in retina. Further studies are required to understand the resulting protein isoforms’ function and cellular localization, and the potential pathogenic role of ARMS2 transcript splice variants in the development of AMD.

ACKNOWLEDGEMENTS

We thank all the individuals and their families who donated eyes for the study. We also thank the Florida Lions Eye Bank which provides the eye tissues for research purposes. We appreciate Dr. Dale Hedges (University of Miami) for his critical suggestions on data interpretation. This research was supported by National Institutes of Health grants (2R01EY012118-11 to M.A.P.-V., J.L.H., W.K.S and A.A.) and was partially supported by NIH center grant P30-EY014801 and by an unrestricted grant to the University of Miami from Research to Prevent Blindness, New York, NY.

S.G.S. is a consultant for Alimera Sciences and Bausch + Lomb, and is co-inventor on a patent related to use of genetic data to detect steroid responders, licensed to IC Labs. W.K.S., M.A.P-V., A.A., and J.L.H. are co-inventors on a patent related to use of ARMS2 genotypes for diagnosis of AMD, licensed to ArcticDx.

Footnotes

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All other authors have no conflicts of interest to declare.

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